KR20020060242A - Method for obtaining nucleic acids from an environment sample, resulting nucleic acids and use in synthesis of novel compounds - Google Patents

Abstract

The present invention relates to a method of preparing nucleic acids from environmental samples, and more particularly to a method of obtaining nucleic acid libraries from samples. The invention also relates to their use in the synthesis of nucleic acids, new compounds, especially new compounds for therapeutic purposes, of nucleic acid libraries obtained by this method. The invention also relates to novel means used in the method for obtaining nucleic acids such as novel vectors and to novel methods for producing such vectors or recombinant host cells containing such nucleic acids. Finally, the present invention relates to a method for detecting a nucleic acid in a nucleic acid library produced by the method, and a nucleic acid detected by the method and a polypeptide encoded by the nucleic acid.

Description

METHODS FOR OBTAINING NUCLEIC ACIDS FROM AN ENVIRONMENT SAMPLE, RESULTING NUCLEIC ACIDS AND USE IN SYNTHESIS OF NOVEL COMPOUNDS

Since discovering the production of streptomycin by actinomycites, the investigation of novel compounds for treatment, more specifically new antibiotics, has increasingly used screening methods for metabolites produced by soil microorganisms.

This method is primarily used to isolate organisms from the ground microflora and incubate them in specially modified nutrient media and, if appropriate, the products found in culture supernatants or cell lysates with one or more prior separation and / or purification steps. Detecting the pharmaceutical activity.

Thus, the in vitro isolation and cultivation methods of the organisms that make up the ground microbiota have enabled the characterization of about 40,000 molecules so far, about half of which show biological activity.

Major products such as antibiotics (penicillin, erythromycin, actinomycin, tetracycline, cephalosporin), anticancer agents, anti-cholesterolemia or insecticides have been characterized according to these in vitro culture methods.

Therapeutic products of therapeutic microbial origin known to date originate mainly from (about 70%) actinomycite, more specifically from Streptomyces genus. However, other therapeutic compounds, such as tecoplanin , gentamicin and spinosine , are known as Micromonospora , Actinomadura , Actinoplanes , Nocardia , Streptosporangium (Streptosporangium), Kita Sato sports Ria (Kitasatosporia), or Saccharomyces Homes in North Fora were separated from the more difficult to culture microorganisms such as (Saccharomonospora).

However, it is empirically explained that characterization of new natural products synthesized by microorganisms in soil microbiota is limited in part due to the fact that the in vitro culture step usually results in the selection of known organisms.

Thus, in vitro isolation and cultivation of organisms from the land to identify new compounds has several limitations.

For example, in actinomycite, the level of rediscovering of known antibiotics is about 99%. Specifically, fluorescence microscopy technology allowed counting more than 10 ¹⁰ bacterial cells in 1 g of soil, whereas only 0.1-1% of these bacteria could be isolated after inoculation into the culture medium.

DNA recombination dynamics techniques have shown that 12,000 to 18,000 bacterial species can be contained in 1 g of soil, while so far only 5000 non-eukaryotic microorganisms have been accounted for in all habitats.

Molecular ecology studies have made it possible to amplify and clone several novel sequences of 16S rDNA from environmental DNA.

The results of these studies tripled the bacterial classification already characterized.

Currently, bacteria are divided into 40 types, some of which consist only of bacteria that cannot be cultured. These recent results provide evidence of the breadth of biodiversity of microorganisms that have not been used to date.

Recent studies have sought to overcome various obstacles in accessing the biodiversity of soil microflora, specifically including the step of culturing in vitro before separating and characterizing industrial, especially therapeutic compounds.

Thus, in some cases, methods have been developed that include extracting DNA from an organism from the land after prior separation of the organism contained in the soil sample.

After lysing bacterial cells without prior in vitro culture, the thus extracted DNA is cloned into a vector used to transfect the host organism to construct a library of DNA originating from soil bacteria.

Such libraries of recombinant clones are used to detect the presence of genes encoding therapeutic compounds or alternatively to detect the production of therapeutic compounds by such recombinant clones.

However, the methods for direct access to soil microbiota DNA described in the prior art provided disadvantages during the implementation of each of the above mentioned steps, which disadvantages the properties that significantly affect the quantity and quality of the genetic material obtained and available. Has

Prior art relating to each step for constructing a library of DNA originating from soil samples has been described in detail below, along with technical disadvantages identified by the applicant and overcome in accordance with the present invention.

1. Extraction of DNA from Soil Samples

1.1 Direct Extraction of Environmental DNA

This is essentially a method using DNA extraction techniques which are usually performed directly with environmental samples after prior in situ dissolution of the organism in the sample.

This technique has been used for samples originating from both aqueous and fresh water. This generally involves the first step of preconcentrating cells present in free or particulate form, consisting of filtering a large amount of water with a different filtration device, for example conventional membrane filtration, tangential filtration or rotary filtration or ultrafiltration. Include.

The pore size is 0.22 to 0.45 mm and often requires prefiltration to avoid clogging due to large volumes of treatment.

In the second step, harvested cells are lysed directly on the filter by enzymatic and / or chemical treatment in small amounts of solution.

This technique is described, for example, in Stein et al., 1996, Journal of Bacteriology, Vol., Which describes the cloning of genes encoding ribosome DNA and encoding transcriptional prolongation factor (EF2) from Archaebacteria in seawater plankton. 178 (3): illustrated by the study of 591-599.

Techniques for direct extraction of DNA from soil or sedimentary layers are also described, based on protocols of physical, chemical or enzymatic lysis performed in situ.

For example, U.S. Patent No. 5 824 485 (Chromaxome Corporation) describes direct chemical lysis of bacteria in samples taken by the addition of hot lysis buffer based on guanidium isothiocyanate.

International Patent Application No. WO 99/20799 (Wisconsin Alumni Research Foundation) describes an in situ lysis step of bacteria using extraction buffer containing protease and SDS.

Other techniques have also been used, such as performing several cycles of freeze-thawing of the sample followed by high pressure compression of the thawed sample. Bacterial dissolution techniques using a series of sonication, heating with microwave and heat shock steps have also been used (Picard et al., 1992).

However, the aforementioned prior art for direct extraction of DNA has very variable efficacy in terms of quantitative and qualitative.

Thus, chemical or enzymatic treatment of samples in situ has the disadvantage of dissolving only a specific category of microorganisms due to the selective resistance of indigenous various microorganisms to the dissolution step due to their heterogeneous form.

Thus, Gram-positive bacteria tolerate hot SDS detergent treatment while substantially all Gram-negative cells are lysed.

In addition, some of the direct extraction protocols described above promote the adsorption of the extracted nucleic acid onto the mineral particles, thereby significantly reducing the amount of available DNA.

In addition, although some of the prior art protocols describe mechanical treatment steps for dissolving microorganisms in a sample taken, this mechanical dissolution step is carried out systematically in liquid medium in extraction buffer, which is the best for the organism diversity present in the sample. It does not allow good homogenization of the starting sample into particulate form, which makes it accessible. Grinding tests were also performed with raw soil samples using glass beads, but the amount of extracted DNA was small.

It has been observed according to the invention that the first step of in situ mechanical lysis in liquid medium adversely affects the amount of DNA that can be extracted.

The amount of DNA that can be used directly for cloning in recombinant vectors also depends on the purification step after extraction.

In the prior art, the extracted DNA is then precipitated in the presence of ammonium acetate or potassium acetate, for example by using polyvinylpolypyrrolidone, by centrifugation with a cesium chloride gradient, or in particular with hydroxyapatite supports, ion-exchange Purification by chromatography on a column or molecular sieve, or by electrophoresis on agarose gels.

The DNA purification techniques already described, in particular when combined with the extraction techniques of environmental DNA described above, can induce co-purification of inhibitory compounds originating from DNA and early samples that are difficult to remove.

Co-extraction of the inhibitory compound with DNA requires an increase in multiple purification steps that induce a significant loss of the initially extracted DNA while simultaneously reducing the diversity and amount of genetic material initially contained in the sample.

Another object of the present invention is to provide a DNA purification step that can overcome the disadvantages of existing purification protocols and on the one hand maintain the optimal level of diversity of DNA in the initial sample while quantitatively promoting its production on the other hand. To develop.

More specifically, the qualitative and quantitative improvements of DNA purification are maximal when the combination of the direct DNA extraction method according to the invention and subsequent purification steps described below is used.

1.2. Indirect Extraction of Environmental DNA

This technique involves the first step of separating the various organisms of the ground microbiota from other components of the starting sample before the actual DNA extraction step.

In the prior art, existing methods of separating microbial parts from soil samples typically involve physical dispersion of a sample by grinding it in a liquid medium using an apparatus such as, for example, Waring Blender or mortar.

Chemical dispersions, for example dispersions on ion-exchange resins or dispersions using non-specific detergents such as sodium deoxycholate or polyethylene glycol, have also been described. Whatever the method of dispersion, soil samples should be suspended in water, phosphate buffer or saline.

Following a physical or chemical dispersion step, centrifugation in a density gradient that allows separation of the cells contained in the sample and particles of the sample can be performed, which is natural for bacteria to have a lower density than most soil particles. .

Alternatively, a slower centrifugation or cell suspension may be performed following the physical dispersion step.

DNA can then be extracted from the cells separated by the available lysis methods and purified by a number of methods, including the purification method described in paragraph 1.1 above. In particular, cells may be included in cold-melt agarose to control lysis.

However, the methods described in the prior art known to the applicant are unsatisfactory because of the presence of the unwanted composition of the starting sample, which significantly affects the final quantity and quality of the DNA in the fraction containing the extracted DNA.

The present invention provides a solution to the technical difficulties encountered in the prior art methods, which will be described later.

2. Molecular Characterization of Extracted DNA

When it is desired to construct DNA libraries from environmental samples, in particular soil samples, it is advantageous to examine the amount and diversity of extracted and purified DNA origin before it is inserted into a suitable vector.

The purpose of this molecular characterization of the extracted and purified DNA is to obtain a profile indicating the proportion of the various bacterial taxa present in the DNA extract. The molecular characterization of the extracted and purified DNA indicates whether or not artifacts are introduced during the execution of the various extraction and purification steps, and in some cases the original diversity of the extracted and purified DNA is initially present in the sample, particularly the soil sample. Allows you to determine whether or not it represents microbial diversity.

To the best of our knowledge, the prior art utilizes a quantitative hybridization process using oligonucleotide probes that are specific for different groups of bacteria and are applied directly to DNA extracted from the environment.

Unfortunately, this approach is relatively inaccurate and incapable of detecting taxa or genera that exist in small amounts.

The prior art also describes quantitative PCR procedures such as MPN-PCR or competitive quantitative PCR. However, this technique has significant disadvantages.

Accordingly, MPN-PCR is complex to perform because of increased dilution and repetition, which makes it unsuitable for multiple samples or primer couples.

In addition, competitive quantitative PCR is difficult to perform because of the need to construct competitors that are specific for the target DNA and that do not compete with any bias or the artifacts themselves.

In accordance with the present invention, the process allows for rapid, simple, reliable, testing of the properties of pre-extracted and purified DNA to determine the value of constructing a library of clones prepared from this purified starting DNA. It has been proposed for prescreening DNA libraries originating from.

3. Vectors for Cloning DNA Purified and Purified from Environmental Samples

Many vectors for cloning DNA extracted from environmental samples have already been described in the prior art.

Therefore, the international patent application No. As described in WO 99/20799, viral vectors, phages, plasmids, phagemids, cosmids, phosmids, vectors of the BAC (bacterial artificial chromosome) type or bacteriophage P1, PAC (artificial chromosome based on bacteriophage P1) type Vector, YAC (yeast artificial chromosome) type, yeast plasmid or any other vector capable of stably maintaining and expressing genomic DNA can be used.

PCT Patent Application No. Example 1 of WO 99/20799 describes the construction of genomic DNA libraries by cloning into vectors of the BAC type.

To the best of our knowledge, DNA libraries originating from environmental samples have not yet been produced effectively as conjugation vectors, and this technique has been first utilized and reproducible by those skilled in the art by the teachings of the present invention.

4. Host cell

In the prior art, it has been described that a number of host cells can be used to receive vectors containing inserts of DNA derived from purified and purified DNA from environmental samples.

Accordingly, PCT patent application No. WO 99/20799 discloses Escherichia coli , in particular DH 10B strain or 294 (ATCC 31446) strain, E. coli B strain, E. coli X 1776 (ATCC No. 31.537), E. coli DH5 α and A number of suitable host cells are cited, such as Coli W3110 (ATCC No. 27.325).

The PCT Patent Application also other suitable, such as Enterobacter (Enterobacter), control Winiah (Erwinia), keulrep when Ella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), Serratia marcescens (Serratia), Shigella (Schigella) host cell or B. subtilis (B. subtilis) and B. Li Kenny Po Ms (B. licheniformis) and Bacillus-type strains and Pseudomonas (Pseudomonas), Streptomyces sheath (Streptomyces) or liquid Martino My sheath (Actinomyces) of The bacteria in the genus were also cited.

U.S. Patent No. 5 824 485 specifically refers to yeast cells, such as Streptomyces lividan TK66 strain or Saccharomyces pombe .

5. Characterization of the genes in DNA libraries derived from environmental samples

PCT Patent Application No. Identification of phenotypes of different clones (respectively hemolysine-producing clones, clones that hydrolyze esculin or clones that produce orange pigments) WO 99/20799 belong to the DNA library of B. cereus It describes.

Mutagenesis techniques based on the use of transposons encoding the pho A enzyme allowed subsequent isolation of mutated clones and characterization of sequences responsible for the observed phenotype.

The aforementioned paper by Stein et al. (1996) describes the use of primers specific for ribosome DNA for amplifying a vector with DNA inserted by a particular clone of a genomic DNA library of seawater plankton acabacteria, and many of the amplified DNAs. Describes the identification of the coding sequence.

A paper by Borschert S. et al. (1992) describes a Bacillus subtilis using several pairs of primers hybridized with a conserved region of known peptide synthase to identify one or more corresponding genes in the genome of Bacillus subtilis. A method for screening genomic DNA libraries is described.

This technique enabled the detection of about 26 kb of chromosomal DNA fragments carrying portions of the serpentine biosynthetic operon.

Kah-Tong S. et al. (1997) describe a method for screening DNA libraries derived from soil through primers hybridized with conserved sequences of operons responsible for the biosynthetic pathways of type II polyketides. Shows the identification of sequences belonging to the PKS-β gene. The paper also describes the construction of hybrid expression cassettes in which the sequences of PKS-β subunits found naturally in operons responsible for polyketide biosynthesis have been replaced with various similar sequences found in DNA libraries.

Likewise, Hong-Fu et al. (1995) describe the construction of expression cassettes containing various open reading frames of operons responsible for polyketide biosynthesis, and the various expression cassettes are described in Streptomyces coelicolor. ) Is artificially constructed by combining an open reading frame that is not naturally found in the genome. This paper demonstrates that the combination of open reading frames originating from different bacterial strains in artificial expression cassettes enables the production of polyketides with different structural features and relatively high antibiotic activity for Bacillus subtilis and Bacillus cereus. Shows.

Polyketides form part of a large class of natural products of various structures with a wide variety of biological activities. Among the polyketides, examples include tetracycline and erythromycin (antibiotic), FK506 (immunosuppressant), doxorubicin (anticancer agent), monensin (coccidiotic) and avermectin (antiparasitic). Can be.

These molecules are synthesized by multifunctional enzymes known as polyketide synthetases that catalyze the repeat condensation cycle between acyl thioesters (typically acetyl, propionyl, malonyl or methylmalonyl thioesters). Each condensation cycle results in the formation of β-keto groups upon growth of the carbon chain, which in turn may undergo one or more series of reduction steps.

Interest in the development of new polyketides by genetic engineering is of primary concern because polyketides, their conventional biosynthetic mechanisms, and the high degree of conservation observed between groups of genes encoding polyketide synthase are of clinical interest. Increased.

As a result, novel artificial polyketides, such as Mederhodin A or Dihydrogranaterhodin, have been produced by genetic engineering. The large quantities of novel polyketide molecules obtained by genetic engineering are very different in structure from corresponding natural polyketides.

From the prior art, it has a particularly increased level of antibiotic activity or a different spectrum of antibiotic activity (which is wider than known polyketides) compared to the corresponding novel polyketides, more specifically their natural homologs, or On the other hand there is a need to obtain more selective therapeutic polyketides.

As will be described later, this need is partially fulfilled in accordance with the present invention.

The present invention relates to a method for preparing nucleic acid from an environmental sample, and more particularly to a method for obtaining a nucleic acid collection from a sample. The invention also relates to the nucleic acids or nucleic acid collections obtained according to the method and their use in the synthesis of novel compounds, in particular novel compounds for treatment.

The present invention also relates to novel means for use in the above method for obtaining nucleic acids, such as novel vectors, and to novel methods for producing recombinant host cells comprising such vectors or, alternatively, nucleic acids of the present invention.

The present invention also relates to a method for detecting a corresponding nucleic acid in a nucleic acid collection obtained according to the above method, and to a nucleic acid detected by such a method and a polypeptide encoded by such a nucleic acid.

The invention also relates to nucleic acids obtained and detected according to the above methods, in particular nucleic acids encoding enzymes participating in the biosynthetic pathway of antibiotics such as β-lactams, aminoglycosides, heterocyclic nucleotides or polyketides, and such nucleic acids. A pharmaceutical composition comprising an enzyme encoded by a polyketide produced by expression of such nucleic acid, and finally a polyketide pharmaceutically effective amount produced by expression of such nucleic acid.

The invention will also be illustrated by the following figures and examples, which are not limiting.

1 illustrates an overview of various dissolution steps performed according to protocols 1, 2, 3n, 4a, 5a and 5b described in Example 1. FIG.

FIG. 2 illustrates electrophoresis on 0.8% agarose gel of DNA extracted from 300 mg of soil No3 (St Andre coast) after various dissolution treatments (Protocols 1 to 5, see FIG. 1). M: lambda phage molecular weight marker.

FIG. 3 illustrates the proportions of various genera of actinomycetes cultured after treatments 1-5 (see FIG. 1). cfu (colony-forming units) values are measured in media selective for bacteria of this group. The total value of about 400 colonies was analyzed.

Figure 4 illustrates the recovery of lambda phage DNA digested with HindIII added to soil at different concentrations prior to grinding (G) and after (G ^* ). Treatments T (heat shock) and S (ultrasonic treatment) are additional dissolution treatments. Quantification is performed by analysis with a phospho-imager after dot-blot hybridization. Each soil sample is used for each concentration of lambda phage added. Soil characteristics are shown in Table 1. Samples corresponding to 10-15 μg of added DNA were not processed.

5 illustrates PCR amplification of DNA extracted from soil No 3 according to protocols 1, 2, 3, 5a and 5b. Primers FGPS 122 and FGPS 350 (Table 2) are used to target indigenous Streptosporangium spp . The extracted DNA is used undiluted or diluted 10 to 100 times. M: 123 bp molecular weight marker (Gibco BRL), C: DNA-free amplification control.

FIG. 6 illustrates the amount of DNA extracted after inoculation of spores (a) or mycelium (b) of S. lipid OS48.3 inoculated into soil at different concentrations. The amount of mycelium added to the soil corresponds to the number of spores inoculated into the germination medium. About 50% of germinated spores and the number of cells or genomes contained in germinated spore mycelia were not measured. Therefore, the amount of spores and mycelium inoculated cannot be directly compared. Extraction protocol is performed according to Protocol 6 (see Materials and Methods section). The symbol (') indicates that RNA is included in the extraction buffer. Target DNA is amplified by PCR using primers FGPS 516 and FGPS 517 and quantification is performed with phospho-imagers after dot-blot hybridization using probe FGPS 518. Each soil sample is used for each concentration of mycelia or spores. The soil characteristics are described in Table 1.

FIG. 7 shows phylogenetic diagrams obtained with the Neighbor Joining algorithm, which locates 16S rDNA sequences contained in soil DNA libraries for cultured reference bacteria. Gray: sequence obtained from a pool of library clones.

Bootstrap values appear at nodes after 100 re-samplings. Scale bars represent the number of substitutions per site. Access numbers of Genbank database sequences are shown in parentheses.

8 shows a schematic of the vector pOSint 1.

9 shows a schematic of the vector pWED 1.

10 shows a schematic diagram of the vector pWE15 (ATCC No 37503).

11 shows a schematic of the vector pOS700I.

12 shows a schematic of the vector pOSV010.

Figure 13 shows a fragment containing the "cos" site inserted in plasmid pOSV010 during the construction of vector pOSV303.

14 shows a schematic of the vector pOSV303.

15 shows a schematic of the vector pE116.

16 shows a schematic of the vector pOS700R.

17 shows a schematic of the vector pOSV001.

18 shows a schematic of the vector pOSV002.

19 shows a schematic of the vector pOSV014.

20 shows a schematic of the vector pBAC11.

21 shows a schematic of the vector pOSV403.

FIG. 22 shows electrophoretic gels of library DNA following digestion of positive clones of libraries screened with PKS-I oligonucleotides with enzymes BamHI and Dral.

Figure 23 illustrates the production of puromycin by S. lividan recombinants compared to the production of S. alboniger wild type strains.

24 illustrates the alignment of soil PKS with conserved active sites of other PKS. References to each peptide are shown. Beta-ketoacyl synthetase domains are aligned using the GCG PILEUP program (Wisconsin Package Version 9.1 of the Genetics Computer Group, Madison, Wisconsin).

25 illustrates the construction of integrated conjugated cosmids.

26 illustrates the construction of an integrated bond BAC.

27 illustrates an overview for the construction of the vector pOSV308.

28 illustrates an overview for the construction of the vector pOSV306.

29 illustrates an overview for the construction of the vector pOSV307.

30 illustrates an overview for constructing a vector PMBD-1.

FIG. 31 shows a detailed map of plasmid pMBD-2 and also an overview for the construction of vector pMBD-3.

32 illustrates a detailed map of plasmid pMBD-4.

33 shows an outline for constructing plasmid pMBD-5 from plasmid pMBD-1.

34 illustrates a detailed map of the vector pBTP-3.

35 illustrates an overview for the construction of the vector pMBD-6 from the vector pMBD-1.

FIG. 36 illustrates a map of cosmid a26G1 in which the DNA insert contains an open reading frame encoding a plurality of polyketide synthase.

FIG. 37 is a schematic showing the DNA insert (+ strand) of cosmid a26G1 in which various read frames encoding multiple polyketide synthase are located.

First, the present invention relates to a method for constructing a DNA library originating from an environmental sample, which sample is evenly distributed in an aqueous medium (fresh water or fresh water, for example, such as a sample originating from a plant, insect or seawater organism and having an associated microflora) Seawater), soil samples (topsoil, subsoil or sediment) or eukaryotic organism samples containing associated microflora.

The development of methods for constructing DNA libraries from environmental samples, more specifically soil samples, involves an important step in which its implementation must be optimized to obtain a library of DNA whose content of nucleic acid satisfies the purpose initially set.

An important first step consists in extracting and then purifying the nucleic acid initially contained in the sample, that is, the nucleic acid contained in the various organisms consisting primarily of the microflora of the sample.

Purification properties of the extracted DNA are factors that determine the achieved results.

An important second step in the construction of nucleic acid library originating from environmental samples is the assessment of the gene diversity of the extracted and purified nucleic acids. The development of a simple and reliable pre-screening step of extracted and purified DNA to determine whether it is at least partly due to the phylogenetic diversity of the organisms initially present in the starting sample has led to the construction of the extracted and purified DNA for the construction of the nucleic acid library itself. Determining or otherwise making use of the value of the initial origin, or, on the other hand, makes the construction of the nucleic acid library impossible due to the large amount of artifacts introduced when extracting and purifying the nucleic acid. It was also confirmed that the nature of the insert introduced into the vector for constructing the library was a determinant. Therefore, it was judged that the use of restriction enzymes for cleaving purified and extracted DNA from environmental samples had the property of introducing artifacts or "biases" into the insert construct. In particular, DNA extracted from soil or other environments and, in most cases, originating from non-cultured organisms, consists of molecules whose content of G and C bases are by definition unknown and further variable as a function of such organism origin.

The main third step is to insert, on the one hand, the extracted and purified nucleic acid into a vector capable of incorporating nucleic acids of the selected length, and on the other hand to transfect or integrate the genome of a given host cell, This step enables expression in the host cell.

Vectors capable of incorporating large nucleic acids, i.e. nucleic acids larger than 100 kb in size, can be used to clone and identify complete operons that can lead to the complete biosynthetic pathway of industrial compounds, especially those of pharmaceutical or cropological interest. Construct the corresponding vector

Justice

For the purposes of the present invention, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to a hybrid RNA / DNA sequence of DNA and RNA sequences and more than two nucleotides in single- or double-stranded form.

The term “library” or “collection” in this description is derived from a set of extracted, optionally purified nucleic acids, recombinant vector sets (each of the recombinant vector set described above is a set of extracted and optionally purified nucleic acids originating from environmental samples). From a set of recombinant host cells comprising one or more nucleic acids originating from the set of extracted and optionally purified nucleic acids described above, wherein the nucleic acid is carried by one or more recombinant vectors or Is integrated into the genome).

The expression “environmental sample” is equally derived from seawater organisms or insects of aquatic origin, for example, samples from fresh or seawater, or from land and samples originating from the topsoil, sedimentary or soil subsoil (soil). Represents a sample of eukaryotic organisms which may be multicellular with associated microbiota (the microbiota constitutes that organism).

According to the present invention, the term “operon” means a set of open reading frames in which transcription and / or translation are simultaneously regulated by a specific set of signals for regulating transcription and / or translation. In accordance with the present invention, the operon may also comprise a signal for modulating transcription and / or translation.

For the purposes of the present invention, the expression "metabolic pathway" or "biosynthetic pathway" means a set of biochemical reactions of assimilation or catabolism which results in the conversion of the first species to the second species.

For example, the biosynthetic pathway of antibiotics consists of a set of biochemical reactions that convert primary metabolites to antibiotic intermediates and then to antibiotics.

The expression “regulatory sequence operably linked to the nucleotide sequence desired for expression” is positioned so that the transcriptional control sequence (s) are capable of expression of that sequence for that nucleotide sequence desired for expression, and control of expression is regulated. It depends on the elements that interact with the nucleotide sequence.

According to another terminology, the corresponding nucleotide sequence for which expression is desired may be said to be "under control" of the transcription-regulating nucleotide sequence.

For the purposes of the present invention, the term "isolated" means a biological material extracted from the original environment (the environment in which it was originally located).

For example, polynucleotides or polypeptides that exist naturally in organisms (viruses, bacteria, fungi, yeasts, plants or animals) are not isolated. Isolation of the same polynucleotide isolated from the same polypeptide isolated from the natural environment or from adjacent nucleic acid naturally inserted into the genome of the organism.

Such polynucleotides may be included in a vector and / or such polynucleotides may be included in a composition, but nevertheless exist in discrete form because the vector or composition does not constitute its natural environment.

The term “purified” does not need to be a substance present in an absolutely pure form, excluding the presence of other compounds. Rather, this is a relative definition.

The polypeptide or polynucleotide is in the form of a tablet after purifying the starting material at least primary, preferably secondary or tertiary, more preferably fourth or fifth.

For the purposes of the present invention, “% identity” between two sequences of nucleotides or amino acids can be determined by comparing two sequences optimally aligned in the comparison window.

Thus, an optimal alignment of the two sequences can be obtained by including additions or deletions (eg, "gaps") in the comparison window when some of the nucleotide or polypeptide sequences include the comparison sequences (which do not include such additions or deletions).

The percentage measures the number of positions observed for two sequences (nucleic acid or peptide) where the same nucleic acid base or the same amino acid residue is compared, then divides the same number of positions between the two base or amino acid residues by the total number of positions in the comparison window, and then Calculated by multiplying by 100 to obtain the percentage of sequence identity.

Optimal alignment of sequences for comparison can be achieved by computer through known algorithms included in packages from the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Doctor (Madison, Wisconsin).

By way of explanation, the percentage of sequence identity is determined by default parameters (SF Altschul et al., J. Mol. Biol. 1990 215: 403-410, SF Altschul et al., Nucleic Acids Res. 1997 25: 3389-3402 ) Can be measured using BLAST software (BLAST version 1.4.9 from March 1996, BLAST 2.0.4 from February 1998 and BLAST 2.0.6 from September 1998). Blast to Blast search for sequences similar / homologous to a reference "request" sequence, with the aid of the algorithm. The search sequence and the database used can have the properties of peptides or nucleic acids, and any combination.

Extraction and Purification of Nucleic Acids Derived from Environmental Samples

1. Direct Extraction of Nucleic Acids

In order to obtain a library of nucleic acids originating from the organisms contained in the soil sample, on the one hand, various organisms in the sample must be available for subsequent extraction of the nucleic acid, and on the other hand, the initial treatment step of the soil sample is performed by the organism in the sample. It is understood from the present invention that it is important to create conditions that allow for the highest mechanical lysis of the organism so that it is characterized by the direct availability of nucleic acids, mainly genomic and plasmid DNA, of such organisms to the buffer used for subsequent extraction steps. Can be.

It was thus demonstrated according to the invention that the highest availability of nucleic acids originating from microorganisms from soil samples was achieved through thorough dry-milling of the soil samples pre-dried to obtain particulates. Applicants have therefore determined that drying of the soil sample prior to any subsequent treatment significantly reduces cohesion of the raw soil sample and consequently promotes subsequent cleavage in particulate form when proper grinding treatment is performed.

Surprisingly, the Applicant has found that the particulates in a dry soil sample actually combine physicochemical properties suitable for the extraction of an optimal amount of nucleic acid which may indicate the genetic diversity of the organisms initially present in the starting soil sample. It has been found that the direct extraction method of nucleic acids according to the invention enables the extraction of DNA originating from rare microorganisms such as microorganisms that form very rare streptomyces or spores.

For the purposes of the present invention, the term “particulates” in soil samples means particles derived from samples having an average size of about 50 μm, ie an average of 45 to 55 μm.

According to the invention, before resuspending the fines obtained in the liquid buffer medium, the fines are pre-dried or pre-dehydrated and then obtained from the ground soil sample until fines having an average size of 2 μm to 50 μm are obtained. do.

Such liquid buffer medium may consist of nucleic acid extraction buffers, especially conventional DNA extraction buffers well known to those skilled in the art.

Crushing the soil sample into fines is a two function that mechanically dissolves most of the organisms present in the initial soil sample and allows the undissolved organisms to be used in any subsequent step of chemical and / or enzymatic dissolution. Has

Accordingly, the first subject of the present invention relates to a method for preparing a nucleic acid collection from soil samples containing organisms, the method comprising the first step I- of obtaining fine particles by grinding a pre-dried or pre-dehydrated soil sample. (a) followed by suspending the particulates in the liquid buffer medium.

In a very preferred manner, the grinding step is carried out using a device with an agate or tungsten beads or alternatively with a device with a tungsten ring. Such a device is preferred because the hardness of the material, such as agate or tungsten, greatly facilitates the generation of the above-mentioned fine particles. For this reason, the use of grinding devices with glass beads that have been found to be much less efficient will preferably be unselected or avoided.

Drying or sorting of soil samples can be carried out by any method known to those skilled in the art. For example, the crude soil sample can be dried at room temperature for 24 to 48 hours.

As mentioned above, the liquid buffer medium may consist of a medium for extracting DNA present as particulates. Most preferably an extraction buffer (pH 9.0), known as TENP, containing Tris 50 mM, EDTA 20 mM, NaCl 100 mM and polyvinylpolypyrrolidone 1% (weight / volume), respectively, will be used.

The method of making a nucleic acid collection from a soil sample is also characterized in that the step of obtaining the microparticles by grinding the pre-dried or pre-dehydrated soil sample followed by the step I- (b) of extracting the nucleic acid present as microparticles. .

It is a general opinion that extraction of nucleic acids requires subsequent purification of the extracted nucleic acid accompanied by co-extraction of unwanted soil components and / or compounds, and such subsequent purification steps may allow removal of unwanted soil components and / or compounds. It needs to be selective enough to allow it, and its production should be sufficient to allow only a slight loss relative to the amount of pre-extracted DNA.

Purification of the DNA extracted from the microparticles of the soil sample that meets the selectivity and yield criteria described above is subject to treatment with a combination of two consecutive chromatography (chromatography and anion-exchange chromatography) steps of the extracted DNA. It can be seen according to the invention that it includes.

According to another feature of the method, extraction step I- (b) of nucleic acid is followed by step I- (c) of purifying the extracted nucleic acid by two chromatographic steps as follows:

Flowing the solution containing the nucleic acid onto the molecular sieve and then recovering the elution fraction enriched in the nucleic acid;

The elution fraction enriched in nucleic acid is flowed onto an anion-exchange chromatography support and then the elution fraction containing nucleic acid is recovered.

The nature and sequence of the chromatography steps are essential for good selectivity and good yield of the DNA purification step pre-extracted from the fines of the pre-dried or pre-dehydrated soil samples.

In a very advantageous manner, in the nucleic acid purification step, the "molecular sieve" type of chromatography support consists of a chromatography support of type Sephacryl ^R S400 HR or a chromatography support of equivalent character.

In a very preferred manner, the anion-exchange chromatography support used in the second step for purifying the extracted DNA is a support of Elutip ^R d type or a chromatography support of equivalent character.

By combining step I- (a) of obtaining fine particles of a dry soil sample, I- (b) of extracting nucleic acids present as fine particles and purification step I- (c) by the above-described chromatography step, Thus extracting DNA from the soil directly without prior purification of the organic cells initially contained in the sample, while at the same time avoiding co-extraction of soil contaminants such as humic acid, as observed in the prior art processes. It is possible.

Contaminants such as humic acid seriously impair subsequent use of nucleic acids for which analysis and purification is desirable.

According to the method, in order to obtain a substantially overall collection of the genetically diverse nucleic acid initially present in the soil sample, it is contained in an organism that has not been mechanically dissolved during step I- (a) of obtaining particulates of the soil sample. Access to nucleic acids is also possible. Thus, the fines of the soil sample may undergo a subsequent step of chemical, enzymatic or physical dissolution treatment or a combination of chemical, enzymatic or physical treatment.

According to a first aspect, the method for preparing a nucleic acid collection from a soil sample according to the invention may also be characterized in that the following steps are carried out following step I- (a):

Sonication of the soil suspension in liquid buffer medium;

ㆍ Extraction and recovery of nucleic acid.

In a preferred manner, for sonication, a titanium micro-point type device such as a 600 W Vibracell Ultrasonicator device or Cup Horn type sonicator sold by Bioblock will be used.

In a very preferred manner, the sonication step is carried out at a power of 15 W for a duration of 7 to 10 minutes and comprises a continuous cycle of sonication, the sonication itself being carried out for 50% of the duration of each cycle.

According to a second aspect, the method may also be characterized in that the following steps are performed following step I- (a):

Sonication of the soil suspension in liquid buffer medium;

Culture of the suspension at 37 ° C. in the presence of lysozyme and acromopeptidase after sonication;

Addition of SDS prior to centrifugation and precipitation of nucleic acids;

Recovery of precipitated nucleic acid.

Preferably, the culturing step in the presence of lysozyme and acromopeptidase will be carried out at a final concentration of 0.3 mg / ml of each of the two enzymes, preferably at 37 ° C. for 30 minutes.

Preferably, SDS will be used at a final concentration of 1% and incubated at 60 ° C. for 1 hour prior to centrifugation and precipitation.

According to a third aspect, the method for preparing a nucleic acid collection from a soil sample is also characterized in that the following steps are performed following step I- (a):

Thorough mixing (vortexing) of the soil suspension, followed by homogenization with simple stirring;

Freezing of the homogeneous suspension, followed by thawing;

Sonication of the suspension after thawing;

Suspension culture at 37 ° C. in the presence of lysozyme and acromopeptidase after sonication;

Addition of SDS prior to centrifugation and precipitation of nucleic acids;

Recovery of nucleic acid.

Preferably, the suspension of soil fines is mixed in a vortex and then homogenized by gentle stirring in a circulating stirrer for a duration of 2 hours and then frozen at -20 ° C.

Preferably, the suspension is thoroughly stirred again with a vortex for 10 minutes after thawing and before the sonication step.

The nucleic acid extracted by the above-described direct extraction method of nucleic acid preferably flows first on a molecular sieve, and then eluted fraction obtained after chromatography on molecular sieve is flowed on an anion-exchange chromatography support. It is obvious that the purification is carried out according to the purification step.

2. Indirect Extraction of Nucleic Acids

According to a second aspect of the method for preparing a nucleic acid collection from an environmental sample according to the invention, the environmental sample undergoes a primary treatment having the property of enabling the separation of the sample of the organism contained in the sample from another macro-constitution. .

This second aspect of the method for producing a nucleic acid collection according to the present invention promotes the production of large nucleic acids, which is substantially impossible to obtain according to the first aspect of the method according to the invention described above, and is carried out to obtain microparticles. The dissolution step also has the effect of physically destroying the nucleic acid in the soil sample or the nucleic acid contained in the organism in the soil sample.

Production of large nucleic acids has been attempted by the applicant to at least partially isolate and characterize nucleic acids comprising all of the coding sequences belonging to the same operon capable of inducing the biosynthesis of industrial compounds.

Preferably, by carrying out the second aspect of the method for preparing a nucleic acid collection from a soil sample according to the invention, nucleic acids larger than 100 kb, preferably 200, 250 or 300 kb, most preferably 400, 500 Or nucleic acids larger than 600 kb are obtained.

This second aspect of the method of making a nucleic acid collection from an environmental sample according to the invention consists of a combination of four consecutive steps for obtaining nucleic acids having the above-mentioned characteristics.

If the environmental sample is a soil sample, the first step of obtaining a suspension by dispersing the soil sample in liquid medium promotes the availability of the organisms contained in the sample without causing significant mechanical lysis of the cells. Became known.

The first step of obtaining a dispersion of the soil sample makes the organism in the sample accessible to the external medium and also enables partial separation of the organism and macro- constructs in the sample. This thus makes it possible to subsequently separate the organisms initially contained in the sample from the other constituents of the sample.

If the environmental sample originates, for example, from plants, seawater organisms or insects, pretreatment by grinding is necessary to make the organisms of the relevant microflora available for subsequent steps in the process.

Thus, the method includes the step of separating the organism from the other inorganic and / or organic constructs obtained above by centrifugation in a density gradient. The organisms thus isolated are then subjected to a dissolution step followed by extraction of the nucleic acid.

The centrifugation step in the density gradient surprisingly makes it possible to separate the organism cells in the soil particles contained in the sample suspension. In fact, it can be foreseen that a certain percentage of the cells will be trapped in the gradient of large particles. In addition, centrifugation of soil samples in density gradients leads to the discovery of populations of organisms that exhibit the diversity of organisms present in the starting sample at the aqueous phase / gradient interface, because these organisms are highly variable in volume, density, and shape. This has not been proven so far. It can be reasonably assumed that it will be found in the aqueous phase at the aqueous phase / density gradient interface or in the density gradient itself.

Thus, those skilled in the art will not be able to recover an organism having a density less than or greater than the density gradient used (the density of 1.2 to 1.5 g / ml, preferably the density of 1.3 g / ml), and the effect thereof is to effectively bias the bias. It can be foreseen that they will be introduced into an isolated representative organism and consequently into the diversity of the extracted nucleic acid.

In addition, in one particular embodiment of the method, in particular the spore germination step of actinomycete is carried out, the effect of which being to significantly increase the amount of actinomycete DNA recovered.

The final step consists in purifying the extracted nucleic acid in a cesium chloride gradient.

Surprisingly, purification of the nucleic acid in the cesium chloride gradient allows for substantial or even complete removal of the material making up the density gradient. Occasionally, this is a feature of subsequent use of the purified nucleic acid because the density gradient is known to be a potent enzyme inhibitor capable of inhibiting the catalytic activity of the enzyme used to prepare the insert of the extracted nucleic acid into the vector. It is a deciding factor.

According to this second aspect, the method for preparing a nucleic acid collection from an environmental sample containing an organism according to the invention comprises the following successive steps:

(i) producing a suspension by dispersing the environmental sample in a liquid medium and then homogenizing by gently stirring the obtained suspension;

(ii) separating the organism from the other inorganic and / or organic constituents of the homogeneous suspension obtained in step (i) by centrifuging in a density gradient;

(iii) dissolving the microorganism isolated in step (ii) and extracting the nucleic acid;

(iv) The nucleic acid is purified in a cesium chloride gradient.

Preferably, a suspension of the soil sample is obtained by dispersing this sample by grinding into a device such as Waring Blender or a device of equivalent character. In a very preferred manner, the sample suspension is obtained after three successive grinding operations, each lasting 1 minute in a device such as Waring Blender. Preferably, the ground sample will be cooled with ice between each grinding operation.

Preferably, the organism is then separated from the soil particles by centrifugation in a "Nycodenz" type density cushion sold by Nycomed Pharma AS., Oslo, Norway. Preferred centrifugation conditions are advantageously 10,000 × g at 4 ° C. for 40 minutes in a rotor with a swing-out bucket of “rotor TST 28.38” sold by Kontron.

After centrifugation, the ring of organisms located at the interface between the top aqueous phase and the bottom Nycodenz phase is then removed and washed by centrifugation prior to taking the cell pellet in the appropriate buffer.

Dissolution step (iii) of the organism separated in step (ii) described above may be carried out in any manner known to those skilled in the art.

Advantageously, the cells are lysed in 10 mM Tris-100 mM EDTA solution at pH 8.0 in the presence of lysozyme and acromopeptidase, advantageously at 37 ° C. for 1 hour.

The actual extraction step of DNA can advantageously be carried out by adding a lauryl sarcosyl (1% of the final weight of solution) solution in the presence of proteolytic enzyme K and incubating the final solution at 37 ° C. for 30 minutes.

The nucleic acid extracted in step (iii) is then purified in cesium chloride gradient. Preferably, the step of purifying the nucleic acid in the cesium chloride gradient is carried out by centrifugation at 35,000 rpm for 36 hours, for example in a rotor of type Kontron 65.13.

According to one particular aspect of the method for producing a nucleic acid collection from a soil sample containing an organism according to the invention, the nucleic acid consists mainly of DNA molecules, not exclusively.

According to another aspect, the nucleic acid can be recovered after the organism separated in the density gradient is contained in the agarose block and dissolved, eg chemically and / or enzymatically dissolved, or after the organism is included in the agarose block. .

Another subject of the invention is a nucleic acid obtained in step II- (iv) of the method for producing a nucleic acid collection according to the present invention, or alternatively obtained in step (c) or a subsequent step of the method for producing a nucleic acid collection according to the present invention. A nucleic acid collection consisting of.

The invention also relates to a nucleic acid characterized by being included in the nucleic acid collection as described above.

According to a first aspect, such nucleic acids making up the nucleic acid collection according to the invention are characterized in that they comprise at least one operon or a nucleotide sequence encoding a portion of the operon.

Most preferably, these operons encode all or part of the metabolic pathway.

Example 9 describes the construction of genomic DNA libraries from Streptomyces alboniger strains and cloning into shuttle cosmids pOS700I and pOS700R, respectively. In the DNA library prepared in the integration vector pOS700I, it can be seen according to the invention that the new clone contains a nucleotide sequence belonging to the operon responsible for the puromycin biosynthetic pathway. Likewise, twelve clones containing the nucleotide sequence of the operon responsible for the puromycin biosynthetic pathway were identified in the DNA library prepared in the replication vector pOS 700R.

In particular, the specific integrative and replicable cosmids of the produced libraries have 12-kb fragments which, after digestion with the restriction endonucleases Clal and EcoRV, may contain all sequences of operons responsible for the puromycin biosynthetic pathway.

Thus, according to another aspect, the nucleic acid according to the invention contains the nucleotide sequence of the operon at least partially responsible for the puromycin biosynthetic pathway.

Example 2 below describes the construction of a DNA library according to the method of the present invention with pBluescript SK ^- vector, starting with soil contaminated with Lindane.

The recombinant vector is transfected with E. coli DH10B cells and the transformed cells are then cultured in the presence of Lindane in a suitable culture medium. Screening of clones in the transformed cells of the library can show that, out of 10,000 screened clones, 35 have a Lindane degradation phenotype. The presence of linA gene in these clones is confirmed by PCR amplification with primers specific for this gene.

Accordingly, according to another aspect, the present invention also relates to a nucleic acid containing a nucleotide sequence for a metabolic pathway that causes biodegradation of Lindane.

Therefore, as described above, a method for preparing a nucleic acid collection from a soil sample containing an organism according to the present invention and a method for preparing a recombinant vector collection containing a constituent nucleic acid of the nucleic acid collection described above is isolated and characterized from the nucleotide sequence included in the operon. It is clearly demonstrated that it is very suitable for identification.

Further evidence of the ability of the encoding nucleotide sequences according to the invention to participate in biosynthetic pathways regulated in operon form is also described below: this is a polyketide primarily for therapeutic use, in particular belonging to a specific class of molecules representing antibiotics. Cloning and characterization of a sequence encoding a polyketide synthase involved in the biosynthetic pathway of

The subject of the present invention is therefore also the constitutive nucleic acid of the nucleic acid collection according to the invention, characterized in that it comprises all of the nucleotide sequences encoding the polypeptide.

According to a first aspect, the constituent nucleic acids of the nucleic acid collection according to the invention are of prokaryotic origin.

According to a second aspect, the constituent nucleic acids of the nucleic acid collection according to the invention are from bacteria or viruses.

According to a third aspect, the constituent nucleic acids of the nucleic acid collection according to the invention are of eukaryotic origin.

In particular, such nucleic acids are characterized as originating from fungi, yeasts, plants or animals.

Molecular Characterization of Nucleic Acid Collections Extracted from Soil

In order to overcome various technical shortcomings of the methods of characterizing DNA libraries extracted and purified from environmental samples described in the paragraphs describing the prior art, Applicants qualitatively and semi- A simple and reliable method of quantitative characterization has been developed.

The method according to the invention thus universally amplifies the 700 bp fragment located within the 16S ribosomal DNA sequence, then hybridizes the amplified DNA with oligonucleotide probes of various specificities, and finally the hybridization strength of the sample is known. Comparison with an external measurement range of DNA of sequence or origin.

Amplification prior to hybridization with oligonucleotide probes allows for the quantification of relatively rare microbial genus or species. In addition, amplification with conventional primers allows the use of a wide range of oligonucleotide probes during hybridization.

Accordingly, the subject of the present invention is also a method of measuring the diversity of nucleic acids contained in a nucleic acid collection, more specifically in a nucleic acid collection originating from an environmental sample, preferably a soil sample, the method comprising the following steps:

Contacting the nucleic acid of the nucleic acid collection to be tested with a pair of oligonucleotide primers which hybridize in the sequence of bacterial 16S ribosomal DNA;

Performing at least three amplification cycles;

Amplified nucleic acid is detected using an oligonucleotide probe or a plurality of oligonucleotide probes (each probe specifically hybridizes with 16S ribosomal DNA sequences common to bacterial systems, throats, subclasses or genus);

Optionally, compare the results from the preceding detection step with the probe of a nucleic acid of known sequence constituting the measurement range or the result of detection using a plurality of probes.

Preferably, the first pair of primers hybridized with the universal conserved region of the gene for 16S ribosomal RNA consists of primers FGPS 612 (SEQ ID NO: 12) and FGPS 669 (SEQ ID NO: 13), respectively.

The second embodiment of the preferred pair of primers according to the present invention consists of a pair of conventional primers 63f (SEQ ID NO: 22) and 1387 r (SEQ ID NO: 23).

According to one particular aspect of the method for measuring the diversity of nucleic acids in a nucleic acid collection, the amplification step using a pair of conventional primers is a hybridization step with an oligonucleotide probe specific for a particular bacterial system, throat, subclass or genus. Previously, nucleic acid from the nucleic acid collection in question can be performed with the inserted recombinant vector collection.

Such methods for measuring the diversity of nucleic acids included in a collection are more specifically applicable to nucleic acid collections obtained in accordance with the teachings of the present description.

Thus, Example 3 comprises an indirect extraction of DNA by dispersing the soil sample before separating the cells from the Nycodenz gradient, lysing the cells and purifying the DNA in a cesium chloride gradient from the soil sample containing the organism. The preparation of nucleic acid collections is described in detail.

The nucleic acid collection thus obtained is used as such or in the form of an insert into a cosmid type vector in an amplification method using conventional primers described above for 16S rDNA, followed by the amplified DNA sequence shown in Table 4. To a detection step using an oligonucleotide probe of SEQ ID NO: 14 to SEQ ID NO: 21.

The results show that the method of preparing a nucleic acid collection starting with a soil sample containing an organism according to the present invention makes available more than 14% of the total microbiota in the ground, ie 2 x 10 ⁸ cells per gram of soil, The total microbiota that can be cultured shows only 2% of the total microbial population.

To determine the phylogenetic diversity of the nucleic acid collections prepared according to the present invention, 47 sequences of 16S rRNA genes are isolated and sequenced. These sequences correspond to the nucleotide sequences of SEQ ID NO: 60 to SEQ ID NO: 106, respectively.

Nucleic acids comprising the sequences of SEQ ID NOs: 60 to 106 also form part of the present invention, and at least 99%, preferably 99.5%, or 99.8% engineered nucleic acids (these nucleic acids include SEQ ID NOs: 60 to Identical to the nucleic acid comprising the sequence of No. 106). Such sequences can be used as probes to identify clones of the library, particularly those containing such sequences, by screening clones of DNA libraries, which sequences are involved in the biosynthetic pathway of antibiotic metabolites such as polyketides. To the corresponding coding sequence, such as the sequence encoding the enzyme.

RDP database (Maidak BL, Cole JR, Parker CT, Garrity GM, Larsen N., Li B., Lilburn TG, McCaughey, MJ, Olsen GJ, Overbeek R) of 16S rRNA sequences from DNA libraries prepared according to the present invention , Pramanik S., Schmidt ™, Tiedje JM, Woese CR (1999) "A new project of the Ribosomal Database Project (RDP)" Nucleic Acids Research Vol. 27: 171-173). It is said that the nucleic acids included in the nucleic acid collection according to the present invention originate from genes associated with α-proteobacteria, β-proteobacteria, δ-proteobacteria, γ-proteobacteria, actinomycetes and ashdobacteria. Make judgment. These results, shown in the phylogenetic diagrams of Table 7 and FIG. 7, take into account the significant phylogenetic diversity of the nucleic acids included in the DNA libraries prepared by the method according to the invention.

Cloning and / or Expression Vectors

Nucleic acids included in the nucleic acid collections prepared according to the present invention may be inserted into cloning and / or expression vectors, respectively.

For this purpose, viral vectors, phages, plasmids, phagemids, cosmids, phosmids, vectors of type BAC, P1 bacteriophages, vectors of type BAC, vectors of type YAC, yeast plasmids or any known to those skilled in the art Any type of vector known in the art, such as other vectors of may be used.

In accordance with the present invention vectors that enable stable expression of DNA library nucleic acids will be advantageously used. For this purpose, such vectors preferably comprise a transcription-regulatory sequence operably linked with a genomic insert to initiate and / or regulate expression of at least a portion of the DNA insert.

The present invention also provides a method for cloning and / or expression vectors for nucleic acids obtained in step II- (iv) or step I- (c) or any other subsequent step of a method for preparing a nucleic acid collection from soil samples containing an organism according to the present invention. It is indicated above that the invention relates to a method of making a recombinant vector collection, characterized in that it is inserted in.

Prior to cloning and / or insertion into an expression vector, the constituent nucleic acids of the nucleic acid collection according to the invention are subjected to electrophoresis in an agarose gel, depending on their size, for example after digestion with restriction endonucleases. Can be separated.

According to another aspect, the average size of the constituent nucleic acids of the nucleic acid collection according to the invention can be made to be substantially uniform in size by performing a physical disruption step prior to cloning and / or inserting it into the expression vector.

This physical or mechanical rupture step of the nucleic acid may consist of continuously passing such nucleic acid in solution into a metal channel of about 0.4 mm diameter, for example a channel of a syringe needle having this diameter.

The average size of the nucleic acid may in this case be 30 to 40 kb in length.

Construction of preferred vectors according to the present invention is schematically illustrated in FIGS. 25 (conjugated integrated cosmid) and 26 (integrated BAC).

Cloning and / or expression vectors which can be advantageously used for inserting nucleic acids contained in a DNA library or collection according to the invention are in particular the vectors described in European Patent No. EP 0 350 341 and US Patent No 5 688 689. Such vectors are particularly suitable for transformation of actinomycete strains. Such vectors contain, in addition to the insert DNA sequence, a linking sequence att and a DNA sequence encoding an integrase ( int sequence) that is functional in the actinomycete strain.

However, it has been observed in accordance with the present invention that certain cloning and / or expression vectors have disadvantages and that their theoretical functional capacity is not actually achieved.

Thus, it is shown that the integration system included in prior art vectors, in particular the vectors described in European Patent No. EP 0 350 341, does not in fact allow good integration of DNA inserts from the library into bacterial chromosomes.

From the hypothesis that functional defects in the integration of such vectors into the bacterial chromosomes are due to expression defects in the integrase genes present in these vectors, we can first increase the initial transcription promoter to significantly increase the number of integrase transcripts. The expression of the integrase gene was increased by replacement with a transcriptional promoter.

The results were disappointing and the integration of these vectors into the chromosome was not improved.

Surprisingly, it has been shown according to the present invention that the integrase expression difficulty found in this class of integration vectors is not due to the amount of transcript expression, but because of the stability of the transcript.

According to the second hypothesis, Applicants have found that the stability defect of the integrase transcript is caused by the transcription termination defect of the corresponding messenger RNA.

Accordingly, Applicant inserted a stop site downstream of the sequence encoding the integrase of the vector to obtain a messenger RNA of a given size. Inserting an additional termination signal downstream of the nucleotide sequence encoding the integrase of the vector made it possible to obtain an integrated vector class of cosmid type and BAC type.

Preferably, the stop site is located downstream of the linking site att .

Applicants have also developed novel conjugation vectors and cosmid type novel replication vectors and BAC type novel conjugation vectors that can be advantageously used to insert constituent nucleic acids of nucleic acid collections prepared according to the methods of the invention.

If insertion of an average size DNA fragment is desired, a cosmid type vector capable of accepting an insert having a maximum size of about 50 kb is preferably used.

Such cosmid vectors are particularly suitable for inserting the constituent nucleic acids of the nucleic acid collections obtained according to the method of the invention, comprising the first step of extracting DNA directly by mechanical lysis of the organisms contained in the initial soil sample. Do.

If insertion of large nucleic acids, in particular nucleic acids larger than 100 kb in size, or even nucleic acids larger than 200, 300, 400, 500 or 600 kb is desired, then preferably of the BAC type which can accommodate DNA inserts of this size Vector will be used.

Such vectors of the BAC type are particularly suitable for inserting constituent nucleic acids of the nucleic acid collection obtained according to the method of the present invention, wherein the first step is to pre-separate the organisms contained in the initial soil sample and then to separate the bulk from the soil sample. Indirect extraction of DNA by removing constructs

In particular, vectors of the BAC type are advantageously used to insert large nucleic acids which at least partly comprise the nucleotide sequence of an operon.

Thus, the method for producing a recombinant cloning and / or expression vector collection according to the invention is also characterized in that the cloning and / or expression vector is plasmid type.

According to another aspect, this method is characterized in that the cloning and / or expression vector is cosmid.

According to the first aspect, this may be cosmid that is replicable in E. coli and integrative in Streptomyces. A very preferred cosmid corresponding to this limitation is the cosmid pOS700I described in Example 3.

According to another aspect, the cosmid vector is conjugative and integrative in Streptomyces.

In general, avoiding dependence on transformation techniques that are difficult to automate in cosmid or BAC-type conjugation vectors containing units recognized by cellular enzyme machinery known as "conjugated origins" in their nucleotide sequences. Used when desired.

For example, transfection of a vector initially possessed by E. coli cells to Streptomyces cells typically involves recovery of the recombinant vector contained in the E. coli cells, followed by transformation of the Streptomyces protoplasts. This requires a step of purification. It is commonly accepted that transfection of 1000 E. coli clone combinations with Streptomyces requires the production of about 8000 clones so that each E. coli clone has a chance to appear.

In contrast, the transfection step by conjugating the vector of E. coli to Streptomyces cells requires an equal number of clones of each microorganism, and the conjugation step occurs as "clones versus clones" and, for example, polyethylene It does not include technical difficulties with respect to the step of delivering the genetic material by transformation of the protoplasts in the presence of glycols.

In order to optimize the construction of DNA libraries in Streptomyces, novel conjugation vectors of cosmid type and BAC type have been developed in accordance with the present invention that have properties that enable the highest efficiency of the conjugation step.

In particular, the novel conjugation vectors according to the invention are constructed by placing the selectable marker gene at the DNA terminus of the vector delivered at the terminus of the recipient bacterium. Improvement of the conjugation vector to this prior art allows the practical selection of only the accepting bacteria that receive all the vector DNA and consequently all of the corresponding insert DNA.

The cosmids conjugated, integrative and preferred in accordance with the present invention in Streptomyces are the cosmids pOSV303, pOSV306 and pOSV307 described in Example 5.

According to another aspect, the method for producing a recombinant vector collection according to the present invention is carried out using a cosmid that is replicable in both E. coli and Streptomyces. This cosmid is advantageously the cosmid pOS700R described in Example 6.

According to another aspect, the method can be performed with cosmids that are replicable in E. coli and Streptomyces and conjugated in Streptomyces.

Such replicable and conjugated cosmids can be obtained from the replicable cosmid according to the present invention by inserting a suitable delivery origin such as RK2, as described in Example 5 for the construction of the vector pOSV303.

According to another advantageous embodiment of the method for producing a recombinant vector collection according to the invention, cloning and / or expression vectors of the BAC type are used.

According to the first aspect, the vector of the BAC type is integrated and conjugated in Streptomyces.

In a very preferred manner, these BAC vectors which are integrative and conjugative in Streptomyces are the vector BAC pOSV403 described in Example 8 or else the vectors BAC pMBD-1, pMBD-2, pMBD- described in Example 15. 3, pMBD-4, pMBD-5 and pMBD-6.

The subject of the invention is also a recombinant vector, characterized in that it is selected from the following recombinant vectors:

a) a vector comprising the constituent nucleic acids of the nucleic acid collection according to the invention;

b) A vector obtained according to a method which avoids the involvement of restriction endonuclease action in the DNA fragment to be inserted as described above.

In a very preferred manner, the invention also relates to a vector selected from the following vectors:

Cosmid pOS700I;

Cosmid pOSV303;

Cosmid pOSV306;

Cosmid pOSV307;

Cosmid pOS700R;

Vector BAC pOSV403;

Vector BAC pMBD-1;

Vector BAC pMBD-2;

Vector BAC pMBD-3;

Vector BAC pMBD-4;

Vector BAC pMBD-5;

Vector BAC pMBD-6.

The invention also relates to a collection of recombinant vectors obtained by one of the methods according to the invention.

Method for producing recombinant cloning and / or expression vector according to the present invention

Conventional techniques for inserting DNA into a vector to produce recombinant cloning and / or expression vectors are typically incubated with a restriction endonuclease-inserted DNA and a recipient vector, resulting in a final ligation that allows for the production of the recombinant vector. And a first step of forming a compatible terminus between the inserted DNA and the vector DNA, allowing for the combination of the two DNAs before the step.

However, such conventional techniques have significant drawbacks when more specifically it is desired to insert large nucleic acids into cloning and / or expression vectors.

In particular, in the DNA fragment to be inserted into the vector, the preceding action of the restriction enzyme cannot be reduced to a detectable size before insertion into the vector. Significant reduction in DNA size prior to insertion into the vector clones large DNA fragments where expression may contain all coding sequences of the operon and, in some cases, regulatory sequences, which constitute the complete biosynthetic pathway of industrial metabolites, particularly therapeutic compounds. It is obvious that this is especially unfavorable when it is hoped to do.

To overcome the shortcomings of the prior art, two methods have been developed in accordance with the present invention for preparing recombinant cloning and / or expression vectors that do not use restriction endonucleases in the inserted DNA prior to introduction into the vector. As a result, this method is well suited for the cloning of long DNA fragments that may at least partially contain the coding sequence of the complete operon responsible for the biosynthetic pathway and, optionally, the regulatory sequence.

According to a first aspect, one method for preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the cloning and / or insertion of the nucleic acid into the expression vector comprises the following steps:

Cloning and / or opening the expression vector using a suitable restriction endonuclease at the selected cloning site;

Adding a first homopolymeric nucleic acid to the free 3 'end of the open vector;

Adding a second homopolymer nucleic acid whose sequence is complementary to the first homopolymer nucleic acid to the free 3 ′ end of the nucleic acid inserted into the vector;

Combining the nucleic acid and the nucleic acid of the vector by hybridizing the first and second homopolymer nucleic acids of the sequences complementary to each other;

-Close the vector by concatenation

This method is described in Examples 10 and 13 below.

Advantageously, the method may include the following features individually or together:

The first homopolymer nucleic acid is a poly (A) or poly (T) sequence;

The second homopolymer nucleic acid is a poly (T) or poly (A) sequence.

In a very preferred manner, the homopolymer nucleic acid has a length of 25 to 100 nucleotide bases, preferably 25 to 70 nucleotide bases.

The above-described methods for producing recombinant cloning and / or expression vectors are particularly suitable for constructing DNA libraries in vectors of the BAC type. Thus, according to one advantageous aspect of the recombinant vector preparation method described above, the method is also characterized in that the size of the inserted nucleic acid is at least 100 kb and preferably at least 200, 300, 400, 500 or 600 kb.

This method of preparation is therefore particularly suitable for the insertion of nucleic acids contained in the nucleic acid collections obtained according to the methods of the invention.

In order to insert large DNA fragments into cloning and / or expression vectors, a second method has been developed in accordance with the present invention that does not require the use of restriction endonucleases in the DNA to be inserted into the vector.

Such a method for preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the step of inserting the nucleic acid into the cloning and / or expression vector comprises the following steps:

Forming a blunt end at the nucleic acid end of the collection by removing the protruding 3 'sequence and filling the protruding 5' sequence;

Opening the cloning and / or expression vector at the selected cloning site using an appropriate restriction endonuclease;

Adding complementary oligonucleotide adapters;

Forming a blunt end at the end of the vector nucleic acid by removing the protruding 3 'sequence and filling the protruding 5' end, followed by dephosphorylation of the 5 'end to prevent recirculation of the vector;

The nucleic acid of the collection is inserted into the vector by ligation.

Preferably, removal of the protruding 3 'sequence is performed using exonucleases such as the Klenow enzyme.

Preferably, filling of the protruding 5 'sequence is carried out in the presence of 4 nucleotides triphosphate using polymerase, most preferably T4 polymerase.

As described above, methods for preparing recombinant cloning and / or expression vectors by removing the salient 3 'sequences and filling the salient 5' sequences are particularly suitable for the construction of DNA libraries from cosmid-type vectors.

This method of obtaining a recombinant vector is described in Example 12.

In one particular method of making a recombinant vector according to the present invention, oligonucleotides comprising one or more rare restriction sites are added to the vector at the cloning site of the inserted DNA, according to the teachings of Example 10. This addition of oligonucleotides facilitates subsequent recovery of the insert without its cleavage.

Host cell

Although any type of host cell can be used for transfection or transformation with a nucleic acid or recombinant vector according to the invention, its physiological, biochemical and genetic properties are well characterized, can be easily cultured on a large scale and metabolized Host cells in which the culture conditions for the production of the products are well known, in particular prokaryotic or eukaryotic host cells, will be preferably used.

Preferably, the host cell containing the nucleic acid or recombinant vector according to the present invention is phylogenetically close to the donor organism initially contained in the environmental sample from which the nucleic acid originates.

In the most preferred manner, the host cell according to the invention should have a codon usage similar or at least in close proximity to the donor organism initially present in environmental samples, more specifically in soil samples.

The size of the DNA fragment that can carry the corresponding nucleotide sequence of interest can vary. Thus, enzymes encoded by genes having an average size of 1 kb can be expressed using small size inserts, whereas expression of secondary metabolites is much larger fragments in the host organism, for example 40 kb to 100 It will require maintenance of kb, 200 kb, 300 kb, 400 kb or 600 kb.

Thus, E. coli host cells are the preferred choice for cloning large DNA fragments.

In the most preferred manner, the E. coli strain known as DH10B and described by Shizuya et al. (1992) will be used so that the protocol for cloning into BAC vectors is optimized.

However, other strains of E. coli, such as E. coli Sure, E. coli DH5 α or E. coli 294 (ATCC No. 31446) strains can be advantageously used to construct DNA libraries according to the present invention.

In addition, according to the present invention, it is also possible to construct a DNA library by transfecting E. coli cells with a recombinant vector, including Bacillus , Thermotoga , Corynebacterium , and Lactobacillus. Gene expression of various prokaryotes, such as) or Clostridium , is described in PCT patent application No WO 99/20799.

In general, E. coli host cells can constitute a temporary host in which in all cases the recombinant vector according to the invention can be maintained very effectively, which allows genetic material to be easily handled and achieved stably.

The most comprehensive for a variety expressing the molecule, Bacillus, Pseudomonas (Pseudomonas), such as Streptomyces sheath, Mick Socorro kusu (Myxococcus), Aspergillus you dulran (Aspergillus nidulan) or Neuro spokes la Klein Inc. (Neurospora crassa) cells Other host cells can also be used to advantage.

It is also known in accordance with the present invention that Streptomyces Libidan cells can be used successfully and constitute an expression system complementary to E. coli.

Streptomyces lividan has constructed a model for studying the genus Streptomyces and has also been used as a host for heterologous expression of many secondary metabolites. Like other actinomycetes, Streptomyces coelicolor , Streptomyces griseus , Streptomyces fradiae and Streptomyces griseochromogene Streptomyces lividans such as precursors required for the expression of all or part of complex biosynthetic pathways, such as, for example, polyketide biosynthetic pathways or biosynthetic pathways of non-ribosomal polyketides representing molecular classes of a wide variety of structures. Have molecular and regulatory systems.

Streptomyces lividan also has the advantage of accepting foreign DNA with high transformation efficiency.

Accordingly, the present invention also relates to a recombinant host cell comprising a nucleic acid according to the invention which is a constituent of a nucleic acid collection produced by the method according to the invention or a recombinant host cell comprising a recombinant vector as described above.

According to the first aspect, it may be a recombinant host cell of prokaryotic or eukaryotic origin.

Advantageously, the recombinant cell according to the invention is a bacterium, most preferably a bacterium selected from E. coli and Streptomyces.

According to another aspect, the recombinant host cell according to the invention is characterized in that it is a yeast or filamentous fungus.

The present invention also relates to a collection of recombinant host cells, each constituent host cell comprising a nucleic acid originating from a nucleic acid collection prepared according to the method for producing a nucleic acid collection from a soil sample containing an organism as described above.

The invention also relates to a collection of recombinant host cells, each constituent host cell of the collection comprising a recombinant vector according to the invention.

Because the size of the insert is large, it is necessary to have the highest transformation efficiency. For this purpose, a receiving strain of Streptomyces lividan which structurally expresses pSAM2 integrase to promote site-specific integration of the vector is preferred. To do this, the int gene, under the control of a strong promoter, is integrated into the chromosome. Overproduction of integrase does not induce cleavage (Raynal et al., 1998).

Production of new metabolites from an insert may be toxic to Streptomyces if the insert does not contain a gene that is resistant to the antibiotic produced or if this gene is not expressed or is only slightly expressed. The ability of various genes to make Streptomyces ambofacien resistant to the produced antibiotics has been studied (Gourmelen et al., 1998; Pernodet et al., 1998). Some of these genes encode ABC-type transporters that can confer a broad spectrum of resistance. Such genes can be introduced and overexpressed in Streptomyces ligand host strains.

In contrast, strains sensitive to antibiotics can be used to detect the presence of resistance genes in the library (Pernodet et al., 1996). Specifically, in antibiotic-producing microorganisms, such resistance genes often associate with genes for the biosynthetic pathway of antibiotics. Selection of resistant clones allows for easy primary sorting before more complex detection tests of novel metabolites produced by the clones.

Isolation and Characterization of Novel Nucleotide Sequences Encoding Polyketide Synthetase

According to the invention, a collection of recombinant host cells is obtained after transfecting the host cells with a collection of recombinant vectors, each containing a nucleic acid insert originating from a collection of nucleic acids produced by the method according to the invention.

More specifically, the DNA fragment obtained according to the method of the present invention, in which an indirect extraction step of DNA from an organism contained in a soil sample is performed, is first cloned into the integrated cosmid pOS700I.

Inserting the DNA fragment into the integrated cosmid pOS700I is carried out according to the method of the present invention in which homopolymer polynucleotide tails poly (A) and poly (T) are added to the 3 'end of the vector nucleic acid and the inserted DNA fragment, respectively. .

The recombinant vector thus constructed is encapsidated in the lambda phage head and the resulting phage is used to infect E. coli cells according to techniques well known to those skilled in the art.

A library of about 5000 E. coli clones is obtained.

This clone's library is screened with primers specific for the nucleotide sequence encoding the I PKS enzyme type, also known as β-ketoacyl synthetase, an enzyme involved in the polyketide biosynthetic pathway.

It is recalled here that the polyketide constitutes a wide variety of structural chemical categories, including molecules of many pharmaceutical subjects such as tyrosine, monensin, vermectin, erythromycin, doxorubicin or FK506.

Polyketides are synthesized by the condensation of acetate molecules under the action of an enzyme known as polyketide synthase (PKS). There are two types of polyketide synthase. Type II polyketide synthase is generally involved in the synthesis of polycyclic aromatic antibiotics and catalyzes the repeat condensation of acetate units.

Type I polyketide synthase is involved in the synthesis of macrocyclic or macrolide polyketides and constitutes a multifunctional enzyme.

If this is for therapeutic use, there is a need to isolate and characterize novel polyketide synthetases that can be used for the production of novel pharmaceutical compounds, especially novel pharmaceutical compounds with antibiotic activity.

Screening libraries of the recombinant clones described above with PCR primers that selectively amplify the nucleotide sequences encoding the Type I polyketase synthetase contains a DNA insert comprising a nucleotide sequence encoding the new polyketide synthase To identify recombinant clones. Nucleotide sequences encoding such novel polyketase synthetase are referred to as SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120.

Another subject of the invention relates to a nucleic acid encoding novel polyketase synthase I, characterized in that it comprises one of the nucleotide sequences of SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120.

Preferably, such nucleic acid is in isolated and / or purified form.

The present invention also relates to a recombinant vector comprising a polynucleotide comprising one of SEQ ID NOs: 34 to 44 and SEQ ID NOs: 115 to 120.

The invention also provides a recombinant host cell comprising a nucleic acid selected from polynucleotides comprising one of the nucleotide sequences of SEQ ID NOs: 34 to 44 and SEQ ID NOs: 115 to 120 and SEQ ID NOs: 34 to SEQ ID NO: 44 and SEQ ID NO: 115 It relates to a recombinant host cell comprising a recombinant vector inserted with a polynucleotide comprising one of the nucleotide sequence of SEQ ID NO: 120.

Advantageously, the recombinant vector containing the DNA insert encoding the novel polyketide synthase type I according to the present invention is a cloning and expression vector.

Preferably, the recombinant host cell as described above is a bacterium, yeast or filamentous fungus.

The amino acid sequence of the novel polyketase synthase originating from the organism contained in the soil sample is derived from the nucleotide sequences of SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120 above. These are polypeptides comprising one of the amino acid sequences of SEQ ID NO: 48 to SEQ ID NO: 59 and SEQ ID NO: 121 to SEQ ID NO: 126.

The present invention also relates to a novel polyketide synthetase comprising an amino acid sequence selected from SEQ ID NOs: 48 to 59 and SEQ ID NOs: 121 to 126.

The nucleotide sequence of SEQ ID NO: 114, which includes 6 open reading frames each encoding a polypeptide of SEQ ID NO: 121 to SEQ ID NO: 126, also forms part of the present invention.

The nucleotide sequence of SEQ ID NO: 113 of a26G1 cosmid containing a sequence complementary to the sequence of SEQ ID NO: 114 also forms part of the present invention.

Streptomyces coelicolor (ATCC No. 101.478), Streptomyces ambopaciene (NRRL No. 2.420), Streptomyces lactamanduran (ATCC No. 27.382), Streptomyces rimosus , Genomic DNA derived from pure bacterial strains such as Bacillus subtilis (ATCC No. 6633) or Bacillus licheniformis and Saccharopolyspora erythrea . Extracted and amplified according to the invention.

PCR amplification of DNA from each of the aforementioned bacterial strains is performed using primers specific for the nucleic acid sequence of polyketide synthase type I.

New bacterial polyketide synthase type I genes can thus be isolated and characterized. These are the nucleic acid sequences of SEQ ID NO: 30 to SEQ ID NO: 32.

Accordingly, the subject of the present invention is also a nucleotide sequence encoding a novel polynucleotide synthase type I selected from polynucleotides comprising one of the nucleotide sequences of SEQ ID NOs: 30-32.

Recombinant vectors comprising a nucleotide sequence encoding the novel polyketase synthase type I described above also form part of the present invention.

The invention also provides a recombinant host cell and a recombinant vector as described above, characterized in that they contain a nucleic acid encoding a novel polynucleotide synthase type I comprising a nucleotide sequence selected from the sequences of SEQ ID NOs: 30-32 It relates to a recombinant host cell comprising a.

Subject of the invention is also a polypeptide encoded by a sequence comprising a nucleic acid of SEQ ID NOs: 30 to 32, more specifically a polypeptide comprising an amino acid sequence of SEQ ID NOs: 47 to SEQ ID NO: 50.

The subject of the present invention is also a method of producing polyketide synthase type I according to the present invention, the production method comprising the following steps:

A recombinant host cell comprising a nucleic acid encoding a polynucleotide synthase type I comprising a nucleotide sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120 To produce;

Incubating the recombinant host cell in a suitable culture medium;

Recover and optionally purify polyketide synthase type I from culture supernatant or cell lysate.

The novel polyketide synthase type I obtained according to the method described above can be characterized by binding an antibody that recognizes this polyketide synthase with a pre-immobilized immuno-affinity chromatography column.

Polyketide synthase type I according to the present invention, more specifically, the above-mentioned recombinant polyketide synthase may also be used, for example, in reverse phase chromatography techniques or anion-exchange or cation-exchange chromatography techniques well known to those skilled in the art. It can be purified by high performance liquid chromatography (HPLC) such as.

Recombinant or non-recombinant polyketide synthase according to the present invention can be used for the preparation of antibodies.

According to another aspect, the subject of the present invention is also an antibody that specifically recognizes a polyketide synthase type I or a peptide fragment of such polyketide synthase according to the present invention.

Antibodies according to the invention may be monoclonal or polyclonal. Monoclonal antibodies are described in Kohler and Milstein C. (1975), Nature, Vol. It can be prepared from hybridoma cells according to the technique described by 256: 495.

Polyclonal antibodies are polyketide synthase type I according to the invention of mammals, in particular mice, rats or rabbits, optionally from complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide or muramyl peptide class. It can be prepared by immunization in the presence of an immuno-adjuvant compound such as a compound of.

For the purposes of the present invention, Fab, Fab ', F (ba') ₂ or Martineau et al. (1998) J. Mol. Biol., Vol. 280 (1): 117-127 or antibody fragments such as single-chain antibody fragments containing the variable moiety (ScFv) described in US Pat. No. 4,946,778 and Reinmann KA et al. (1997), AIDS Res. Hum. Retroviruses, Vol. 13 (11): 933-943 or Leger OJ et al. (1997), Hum. Antibodies, Vol. The humanized antibody described by 8 (1): 3-16 also constitutes an “antibody”.

The method for producing an antibody according to the present invention is particularly useful for the simple detection of the presence of polyketide synthase type I according to the present invention or for example in the culture of supernatant or cell lysate of bacterial strains capable of producing such enzymes. Useful for qualitative or quantitative immunoassays to quantify the amount of ketase synthase.

Another subject of the invention relates to a method for detecting a polyketide synthase type I or a peptide fragment of the enzyme in a sample, said method comprising the following steps:

a) contacting the antibody according to the invention with a sample to be tested;

b) detect the antigen / antibody complex formed.

The invention also relates to a kit or a device for detecting a polyketide synthase type I according to the invention in a sample comprising:

a) an antibody according to the invention;

b) optionally a reagent required to detect the antigen / antibody complex formed.

Antibodies to polyketide synthase type I according to the present invention can be labeled using detectable labels of isotopes or non-isotopes, according to methods well known to those skilled in the art.

The present invention uses a pair of primers hybridized with a desired target sequence, such as the sequence of the puromycin biosynthetic pathway, the linA gene sequence involved in the biodegradation of lindene, or the sequence encoding polyketase synthase type I. Screening of the DNA library according to has been described in detail above.

A subject of the present invention is therefore a method for detecting a given nucleotide sequence, or a nucleic acid structurally similar to a given nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:

Contacting the recombinant host cell collection with a pair of primers hybridized with a given nucleotide sequence or a nucleotide sequence structurally similar to a given nucleotide sequence;

Performing at least three amplification cycles;

Detecting any nucleic acid that has been amplified.

For suitable amplification conditions depending on the target sequence of interest, one skilled in the art can advantageously refer to the following examples.

According to another aspect, the invention also relates to a method for detecting a nucleic acid, a given nucleotide sequence or a nucleotide sequence structurally similar to a given nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps: will be:

Contacting the recombinant host cell collection with a probe hybridized with a given nucleotide sequence or a nucleotide sequence structurally similar to a given nucleotide sequence;

Detecting a hybrid formed between the probe and the nucleic acid included in the vector of the collection.

In order to perform the screening of the DNA libraries according to the invention for detecting the presence of nucleotide sequences encoding polypeptides capable of degrading Lindane, the corresponding recombinant clones are detected based on the phenotype corresponding to their Lindane resolution. do. For this purpose, isolated clones and / or clone sets of the prepared DNA libraries are cultivated in the culture medium in the presence of Lindane and Lindane degradation is observed by faint halo formation in the environment around the cells.

The invention also relates to a method for the identification of a compound of interest by one or more recombinant host cells in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:

Incubating the collection of recombinant host cells in a suitable culture medium;

Detecting the compound in culture supernatant or cell lysate of one or more recombinant cells in culture.

A subject of the invention is also a method for screening recombinant host cells for producing the compound in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:

Incubating the collection of recombinant host cells in a suitable culture medium;

Detecting the compound in culture supernatant or cell lysate of the cultured one or more recombinant host cells;

Select recombinant host cells that produce the compound.

The invention also relates to a process for the production of the compound, characterized in that it comprises the following steps:

Culturing selected recombinant host cells according to the methods described above;

Recovery to optionally purify the compound produced by the recombinant host cell.

The invention also relates to the corresponding compounds, characterized in that they are obtained according to the process described above.

The compound according to the present invention is a polyke produced by expressing at least one nucleotide sequence comprising a sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120 It can be made of tide.

The present invention also includes a polyketide produced by expressing at least one nucleotide sequence comprising a sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120 It relates to a composition to be.

The polyketide produced by expressing the at least one nucleotide sequence is preferably a product having the activity of a plurality of coding sequences contained in a functional operon in which the translation product is various enzymes required for the synthesis of the polyketide, and One of the sequences is included in and expressed in the operon. Such operons comprising a nucleic acid sequence according to the invention encoding a polyketide synthetase can be constructed, for example, according to the teachings of Borchert et al. (1992).

The invention also relates to a pharmaceutical composition comprising a polyketide pharmaceutically effective amount according to the invention, optionally in combination with a pharmaceutically compatible vehicle.

Such pharmaceutical compositions advantageously comprise the polyke according to the invention in the range of 1 μg / kg to 10 mg / kg per day, preferably at least 0.01 mg / kg per day and most preferably 0.01 to 1 mg / kg per day. It will be modified for the administration of polyketide amounts synthesized by tide synthase type I, for example parenteral administration.

The pharmaceutical compositions according to the invention can be administered orally, rectally, parenterally, intravenously, subcutaneously or transdermally.

The invention also relates to the use of a polyketide obtained by expressing a polyketide synthase type I according to the invention for the manufacture of a medicament, in particular a drug with antibiotic activity.

Example 1 Method for Preparing Nucleic Acid Collections from Soil Samples Containing Organisms, Including Direct Extraction of DNA from Soil Samples

1. Materials and Methods

1.1 Soil : The characteristics of the six soils used in this study are listed in Table 1.

Clay content and organic content range from 9 to 47% and 1.7 to 4.7%, respectively, and pH range from 4.3 to 5.8.

Soil samples are collected from surface layers 5-10 cm deep. All visible roots are removed and the soil stored for several days at 4 ° C. after which it is dried at room temperature for 24 hours and then screened (mean mesh size: 2 mm) and then stored at 4 ° C. for several months.

1.2 Bacterial Strains and Culture Conditions : Bacterial strains that provide feeder cells, spores or mycelium used to inoculate extracellular DNA and soil samples are selected so that their presence is specifically monitored.

To obtain a large amount of extracellular DNA, a lysogenic strain of E. coli 1192 Hfr P4X (metB) containing lambda phage CI857 Sam7 was incubated for 2 hours at 30 ° C. for 30 hours at 30 ° C. in Luria-Bertani (LB) medium. Incubate at 37 ° C. for 3 hours. Lambda phage DNA was described in Sambrook J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd, ed. Extract according to the technique described by Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.

Non-toxic strains of Bacillus anthracis (STERNE 7700) are used as bacterial cell inoculum. Bacillus anthracis is grown for about 6 hours in a "trypticase soy broth" (TSB) (Biomerieux, Lyon, France) culture broth and confirmed that the OD ₆₀₀ is maintained below 0.6. This condition allows feeder cells to grow without the formation of spores (Patra et al., (1996), FEMS Immunol. Medical Microbiology, vol. 15: 223-231). Spores of Streptomyces lividane OS48.3 (Clerc-Bardin et al., Undisclosed) are mechanically removed from organism cultures in R2YE medium (Hopwood et al., (1985), Genetic Manipulation of Streptomyces-A Laboratory Manual. The John Innes Foundation, Witch. Mycelium of S. lividan OS48.3 is obtained from pre-germinated spores because the use of short mycelium is predicted to minimize rupture and subsequent loss of DNA. Spores in TES buffer (N-tris [hydroxymethyl] methyl-2-aminoethane-sulfonic acid; Sigma-Aldrich Chimie, France) (0.05 M; pH 8) (Holben WE et al., (1998), APPL.Environ Microbiol. Vol. 54: 703-711) and then heat shock (heat shock at 50 ° C. for 10 minutes and then cooling with cold running water, followed by pre-germination medium (1% yeast extract, 1% casamino acid, 0.01 M CaCl ₂ ) to the same amount).

The solution is incubated in a stirrer at 37 ° C. The proportion of germinated spores is estimated to be about 50% according to the results of Hopwood et al. (1985). After centrifugation, the pellet is resuspended in TES buffer added to 3% TSB medium and incubated at 37 ° C. until an OD ₄₅₀ of 0.15 is achieved (Hopwood et al. (1985)). Streptomyces hygroscopicus SWN 736 and Streptosporangium fragile AC1296 (Institute Pushino, Moscow) are incubated according to the techniques described by Hickey and Tresner (1952).

Spores and hyphae DNA of S. lividan are extracted from pure cultures (except that no pulverization is performed) according to the dissolution protocol 6 described below, while spores of S. hygroscopicus and S. praxyl Extraction by chemical / enzymatic lysis (Hintermann et al., 1981).

1.3 Selection of Extraction Buffer TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% weight / volume polyvinylpolypyrrolidone) developed by Picard (1992) is used. Similar buffers are subsequently used by others (Clegg et al., 1997; Kuske et al., 1998; Zhou et al., 1996).

Tris and EDTA protect DNA from nuclease activity, NaCl provides dispersibility and PVPP absorbs humic acid and other phenolic compounds (Holben et al. (1998); Picard et al. (1992)).

In this study, the extractability of this buffer was assessed using 20 different soils with pH ranging from 5.8 to 8.3 and organic content between 0.2 and 6.3% at different pH values (6.0-10.0). These 20 soils (other features are not shown) are used only in this experiment. The amount of DNA is measured by the colorimetric method described by Richard (1974) and described below.

1.4 In situ Lysis and DNA Extraction Protocols : A number of protocols with increasing number of steps have been tested to evaluate the efficacy of various techniques to dissolve soil microorganisms in situ. For this experiment, indigenous soil microflora was targeted in six soils. Additional experiments were conducted to study the efficacy of lysis treatment of released DNA by analyzing the quantity and quality of recovered DNA originating from lambda phage DNA pre-added to the soil.

Once an optimized protocol (referred to as Protocol 6) is developed, this protocol is used to quantify DNA originating from indigenous actinomycetes and DNA originating from Gram-positive bacteria inoculated into selected soils. In all cases, soil samples are dried and screened as described above.

After grinding, 0.5 ml of TENP buffer is added to the dry 200 mg soil, except for Protocol 1, where the buffer is added to the unground soil.

For various dissolution treatments (see below), the soil suspension is vortexed for 10 minutes and centrifuged (4000 x g for 5 minutes), then aliquots (25 μl) of supernatant are analyzed by gel electrophoresis (0.8% agarose). .

Another aliquot of the supernatant, usually having a known volume of 350 μl, is precipitated with isopropanol.

5 aliquots (representing DNA derived from 1 g of soil) were combined to purify (Protocol D, see below) and quantification by hybridization (Dot-Blot) or total amplification of PCR amplification products (Dot-Blot) Resuspend in 100 μl of sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) before (see below).

Hybridization signals are quantified by phosphorescence imaging (“phospho-imaging” technology, see below).

1.5 Evaluation of in situ cell lysis method : Extracellular DNA obtained after washing the soil with extraction buffer for the quality and quantity of DNA extracted after several lysis treatment steps (Protocol 2-5b) (Protocol 1: see FIG. 1). Compare with that of.

Protocol 1: No Dissolution

TENP buffer is added to the unground soil and the DNA extraction step is performed as described above.

Protocol 2: DNA Extraction Following Soil Crushing

Two different types of apparatus are used to grind the soil.

To compare their respective efficacy, 5 g of dry soil is ground in a mill containing tungsten rings for 30 seconds or in a soil mill containing mortar and agate beads (20 mm diameter) for various times up to 60 minutes.

TENP buffer is then added and the DNA is extracted as described above.

Gel electrophoresis results show that 40 minutes of grinding using agate beads is necessary to obtain the same amount of extracted DNA after 30 seconds of grinding using a tungsten ring.

The size distribution of the DNA fragments is similar no matter which method is used.

Accordingly, such processing is considered equivalent and as a result will not be specifically specified for use in the protocol described below.

In protocols 3 to 5, after grinding the soil, a number of different dissolution treatment efficiencies are tested individually or in different combinations.

Protocol 3:

This protocol is identical to Protocol 2, except that it includes a homogenization step using a set of Ultra-turrax type mixers (Janker and Kunkel from IKA Labortechnik, Germany) at half the maximum speed for 5 minutes.

Protocol 4a and 4b:

This protocol is the same as Protocol 3 except for further sonication steps.

Two types of sonicator devices were compared: a titanium micropoint sonicator (600 W Vibracell Ultrasonicator, Ilkirche Bioblock, France) (protocol 4a) and a cup horn type sonicator (protocol 4b).

Vibracell micropoints that generate ultrasound are in direct contact with the soil solution.

For Cup Horn type devices, the soil solution is stored in a tube placed in a bath through which ultrasonic waves pass.

Preliminary experiments are performed to determine the optimal conditions for both sonicators (results not shown).

The best compromise, in terms of the amount and fragment size of the extracted DNA, consists of sonication for 7 and 10 minutes by titanium micropoint and Cup Horn type sonicator respectively (power is adjusted to 15 W and 50% active cycle).

Protocol 5a and 5b:

After sonication with a titanium micropoint or Cup Horn type device (protocols 4a and 4b, respectively), lysozyme and acromopeptidase are added to each enzyme at a final concentration of 0.3 mg / ml.

After the soil suspension is incubated at 37 ° C. for 30 minutes, lauryl sulfate is added to a final concentration of 1% and then the suspension is incubated at 60 ° C. for 1 hour before centrifugation and precipitation as described above.

In addition to the protocol described above, sonication (see Cup Horn, protocol 4b) and heat shock (30 seconds in liquid nitrogen, followed by 3 minutes in boiling water) and 3 treatments on lambda phage DNA digested with HindIII previously added to the soil Examine the effect of the repetition (see below).

Heat shock has been proposed in the prior art as a means for in situ cell lysis (Picard et al., (1992)). However, due to the fact that such treatment has a deleterious effect on the free DNA (see results section), it has not been included in the above protocol.

Optimized Protocol

After evaluating the various dissolution treatments, an optimized protocol is defined and referred to as Protocol 6. Protocol 6 is identical to Protocol 5b, except that before sonicating, the soil suspension is vortexed and then stirred by rotation on a wheel for two hours before freezing at -20 ° C.

After thawing, the soil suspension is vortexed for 10 minutes before sonication. Protocol 6 was used for experiments in which soil was incubated with bacterial cells and for quantifying indigenous actinomycetes (see below).

1.6 Microscopic Counting : As a method of lysis of bacterial cells, the soil grinding effect is examined under a microscope.

5 g of the dried raw soil was mixed with 50 ml of ultrapure sterile water for 1.5 minutes in a Waring Blender apparatus; At the same time, 1 g (dry weight) of ground soil (protocol 2) is suspended in 10 ml by stirring for 10 minutes. The soil suspension is serially diluted and then acridine orange is added to a final concentration of 0.001%.

After 2 minutes, the suspension is filtered through a Nucleopore brand membrane of 0.2 μm black type. Each filter is washed with lysed sterile water, treated with 1 ml of isopropanol for 1 minute to fix bacterial cells, and then washed again.

Count using a Zeiss Universal epifluorescence microscope with a 100x objective. For each soil type, 3 liters were counted and at least 200 cells were counted in each of the filters.

1.7 Counting of cultivable actinomycetes and total number of colony-forming units (CFUs) : Actinomycetes that survived dissolution treatment (protocols 1-5) were loaded into soil No. Check specifically with 3 (St. Andre shores, see Table 1).

After 10-fold dilution of a solution of yeast extract (6% weight / volume) and SDS (0.05%) to induce germination (Hayakawa et al., (1988)), the soil suspension was serially diluted in sterile water and 40 ° C. Incubate for 20 minutes at and then inoculate in HV medium (Hayakawa et al., 1987).

Replenish HV medium with activinone (50 mg / l) and nystatin (50 mg / l).

After incubation at 28 ° C. for 15 days, the actinomycete colonies are counted.

In total, about 400 colonies were examined. Identification was based on the morphological characteristics of macro and micro and diaminopimelic acid content analysis of isolates (Shirling et al., 1996); Staneck et al., 1974; Williams et al., 1993).

The total amount of cultureable bacteria (total CFU) was also measured for each of the lysis protocols 1-5. The soil suspension is serially diluted and inoculated in pairs in Bennett agar medium (Waksman et al., 1961) supplemented with nistatin and activion (50 mg / l each).

Each Petri dish is covered with a cellulose nitrate filter (Millipore) and incubated at 28 ° C. for 3 days. After counting colonies on the membrane, the filter is removed and the Petri dishes are rearranged at 28 ° C. for 7 days and then counted again.

1.8 Recovery of Lambda Phage DNA Added to Soil :

Lambda phage DNA is digested with HindIII, extracted with a phenol-chloroform mixture, precipitated and resuspended in ultrapure sterile water according to standard protocols (Sambrook et al., 1989).

Dilutions corresponding to 0, 2.5, 5, 7.5, 10 and 15 μg of DNA per g dry weight of the soil are prepared in 60 μl volumes, respectively. These DNA dilutions are added to a 5 g batch of dry soil and then vigorously vortexed for 5 minutes prior to grinding.

In addition, lambda phage DNA is added to the soil at concentrations corresponding to 0, 10 and 15 μg of DNA per gram dry weight of the soil.

After grinding, extraction buffer is added and DNA is extracted according to protocol 2 (see above).

1.9 Saturation of Adsorption Sites by RNA: To determine whether saturation of the nucleic acid adsorption sites of soil colloids can increase the level of DNA recovery, sand mix (Soil No. 4) and clay soil (Soil No. 5). ) Is incubated with RNA solution before any other treatment.

Commercially available Saccharomyces cerevisiae RNA (Boehringer Mannheim, Meylang, France) was diluted in phosphate buffer (pH 7.1), and then dried and sieved soil samples (2 ml per gram soil) RNA per gram dry weight of soil To final concentrations of 20, 50 and 100 mg.

The tube containing the soil suspension is stirred by rotation at room temperature for two hours. After centrifugation, the soil pellets are dried overnight in an oven (50 ° C.). Lambda phage DNA is then added to the soil (0, 20 or 50 μg per gram dry weight of soil) to simulate the fate of the DNA released after cell lysis.

DNA is extracted according to protocol 2. It was then determined that the same effect of RNA addition on the recovery of DNA can be achieved by adding RNA directly to the extraction buffer.

This simplified procedure was performed in clay soil no. Use for five.

RNA is then added at a concentration corresponding to 50 mg of RNA per gram dry weight of soil.

1.10 Qualitative and Quantitative Determination of the Efficacy of the Extraction Protocol : DNA quality (no degradation) is assessed based on the size of the DNA fragment or the relative position of the DNA transfer band after electrophoresis of an aliquot fraction of the DNA solution on 0.8% agarose gel. .

Fluorescence intensity allows for semi-quantitative evaluation of extraction yield.

Another aliquot fraction is used for quantitative determination of DNA content by Dot-Blot and analysis by phospho-imager. The Dot Blot hybridization protocol was described by Simonet et al. (1990).

Hybridization membranes (GeneScreen plus, Life Science Products, Boston, USA) were placed in 20 ml of a solution containing 6 ml of 20 x SSC, 1 ml of Denhardt's solution, 1 ml of 10% SDS and 5 mg of salmon sperm DNA. Prehybridize for at least 2 hours.

Prior to washing the membrane twice in SSC 2 × buffer for 5 minutes at room temperature, hybridization was carried out overnight in the same solution in the presence of labeled probes, followed by a third wash in SSC 2 ×, 0.1% SDS buffer and SSC 1 × , A fourth wash in 0.1% SDS buffer is performed at hybridization temperature for 30 minutes.

Hybridization signals are quantified with a Biorad radiometric imaging system (Molecular Analyst Software, BIORAD, Ibri-sur-Seine, France).

In order to quantify the total amount of DNA derived from the indigenous microflora, various soils are extracted according to protocols 1-5. Non-amplified DNA is applied to the Dot-Blot membrane and hybridized using the universal probe FGPS431 (Table 2).

This probe, which hybridizes with position 1392-1406 of the E. coli 16S rDNA gene (Amann et al., (1995)) at its end, uses polynucleotide T4 kinase (Boehringer Mannheim, Melang, France) to ³² P ATPα ( Amann et al., (1995).

A calibration curve is constructed using E. coli DH5α DNA. As for E. coli, starting from the assumption that the average number of copies (rrn) is 7, the conversion to calculation for soil bacteria requires simplification.

The recovery of extracellular DNA is quantified using lambda phage DNA digested with HindIII. Non-amplified extracts from soil to which lambda phage DNA was added are digested with HindIII and hybridized with randomly labeled lambda phage DNA using Klenau fragment (Boehringer Mannheim, Melang, France).

The amount of DNA is calculated by interpolation using a calibration curve constructed with purified DNA.

The total amount of DNA extracted from soils 1, 2, 3, 4 and 6 according to protocol 2 (crushing) is also quantified by colorimetric means according to the technique described by Richard (1974).

In short, the DNA is mixed with concentrated HClO ₄ (the final concentration of HClO ₄ is 1.5 N). 2.5 volumes of this solution are mixed with 1.5 volumes of DPA (diphenylamine, Sigma-Aldrich, France) and then the mixture is incubated at room temperature for 18 hours before measuring the OD at 600 nm. Soil DNA extracts are quantified against standard curves constructed with DNA extracted from E. coli DH5α according to standard protocols (Sambrook et al., (1989)).

1.11 Development of DNA Quantitation Using PCR Amplification and Hybridization: For PCR amplification, DNA Taq polymerase (Appligene Oncor, France) is used according to the manufacturer's instructions.

The PCR program used for all amplifications was as follows: initial denaturation at 95 ° C. for 3 minutes followed by 35 cycles of 1 minute at 95 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C. and final extension at 72 ° C. for 3 minutes. .

DNA purified from Streptosporangium praxyl is used as a control at concentrations ranging from 100 fg to 100 ng.

To specifically amplify the DNA in this bacterium, the sequences of the actinomycete 16S rDNA are aligned and the primers FGPS122 and FGPS350 (Table 2) complementary to a portion of the 16S rDNA are selected. Their specificity is tested in the Actinomycete strain collection (Streptomyces, Streptosporangium and other highly similar genera).

PCR products are hybridized with oligonucleotide probe FGPS643 (Table 2). To simulate the purity levels routinely obtained with DNA extracted from soil, a control of pure DNA from S. fragile was obtained by treatment according to dissolution protocols 4b and 5b, followed by soil extracts purified according to high protocol D. Mix.

Prior to use, the soil extract is treated with DNase (DNase / ml 1 unit, Gibco BRL) for 30 minutes at room temperature. The DNase is then inactivated by heating at 65 ° C. for 10 minutes. Demonstration of inactivation is performed by PCR. Humic acid concentrations are determined by spectrophotometry (OD ₂₈₀ nm) against the standard curve of commercial humic acid (Sigma).

Soil solutions treated with undiluted, 10-fold and 100-fold diluted DNase are mixed with 100 fg to 100 ng of S. fragile DNA prior to PCR amplification. In another series of experiments, increasing concentrations of Streptomyces hygroscopicus DNA (100 pg to 1 μg) are added to S. fragile DNA to simulate the presence of non-target DNA and its effect on the PCR process. .

1.12 Purification of Raw DNA Extraction: Compare the four DNA purification methods. DNA is extracted from 1 g (dry amount of soil) according to protocol 4a and resuspended in 100 μl of buffer TE8 (50 mM Tris, 20 mM EDTA, pH 8.0).

Protocol A

Elution through two consecutive Elutip d columns (Schleicher and Schuell, Dassel, Germany) (Picard et al., (1992)).

Protocol B :

Elution through Sephacryl S200 column (Pharmacia Biotech, Uppsala, Sweden) followed by Elutip d column (Nesme et al., (1995)).

Protocol C:

Separation using a 2-phase aqueous system with PEG 8000 (Merck, Darmstadt, Germany) 17.9% (w / w) and (NH ₄ ) ₂ SO ₄ (Zaslavsky, (1995)) 14.3% (w / w).

After vigorous vortex mixing, the two phases are left to stand at room temperature to separate.

Transfer 1 ml of each phase to another tube, mix with 100 μl of sample and leave to separate at 4 ° C. overnight.

Excess salt is removed by dialysis through a Millipore membrane for 1 hour in the presence of excess TE 7.5 buffer (10 mM Tris, 1 mM EDTA and 1M MgCl ₂ at pH 7.5).

Protocol D:

Elution through Microspin Sephacryl S400 HR column (Pharmacia Biotech, Uppsala, Sweden) followed by Elutip d column.

Each protocol is completed with an ethanol precipitation step and the DNA is resuspended in 10 μl of TE 7.5 buffer. The efficacy of the purification protocol is examined by PCR amplification of undiluted aliquots and 10-fold and 100-fold dilution aliquots of DNA solution using standard protocols (see below).

1.13 Recovery of DNA from Inoculated Microorganisms :

Cells, spores and hyphae are washed twice and counted on the plate by counting or by direct microscopic counting. 5 g batches of dried and sifted soil (Soils 2, 3 and 5) were subjected to S. libidans spores and mycelium suspension at concentrations corresponding to 0, 10 ³ , 10 ⁵ , 10 ⁷ and 10 ⁹ spores per gram dry soil 100 Inoculate with μL or with B. anthracy vegetative cells at concentrations corresponding to 0, 10 ⁷ and 10 ⁹ cells per gram dry soil.

Calculate the amount of S. lividans hyphae based on the number of spores from which they originate. After the bacterial suspension is added, the soil sample is vigorously vortexed for 5 minutes before grinding. DNA is extracted according to protocol 6 (see below).

PCR amplification followed by Dot-Blot hybridization and phosphorescence imaging (phospho-imaging) is used to quantify the amount of DNA recovered from cells and spores and from bacterial mycelium inoculated into soil.

DNA extraction is performed according to Lysis Protocol 6. PCR amplification and hybridization are performed as described above. Primers and probes are located outside the 16S region and targeted on chromosomal regions that are highly specific for each organism to avoid background signals.

B. For soil inoculated with anthracis, amplification products are hybridized with oligonucleotide probe C501 using primers R499 and R500 (Patra et al., (1996)) (Table 2).

For soil inoculated with S. lividans, PCR reactions are performed using primers FGPS516 and FGPS517, and the amplification products are hybridized with oligonucleotide probe FGPS518 (Table 2).

The amplified region is part of a cassette constructed to specifically obtain strain OS48.3 (Clerc-Bardin et al., Unpublished).

Calibrated counts are obtained in all cases using purified DNA from target organisms.

2. Results

2.1 Selection of Extraction Buffer:

Twenty different soils are used to determine the optimal pH of the DNA extraction buffer. For all solids, the DNA yield increases with increasing buffer pH. The yield (± sd) for each pH, calculated as% of the highest for each soil, is as follows: pH 6.0: 31 ± 13; pH 7.0: 43 ± 16; pH 8.0: 60 ± 14; pH 9.0: 82 ± 12; pH 10.0: 98 ± 3.

For 16 of 20 soils, the highest yield was obtained at pH 10.0, while for the remaining 4 soils, the highest yield was obtained at pH 9.0. However, at pH 10.0 compared to at pH 9.0 (not shown), higher amounts of humic acid were released. As a result, pH 9.0 was chosen for all the experiments presented below.

2.2 Efficacy of DNA Extraction Protocol:

Whole DNA from indigenous soil organisms is extracted and quantified to assess the efficacy of several in situ cell lysis protocols. Soil samples 1-6 (Table 1) are treated according to protocols 1-5 described in the Materials and Methods section (FIG. 1).

After DNA extraction, the soil suspension is precipitated with isopropanol and an aliquot of the resuspended pellet is analyzed by gel electrophoresis in the first step to assess the quality and amount of released DNA.

However, due to the co-extraction of DNA with a compound such as humic acid, the color of the DNA extract became darker as the number of elution steps increased.

Some of these dark colored crude extracts do not migrate in the way expected in agarose gels.

Thus, the raw DNA solution was purified before quantification (protocol B). Gel electrophoresis of the purification solution obtained after various dissolution treatments is shown as an example for soil 3 (FIG. 2).

Visible comparison by ultraviolet irradiation of the intensity of colored DNA allows for a semi-quantitative assessment of treatment efficacy. In addition, the presence of various size migration profiles (discontinuous bands) of DNA fragments and the disappearance of long length fragments indicate that DNA degradation has occurred.

Clay soil No. At 5 no DNA could be extracted.

More accurate quantification of DNA from all soils, extracted according to protocols 1 to 5, was performed using an oligonucleotide probe (probe FGPS 431, Table 2), which is complementary to the highly conserved sequence of the 16S rDNA region and without a pre-PCR amplification step. It was performed by Dot-Blot hybridization.

Clay soil No. Except for 5, DNA was detected in extracts of all soils after each of the various lysis steps.

The results are consistent with the assessments made after gel electrophoresis.

For comparison with independent quantification, DNA extracted from Protocol 2 (from all soils except soil No. 5) was also quantified using colorimetric DNA detection (Richard, 1974).

Good correlation was found between DNA quantified using this colorimetric technique and results obtained with Dot-Blot hybridization / radiation-imaging (r = 0.88), which is a hypothesis that the average copy number (rrn) of soil bacteria is 7 To confirm.

Hybridization (Dot-Blot) was confirmed by extraction without lysis treatment (Protocol 1), the amount of extracellular DNA is 4 μg / g to soil No. 6 for acidic soil (No. 6). 3 showed a range of 36 μg / g (Table 3).

Soil grinding (protocol 2) increased the amount of DNA extracted from all soils (eg, soil 26 μg / g for soil No. 6 and soil 59 μg / g for soil No. 3) (Table 3 2).

For both milling treatments (see section Materials and Methods), discontinuous DNA migration was detected on agarose gels, indicating that DNA molecules were partially degraded (FIG. 2).

The size of the DNA fragment is 20 to 0.2 kb. The band strength of the minimum fragments is very low, indicating that most fragments are much larger than 1 kb.

Protocol 3 involves homogenization in an Ultra-turrax mixing device after adding extraction buffer to soil samples. This step results in an increase in the amount of DNA extracted, as measured by Dot-Blot hybridization on two of the soils (sand soil No. 3 and acidic soil No. 6), while two organic-rich soils (soil No. .1 and No. 2) resulted in the production of small amounts of DNA.

Protocols 4a and 4b allow to evaluate the effects of both types of sonication on DNA yield from pre-milled and pre-homogenized soil.

Compared with Protocol 3, soil No. Except for 6, sonication had no positive effect on DNA yield. However, the dissolution efficacy for the two types of sonicators is different. For soils 2, 3 and 4, the maximum amount of extracted DNA was obtained using titanium micropoints (Table 3; FIG. 2), while soil No. In cases 1 and 6, the DNA yield was higher using the Cup Horn apparatus.

Contrary results were also obtained when the enzyme / chemical dissolution step was added after the sonication step (protocols 5a and 5b); In certain cases, the amount of DNA extracted was greater than those recovered according to protocols 4a and 4b, while in other cases the yield was lower (Table 3).

2.3 Direct Counting of Microorganisms :

Microscopic counting of bacterial cell counts after staining with acridine orange is performed before and after grinding on all soils.

Prior to crushing, the number of bacteria per gram of dry soil was determined by tropical soil No. 5 ranged from 1.4 x 10 ⁹ (± 0.4) to 10 x 10 ⁹ (± 0.7) in soils obtained from the coast of St. Andre (soil No. 3) (Table 1).

After grinding, the cell numbers were respectively determined by soil No. 45, 74, 75, 54, 34 and 75% of the initial values for 1-6.

2.4 Counting of cultivable actinomycetes belonging to different genera :

Soil No. Modifications in the intratrial actinomycete population were noted after various dissolution treatments (FIG. 3).

For example, when no lysis treatment was applied (Protocol 1), the colonies of Streptomycin species predominant over the surviving actinomycete flora and represented 65% of the total colonies identified. After grinding, the percentage of Streptomyces colonies fell to 51%, while the proportion of colonies belonging to the micromonospora increased to 14-41%.

Chemistry / enzyme dissolution (protocols 5a and 5b) appears to be particularly effective for dissolution of streptomycin. When all lysis treatments were applied, including chemical / enzyme lysis (protocols 5a and 5b), the actinomycete microflora, still containing more than 10 ⁶ cfu per gram of soil, predomined in species belonging to the micromonospora, In contrast, few Streptomyces colonies were recovered or not recovered at all.

Organisms belonging to the genus, such as Streptosporangium, Actinomadura, Microbispora, Dactylosporangium and Actino Plains, appeared in small numbers on the plate after grinding, homogenization by Ultra-turrax apparatus and sonication (identification). 2-8% of the total number of colonies), which was generally absent when combined with chemical / enzyme dissolution.

The total number of cultivable bacteria remaining after each lysis treatment (protocols 2 to 5) was also determined by soil no. 4 was investigated. The results suggest that the number of cultivable bacteria does not decrease with the intensity of the lysis treatment (in all cases, and also when no treatment is applied, such as when done according to Protocol 1, about 2 x 10 ⁶ cfu per gram of soil). ).

The production of these low cfu values is likely due to the fact that dry soil is used and that only the most resistant bacteria are amplified on the plate. The number of actinomytes forming colonies is generally greater than that of total cfu (all bacteria) due to the fact that the spore-germination stage included in the actinomycete detection protocol was lost during the control of the total bacteria.

2.5 Recovery of Added Lambda Phage DNA :

The purpose of these experiments is to assess how the continuous lysis treatment can affect the recovery of naked DNA and to evaluate whether this continuous lysis treatment contributes to its degradation.

DNA can be extracellular DNA released from an already dead organism that can last for several months in the soil (Ward et al., 1990), or a fraction of DNA released from an easily dissolved organism during the first stage of treatment. To simulate this situation, lambd phage DNA digested with HindIII is added to the soil at various concentrations before and after grinding. In addition to grinding, combinations of other dissolution treatments are also tested, including sonication (see Cup Horn apparatus, protocol 4b) and heat shock (see Materials and Methods section).

After extraction, the aliquot fractions theoretically required to contain 25 to 150 ng of lambda phage DNA are analyzed by gel electrophoresis. Regardless of the dose or type of soil, no DNA fragments specific for lambda phage could be observed when DNA was inoculated into soil samples prior to milling.

When the DNA was added after grinding and extracted without additional lysis treatment steps, specific lambda phage DNA profiles were detected in 4 extracts of 5 soils tested.

In all these cases, a direct cause-and-effect relationship was obtained between the amount of DNA added and the signal intensity on the agarose gel. However, the signal intensity was less than the expected signal intensity when compared to that of the molecular standard.

Moreover, the band at 23 kb was in many cases absent, indicating that longer fragments preferentially adsorb on soil particles or are more susceptible to degradation compared to shorter fragments.

Tropical soils characterized by very high clay content. No bands were detected in the sample of 5 (Table 1).

For more accurate quantitation, DNA recovery was measured on a phosphorescence imaging device (Phospo-Imager) after Dot-Blot hybridization. According to this technique, the soil No. where DNA could not be detected. Except for 5, DNA was detected in all samples, including those inoculated prior to grinding.

In all other soils, the amount of extracted DNA increases with increasing inoculation size (FIGS. 4A-D).

However, the recovery of lambda phage DNA was low. When crushing was the only lysis treatment applied, the recovery was 0.6-5.9% of the DNA added when this DNA was added before grinding, and 3.6-24% of the DNA added when the latter was added after grinding. The highest level of recovery is soil no. Obtained from 2.

Gel electrophoresis of aliquot fractions of samples treated by heat shock and sonication did not allow for the observation of DNA bands in any sample, including tests in which DNA was added after grinding. Dot-Blot hybridization experiments confirm these results.

Hybridization signals obtained from heat shock and sonicated soil suspensions were at most low.

The sample showing the maximum amount of DNA (15 μg DNA per gram dry soil) was the only one whose signal was significantly different from the background level.

There was no difference (or only a small difference) between the heat shocked samples and those heat shocked and sonicated, suggesting that heat shock has a deleterious effect on DNA. The best recovery was obtained for soil No. 2 was observed (Table 1), whereas clay soil No. DNA was not recovered from 5.

Soil No. inoculated with 20-50 μg of lambda phage DNA per gram of soil. 4 and No. Further experiments are performed with 5 non-milled samples.

Samples are extracted immediately or after 1 hour incubation at 28 ° C., and then DNA extracts are purified and analyzed by gel electrophoresis.

Soil No. 1 hour after inoculation The culture of 4 did not give a qualitatively or quantitatively different profile from those obtained without culturing or those previously observed in which DNA was added after grinding.

These results suggest that enzymatic degradation by soil nuclease is not thought to be involved in low levels of DNA recovery. Moreover, the absence of the grinding step is determined by soil No. It does not allow for increased recovery of DNA from 5, which suggests that structural changes in soil due to grinding do not significantly increase the adsorption of nucleic acids to colloids.

2.6 Saturation of Adsorption Sites by RNA :

Most of the profiles obtained on agarose gels are not significantly different from the preceding profiles without RNA treatment.

For example, regardless of RNA concentration and lambda phage DNA concentration used, clay-rich soil No. No band from 5 was detected.

In addition, specific bands of lambda phage DNA digested with HindIII remained undetectable in the sandy mixed soil treated with RNA (soil No. 4) when RNA was added before grinding.

The intensity of the bands obtained from samples inoculated with DNA after grinding increases with increasing RNA concentration, suggesting that the treatment can have a positive effect.

However, the results after analysis by hybridization and phosphorescence imaging did not confirm the electrophoretic results. For example, when the DNA was added after grinding, the positive effect of RNA treatment on the recovery of DNA from clay mix was not apparent.

On the other hand, the positive effect of RNA was observed for clay-rich soils (No. 5) when DNA was added after grinding.

Although the hybridization signal for the control sample was not different from the background noise level, a significant amount of DNA was released from the RNA treated sample and the signal increased with increasing amount of added DNA and with increasing RNA concentration.

However, even for the highest RNA concentration (100 mg per gram of dry soil), the recovery level never exceeded 3%.

2.7 Purification of Crude DNA Extracts :

Of the four protocols tested, the best amplification of the undiluted DNA extract (1 μl of extract in 50 μl of PCR mixture) was eluted through the Microspin S400 column followed by the Elutip d column as shown by gel electrophoresis of the PCR product. Observed after elution.

DNA purified by a two-phase aqueous system (protocol C) was given a smaller amount of PCR product after amplification using the non-diluted DNA extract as a starting material.

No amplification products can be obtained from the undiluted extract after amplification following the use of protocol A or B. Thus, Protocol B (see Materials and Methods section) was used for all experiments that performed PCR amplification and / or Dot-Blot hybridization.

2.8 Quantitation by PCR and Hybridization:

The first step measures whether the amount of PCR product is proportional to the number of target DNA molecules initially present in the reaction tube. DNA from Streptosporangium praxyl is used as a target (see section Materials and Methods).

Primers used are primers FGPS122 and FGPS350 (Table 2). Gel electrophoresis of the PCR product shows that the band intensity increases with increasing target concentration. PCR products are hybridized with oligonucleotide probe FGPS643 (Table 2) and signals are quantified by phosphorescence imaging (phospho-imaging).

A good correlation (r ² = 0.98) was found between log [number of targets] and log [strength of hybridization signal].

The investigation is then performed to see if the efficacy of PCR amplification is affected by humic acid and non-target DNA. When analyzed by gel electrophoresis, extracts of DNase-treated soil were added to the various amounts of target DNA when amplification was performed with DNA solutions containing humic acid at concentrations ranging from 8 ng to 50 μl of PCR mixture. The increased intensity of the band for the corresponding PCR product was preserved.

With 20 ng of humic acid in the PCR mixture, the band corresponding to a small level of target DNA disappeared, and no band was seen at 80 ng of humic acid concentration and higher concentration.

Various amounts of target DNA from S. fragile are expected when mixed with S. fragile DNA with Streptomyces hygroscopicus DNA and added to 50 μl of PCR mixture in the range of 100 pg to 1 μg prior to amplification. PCR products were supplied to enable simulation of non-target DNA released from soil microbial guns.

2.9 Quantification of Indigenous Soil Actinomytes After Different Dissolution Treatments :

Purification protocol D was applied as described above, followed by PCR amplification, followed by extraction according to protocols 1, 2, 3, 5a and 5b. Actinomycete belonging to the genus Streptosporanium in 3 is quantified (FIG. 5).

After grinding (Protocol 2), the amount of target DNA originating from this actinomycete is 2.5 ± 1.3 ng / g per gram dry weight, as assessed by Dot-Blot and radio-imaging.

If the DNA content is assumed to be 10 fg per cell for Streptomyces (Gladek et al., 1984), this value corresponds to approximately 2.5 × 10 ⁵ genomes. Similar values were obtained after different lysis treatments (protocol 2.6 ± 1.1 and 1.8 ± 1.3 ng DNA per gram of dry soil, respectively, using protocols 3 and 4b).

2.10 Efficacy of DNA recovery from soil pre-inoculated with bacteria :

Three soils (Nos. 2, 3 and 5) are seeded with different concentrations of Streptomyces lividans spores or mycelia (see Materials and Methods section). The amount of mycelium added to the soil (FIG. 6B) corresponds to the number of spores inoculated into the germination medium. Approximately 50% of these spores germinated. The exact number of cells in the mycelia of germinated spores was not determined. Therefore, the amount of spores and mycelium inoculated into the soil could not be directly compared.

For each soil sample, extraction protocol No. in parallel with Dot-Blot hybridization and phosphorus imaging (phospho-imaging) 6, Purification Method D and PCR amplification are used to count the specific target DNA released. The extracted DNA contained soil number of spores added. 3 and No. For ⁵ , exceed 10 ⁵ and soil No. For 2 it can only be clearly distinguished from the background noise when it exceeds 10 ⁷ (FIG. 6A).

When the mycelium is added, the extracted DNA is divided into soil No. 2 and No. For 3, at a quantity equivalent to 10 ³ spores per gram of soil, and soil No. More than 10 ⁷ spores per gram of 5 can be detected (FIG. 6B).

Above the detection level, the hybridization signal increases as the amount of inoculated cells increases.

For spore inoculation, a 100-fold increase in the number of inoculated cells results in a value close to a 100-fold increase in DNA yield. This increase is especially true for mycelial soils. 2 and No. Obviously less than when inoculated at 3 (FIG. 6).

In contrast, in the results obtained when lambda phage DNA is used as the inoculum, the DNA is also recovered from clay-rich soil (No. 5) when bacterial cells are used as the inoculum. However, even for the latter inoculum, RNA treatment increased the recovery of Streptomyces DNA from this soil for both spores and mycelium (FIG. 6).

Inoculation into soil with Bacillus anthracis feeder cells confers recovery levels similar to those obtained for Streptomyces.

Moreover, the soil No. DNA recovery levels from 5 were increased.

Example 2: Construction of a low molecular weight DNA (<10 kb) library using Lindane contaminated soil, cloning and expression of linA gene

This example describes the construction of an E. coli DNA library. This demonstrates the cloning and expression of small genes obtained from non-cultured microflora.

Lindane is an organic chlorine pesticide that is difficult to break down and is persistent in the environment. Under aerobic conditions, biodegradation is catalyzed by dehydrochlorinase encoded by the linA gene, causing the conversion of lindene to 1,2,4-trichlorobenzene. The linA gene was identified only from two strains isolated from the soil: sphingmonas posimobilis (Seeno and Wada 1989; Imai et al., 1991; Nagata et al., 1993) isolated from Japan and rhoda isolated from France Novbacter Lindani Classicus (Thomas et al., 1996, Nalin et al., 1999).

However, the resolution of lindene, as demonstrated by analysis of released chloride ions and PCR amplification of the linA gene from soil with or without contact with lindenin, appears to be more prevalent in the environment (Biesiekierska-Galguen, 1997). ).

1. Direct Extraction of Soil DNA

Dry soil is ground in a Restch centrifugal grinder with six tungsten bis for 10 minutes. 10 grams of ground soil are suspended in 50 ml of pH 9 TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% w / v polyvinylpolypyrrolidone) and homogenized by vortexing for 10 minutes.

After centrifugation at 4000 × g and 4 ° C. for 5 min, the supernatant is precipitated with sodium acetate (3 M, pH 5.2) and isopropanol and then taken up in sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The extracted DNA is then purified on S400 molecular sieve column (Pharmacia) and Elutip d ion exchange columns (Schleicher and Schuell) according to the manufacturer's instructions and then stored in TE.

2. Construction of DNA Library Extracted from Soil in Vector pBluescript SK-

DNA extracted from the vector pBluescript SK- and soil, respectively, is digested with enzymes HindIII and BamHI (Roche) at a rate of 10 units of enzyme per μg of DNA (incubated at 37 ° C. for 2 hours). The DNA is then linked at 15 ° C. overnight by the action of T4 DNA Ligase (Roche) at a rate of 1 enzyme unit per 300 ng of DNA (about 200 ng of DNA insert and 100 ng of degraded vector). Electrocompetent E. coli cells, ElectroMAX DH10B ^™ (Gibco BRL), are transformed into ligation mixture (2 μl) by electroporation (25 μF, 200 and 500 μs, 2.5 kV) (Biorad Gene Pulser).

After 1 hour incubation in LB medium, the transformed cells were diluted to yield about 100 colonies per dish, followed by ampicillin (100 mg / l), γ-HCH (500 mg / l), and X-gal (5-bro). LB medium supplemented with parent-4-chloro-3-indolyl-α-D-galactoside, 60 mg / l), and IPTG (isopropylthio-β-D-galactoside, 40 mg / l) (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl) and incubate overnight at 37 ° C. Since γ-hexachlorocyclohexane (Merck-Schuchardt) is insoluble in water, a 50 g / l solution is prepared in DMSO (dimethyl sulfoxide) (Sigma).

A library of 10,000 clones is obtained.

3. Cloning and Expression of linA Gene

Screening of the library is performed by visualization of the Lindane digestion halo (precipitation of Lindane in the culture medium) around the colonies. Of the 10,000 clones screened, 35 showed Lindane degrading activity. The presence of the linA gene in these clones was confirmed by PCR with the help of specific primers described by Thomas et al. (1996). The degradation performed on the inserts and amplification products showed the same profile between all screened clones and the reference control R. lindaniclassicus. Clones carrying the linA gene also had inserts of the same size (about 4 kb).

Thus, it has been demonstrated that soil DNA can be cloned and expressed in heterologous hosts: E. coli and genes derived from microbial flora that are difficult to culture. Libraries prepared by partially digesting DNA extracted from soil with restriction enzymes such as Sau3AI can also be envisioned.

Example 3:

A method of making a nucleic acid collection from a soil sample, comprising an indirect DNA extraction step

1. Materials and Methods

1.1 Extraction of Bacterial Fractions from Soil

5 g of soil are dispersed in 50 ml of sterile 0.8% NaCl by grinding in Waring Blender for 3 x 1 min, cooling on ice between each grinding. The bacterial cells are then separated from the soil particles by centrifugation on a density cushion of Nycodenz (Nycomed Pharma AS, Oslo, Norway). In a centrifuge tube, 11.6 ml (8 g of Nycodenz suspended in 10 ml of sterile water) with a density of 1.3 g · ml ⁻¹ are placed below 25 ml of the soil suspension obtained previously. Centrifuge for 40 min at 4 ° C in Roto (TST 28.38 Roto, Kontron) with a swing-out bucket, take a cell ring located at the interface between the aqueous and Nycodenz phases, wash in 25 ml of sterile water and then 10,000 Centrifuge at xg for 20 minutes. Cell pellets were then 10 mM Tris; Taken in 100 mMn EDTA pH 8.0 solution.

Prior to soil dispersion in Waring Blender, a step of concentrating the soil in the yeast extract solution may be included to allow the germination of soil bacterial spores in particular. Therefore, 5 g of soil is incubated in 50 ml of sterile solution of 0.8% NaCl-6% yeast extract at 40 ° C. for 30 minutes. Yeast extract is removed by centrifugation at 5000 rpm for 10 minutes to avoid foam formation during grinding.

1.2 Solubility of Soil Bacteria Cells

Lysis of cells in liquid medium and purification in cesium chloride gradient

Cells are lysed for 1 hour at 37 ° C. in 10 mM Tris, 100 mM EDTA, pH 8.0 solution containing 5 mg.ml ⁻¹ of lysozyme and 0.5 mg.ml ⁻¹ of acromopeptidase. Then a solution of lauryl sacosyl (final 1%) and proteinase K (2 mg.ml- ¹ ) is added and incubated at 37 ° C. for 30 minutes. The DNA solution is then purified on a density gradient of cesium chloride by centrifugation for 36 hours at 35,000 rpm on a Kontron 65.13 rotophase. The cesium chloride gradient used is a gradient of 1 g / ml CsCl with a refractive index of 1.3860 (Sambrook et al., 1989).

Lysis of cells after inclusion in agarose block

Cells are mixed with agarose isovolume containing 1.5% (weight / volume) Seaplaque (Agarose Seaplaque FMC Products. Blocks are then incubated for 3 hours at 37 ° C. in lysis solution: 250 mM EDTA, 10.3% sucrose, 5 mg.ml ⁻¹ lysozyme and 0.5 mg.ml ⁻¹ acromopeptidase. The block is then washed in 10 mM Tris-500 mM EDTA solution and incubated overnight at 37 ° C. in 500 mM EDTA containing 1 mg.ml ⁻¹ proteinase K and 1% lauryl sacosyl. After several washings in Tris-EDTA, the blocks are stored in 500 mM EDTA.

The amount of DNA thus extracted is examined by pulse-field electrophoresis.

The amount of DNA extracted is assessed on an electrophoretic gel for the calibration range of calf thymus DNA.

1.3 Molecular Characterization of DNA Extracted from Soil

DNA extracted from soil is characterized by PCR hybridization, which method involves amplification of DNA using primers located in a universally conserved region of the 16S rRNA gene in the first step, followed by different oligos of known specificity for the amplified DNA. And hybridization with the nucleotide probe (Table 4) (to quantify the strength of the hybridization signal against the external calibration range of the genomic DNA).

DNA extracted from soil and genomic DNA extracted from pure cultures are amplified with primers FGPS 612-669 (Table 1) under standard PCR amplification conditions. The amplification product is then denatured with an equal volume of 1N NaOH deposited on a nylon membrane (GeneScreen Plus, Life Science Products) and hybridized with an oligonucleotide probe labeled g ³² P ATP at the end by the action of T4 polynucleotide kinase. . After preliminary hybridization of the membrane in 20 ml of solution containing 6 ml of SSC 20X, 1 ml of Denhardt solution, 1 ml of 10% SDS and 5 mg of heterologous salmon sperm DNA, hybridization was defined by the probe. Overnight at elevated temperature. Membranes are washed for 30 minutes at SSC 2X twice for 5 minutes at room temperature, then once in SSC 2X 0.1% SDS and twice in SSC 1X, 0.1% SDS for 30 minutes. Hybridization signals are quantified using Molecular Analyst software (Biorad, Ibri sur Seine, France) and the amount of DNA is assessed by interpolation of calibration curves obtained from genomic DNA.

2. Results and discussion

2.1 Extraction and Dissolution of Soil Bacterial Fractions

Prior to DNA extraction, microbial cell separation from soil particles is an alternative with a number of advantages over direct extraction of DNA in soil. Specifically, the extraction of the microbial fraction limits the contamination of the DNA extract with extracellular DNA that is freely present in the soil or with DNA of eukaryotic origin. First of all, DNA extracted from microbial fractions of soils has fragments that are longer in size and better conserved than those extracted by direct lysis (Jacobson and Rasmussen (1992)). Moreover, the separation of soil particles makes it possible to avoid contaminating DNA extracts with humic acid and phenolic compounds, which, if contaminated, can seriously impair cloning efficacy.

One of the determining factors for cell extraction from soil is the dispersion of soil samples to dissociate cells that adhere to or within aggregate surfaces of soil particles. Three consecutive cycles of crushing for 1 minute each make it possible to obtain better cell extraction efficacy and higher recovered DNA compared to a single cycle of crushing for 1 minute 30 seconds.

Table 5 shows Nycodenz gradient, total viable microflora (stained with acridine orange and counted under microscope), total cultivable microflora (counted in solid 10% trypticase-Soja medium), and cultureable solution on HV agar medium Report the extraction efficacy obtained after centrifugation on tinomysheet microbiota (after incubation at 40 ° C. in a solution of 6% yeast extract-0.05% SDS to cause germination of spores). Furthermore, the extracted DNA is quantified after lysing the cells in liquid medium (without purification on the cesium chloride gradient) or after lysing the cells contained in the agarose block (after digesting agarose with β-agarase). .

The results show that more than 14% of the total microbiota on land is recovered by this method (ie 2 x 10 ⁸ cells per gram of soil) and that only 2% of the total microbial population can be cultured.

Moreover, the amount of DNA extracted from the cells is 330 ng per gram of dry soil. Given that the DNA content per soil microbial cell is estimated to be 1.6 to 2.4 fg, and given the amount of extracted cells (2 x 10 ⁸ cells per gram of soil), almost all cells are lysed and this lysis is not a major bias in this study. Can be evaluated.

Pulsed-field electrophoresis shows that DNA from soil extracted after Nycodenz and CsCl gradients can be up to 150 kb in size and agarose block lysis allows for fragments of 600 kb or more to be extracted.

These results confirm the benefit of this study, which is independent of cultures for the construction of environmental DNA libraries as an alternative to direct DNA extraction.

2.2 Molecular Characterization of DNA Extracted from Soil

The purpose of characterizing the molecular weight of DNA extracted from soil is to obtain a profile indicating the proportion of various bacterial taxa present in the DNA extract. This also involves the problem of knowing the extraction bias induced by the prior separation of the cellular responses of the soil, compared to the direct extraction method in the absence of direct visualization of the microbial diversity present in the soil. Specifically, little information has been collected about cell extraction for the Nycodenz gradient as a function of morphological structure (cell diameter, filamentous or spore-formed).

The method so far is based on the following method:

Quantitative hybridization using oligonucleotide probes specific for different groups of bacteria, applied directly to DNA extracted from the environment. Unfortunately, this method is not very sensitive and does not allow detection of taxa or genus that are present in low abundance (Amann (1995)).

Quantitative PCR or competitive quantitative PCR (Diviacco et al., (1993)) such as Most Probable Number (MPN-PCR) (Sykes et al., (1992)). Each drawback of these methods is: (i) effort due to numerous dilutions and repetitions, thus making the technique unsuitable for multiple sample or primer pairs, and (ii) specific to target DNA and not competing for bias. It is a necessity to construct competitive materials.

The method introduced according to the invention universally amplifies the 700 bp fragment inside the 16S rDNA sequence, hybridizes this amplification with an oligonucleotide probe of variable specificity (systemic, throat, subclass or genus) and hybridizes the sample. Comparing the intensity against an external calibration range. Amplification before hybridization allows the quantification of relatively rare microorganisms or species. In addition, amplification with universal primers allows the use of a broad series of oligonucleotide probes during hybridization. This allows a comparison between different dissolution modes (direct or indirect extraction) for well-defined taxa.

The results are compared in Table 6.

They show a similar profile between the two extraction methods (direct and indirect). Thus, prior extraction of land-borne microbial fractions does not introduce any true bias between the taxa tested. The only significant difference between the two extraction methods seems to be the greater abundance of rDNA sequences belonging to γ-proteobacteria in the extract by indirect extraction.

In addition, significant effects of cultivating soil samples in yeast extract solutions are observed in spore forming soil populations (gram positive, low% GC and actinomycite). This step results in the germination of the spores, first allowing better recovery of this type of cell, and secondly allowing greater lysis efficacy for the germinating cells.

This method allows for semi-quantitative analysis targeting the main taxa defined using microorganisms cultured and commonly found in soil. The only molecular tool allows to assess the size of the various taxa because the culture method is also limited and depends on the specificity of the medium used.

The results show that most of the microbial population does not appear in the described phylogenetic group, thus demonstrating the existence of a new group of microorganisms that have not been or have not been cultured to date.

Thus, new probes may be defined using a given sequence starting with DNA extracted from soil (new door consisting of non-cultured microorganisms, Ludwig et al. (1997)) to obtain a more accurate image of the composition of the DNA extract. Can be.

Example 4: Construction of Cosmid POS7001

POS7001 features:

Replication in E. coli

Integration in Streptomyces

Selectivity in E. coli AmpR, HygroR and Streptomyces HygroR

The nature of the cosmids makes it possible to insert large DNA fragments of 30 to 40 kg.

this is

1-Inducible promoter tipA of Streptomyces lividans

Integrated system specific for 2-element pSAM2

3-hygromycin resistance gene

4-cosmid pWED1 derived from pWED15

1)-inducible promoter of the A gene of S. libidans

The tipA gene is a 19 KD protein whose transcription is induced by antibiotic thiostrepton or nociheptide. tipA is well regulated: induction (200X) in logarithm and stationary phase (Murakami T, Holt TG, Thompson CJ., J. Bacteriol 1989; 171: 1459-66).

2) -hygromycin-resistant gene

Hygromycin: an antibiotic produced by S. hygroscopicus

The resistance gene encodes phosphotransferase (hph).

The gene used is derived from a cassette constructed by Blondelet et al. Wherein the hyg gene is under the control of its promoter and IPTG-induced plac promoter (Blondelet-Rouault et al .; Gene 1997; 190: 315-7 ).

3)-site-specific integration system

Element pSAM2 is integrated into the chromosome by site-specific integration mechanisms. Recombination occurs between two identical 58 bp sequences present on plasmid (attP) and chromosome (attB).

The int gene, located adjacent to the attP site, is involved in site-specific integration of pSAM2, the product of which is similar to the integrase of the yong bacteriophage of enterobacteria. It has been demonstrated that pSAM2 fragments containing only the attP attachment site and the int gene can be integrated in the same way as complete elements (French Patent No. 88 06638 (May 18, 1988) and Raynal et al., Mol.Microbiol. 1998 28: 333-42).

4)-Construction of cosmid pOS7001

Step 1 / Promoter TipA was isolated from plasmid pPM927 (Smokvina et al., Gene 1990; 94: 53-9) on a 700-base pair HindIII-BamHI fragment and digested with HindIII / BamHI vector pUC18 (Yannish-Perron et al. , 1985).

Step 2 / This HindIII-BamHI fragment is subsequently delivered from pUC18 to pUC19 (Yannish-Perron et al., 1985).

Step 3 / 1500-base pair BamHI-BamHI inserts carrying the int gene and attP site of pSAM2 were isolated from pOSint1 and shown in FIG. 8 (Raynal A et al., Mol Microbiol 1998 28: 333-42). The BamHI site of (pUC19 / TipA) is cloned in an orientation so that the int gene is placed under the control of promoter TipA.

Step 4 The BamHI site located on the 5 ′ side of the / int gene is partially digested with BamHI and then deleted by treatment with Klenow enzyme. Thus the HindIII-BamHI fragment carrying TipA-int-attP was isolated from pUC19 and transferred into pBR322 HindIII / BamHI.

Step 5 / HindIII-HindIII Fragment The hygromycin cassette isolated from pHP45Ωhyg (Blondelet-Rouault et al., 1997) is cloned into the HindIII site located upstream of promoter TipA.

Step 6 / Ω HindIII site located between the Heyg cassette and promoter TipA is deleted by Klenow treatment after partial HindIII digestion.

The plasmid obtained after step 7 / previous step allows to isolate a single HindIII-BamHI fragment carrying all VIIIHyg / TipA / int attP elements, which are cloned into the EcoRV site of cosmid pWED1 after Clenau treatment. The cosmid pWED1 shown in FIG. 9 is the cosmid pWE15 (Wahl GM, et at., Proc. Natl. Acad. Sci. USA 1987) shown in FIG. 10 by deletion of the neomycin gene and the Hpal-Hpal fragment carrying SV40 origin. 84: 2160-4).

A map of the vector pOS 7001 is shown in FIG. 11.

Example 5 Construction of Conjugate and Integrative Cosmid, Vectors pOSV 303, pOSV306, and pOSV307 in Streptomyces

5.1 Construction of the vector pOSV303

Assuming packaging selects clones larger than 30 kb, only 10-15% of the clones do not contain inserts, thus eliminating the need to actually build a screening system of recombinants, thus enabling smaller vectors to be constructed. .

Construct:

Step 1: Vector pOSV001

The 800 base pair PstI-PstI fragment carrying OriT (Guiney et al., 1983), the origin of delivery of replicon RK2, is cloned into plasmid pUC19 opened with PstI. This cloning step makes it possible to obtain vectors transferable from E. coli to streptomyces by conjugation.

A map of the vector pOSV 001 is shown in FIG. 17.

Step 2: Vector pOSV002

Insertion of a selectable hygromycin marker (Ωhyg cassette) in Streptomyces finally delivers the hygromycin resistance gene to ensure complete delivery of BAC by soil DNA inserts.

The hygromycin cassette isolated at pHP45Phyg is cloned on the HindIII-HindII fragment carrying the hygromycin resistance gene. This fragment is cloned into the PstI site (position 201) of the vector pSOV001. Given the direction of delivery, this PstI site is chosen such that the Hygro marker is delivered last during conjugation. PstI and HindIII ends are made compatible after treatment with Klenow fragments of DNA polymerase, resulting in "smooth ends". The orientation of the hyg fragment is determined at the end of the construct.

A map of the vector pOSV002 is shown in FIG. 18.

Step 3: Vector pOSV010

Xbal-HindIII fragments isolated from plasmid pOSV02 and containing hygromycin resistance markers and delivery origin are cloned into plasmid pOSint1 digested with Xbal and HindIII. The orientation of the site is the orientation that ensures that hygromycin markers are always delivered last.

The plasmid pOSint1 shown in FIG. 8 is described in the paper by Raynal et at. (Raynal A et al., Mol. Microbiol. 1998 28: 333-42).

Such constructs allow expression of integrase in E. coli and Streptomyces.

Step 4: Insert the "cos" site

The principle is to insert the "cos" site into plasmid pOSV010, which is thus packaged into plasmid pOSV010 shown in FIG.

Generation of the "cos" fragment is shown in FIG. 13.

This fragment is obtained by PCR. PCR amplification is carried out using oligonucleotides corresponding to the sequence -50 / + 130 for the cos site, with the fragment carrying the cohesive end of λ (bacteriophage lambda or cosmid pHC79) as a starting material. Such oligonucleotides also contain Nsil cloning sites, Pstl compatibility, XhoI sites, SalI compatibility, and EcoRV sites, which are sites for obtaining "smooth ends".

The addition of rare SwaI and PacI sites makes it possible to isolate and / or map cloned inserts.

PCR fragments are bounded by the PstI site at the 5 'end and by the HincII site at the 3' end, thereby allowing cloning into the vector pOSV010 (Figure 12), pre-digested with the enzymes Nsil and EcoRV, thereby allowing laclq repress. The fruit of the book will come about.

A map of the vector pOSV303 is shown in FIG. 14. The vector pOSV303 contains a cloning site such as an Nsil site, PstI compatibility, XhoI site, SalI compatibility or EcoRV site for obtaining “smooth ends”.

5.2 Construction of the vector pOSV306

Step 1: Construction of the Vector pOSV308

The vector pOSV308 is constructed according to the process illustrated in FIG. 27. Primer pairs of SEQ ID NO: 107 and SEQ ID NO: 108 sequences from cosmid vector pHc79 described by Hohm B and Collins (1980) are used to amplify the 643-bp fragment containing the cos region.

This amplified nucleotide fragment is directly cloned into a pGEMT-easy vector sold by Promega as illustrated in FIG. 27 to generate vector pOSV306.

Step 2: Construction of the vector pOSV306

The vector pOSV010 is constructed as described in step 3 of constructing the vector pOSV303, as described in paragraph 5.1 of this embodiment.

As illustrated in Figure 28, the vector pOSV10 is digested with enzymes EcoRV and Nsil to cleave the 7874-bp fragment and then continue to purify.

Subsequently, the vector pOSV308 obtained in step 1) is digested with the enzymes EcoRV and PstI to cleave the 617-bp fragment and then continue to purify.

28, the 617-bp cos fragment obtained from the vector pOSV308 is integrated by ligation into the vector pOSV10 to obtain the vector pOSV306.

5.3 Construction of the vector pOSV307

Cosmid pOSV307 still contains the Laclq gene for improving the stability of cosmid in S17-1 strains of Streptomyces, for example Streptomyces.

To construct vector pOSV307, vector pOSV010 is digested with enzyme PvuII to yield 8761-bp fragment, which is purified and then dephosphorylated.

Subsequently, the vector pOSV303 obtained as described in step 1) of paragraph 5.2 above is digested with enzyme EcoRI to yield 663-bp fragment, which is purified and then treated with Klenow enzyme.

As illustrated in FIG. 29, the nucleotide fragment thus treated is integrated into the vector pOSV010 after ligation to obtain the vector pOSV307.

Example 6 Construction of E. coli-Streptomyces Replication Shuttle Cosmid pOS700R

Fragments of plasmid pEI16 (Volff et al., 1996) shown in FIG. 15 are isolated and treated with Klenow. These fragments contain sequences necessary for replication and stability originating from plasmid SCP2.

Inserting these two fragments separately into the EcoRV site of cosmid pWED1 results in two different clones.

Hygromycin cassettes isolated from pHP45Ωhyg on HindIII-HindIII fragments are cloned into the HindIII site of pWED1 cosmid containing ScP2 inserts in the form of PstI-EcoRI or XbaI fragments. This confers hygromycin resistance, which can be selected from both E. coli and Streptomyces.

Determination of Transformation and Transformation Efficacy of S. lividans

Cosmids containing XbaI inserts are less stable than those containing PstI EcoRI fragments. It is therefore the latter cosmid selected under the name pOS700R.

A map of the vector pOS 700R is shown in FIG. 16.

Example 7: Integrity (pOS700I) and Transformation Efficacy of Replication Vectors

Possibility

Incorporation into plasmid pTO1 carrying thiostrepton-resistant markers confers thiostrepton resistance to S. lividans strain.

Preparation of Protoplasts from S. lividans incubated in the presence of thiostrepton.

With the pOS700I vector, the transformation efficacy is about 3000 transformants per μg DNA.

With the vector pOS700R, the transformation efficacy is about 30,000 transformants per μg DNA.

Example 8 Construction of Integrative and Conjugated BAC Vectors in Streptomyces

Characteristic:

Replication in E. coli

Can be delivered by conjugating E. coli with streptomyces

Integration in Streptomyces

Selectable from E. coli and Streptomyces

Insertion of large DNA fragments; It should be pointed out that the need to have available soil DNA of size from 100 to 300 kb and not contaminated with small fragments. This is because small fragments are very preferably integrated.

Screening allows selection of a plasmid carrying the insert. This screening allows them to work at high rates between the vector and the DNA to be inserted by removing the closed and undigested vectors from each other, thus having better cloning efficacy in library construction.

Construct:

Step 1: Vector pOSV001

Cloning an 800 base pair PstI-PstI fragment (Guiney et al., 1983) carrying OriT of the origin of delivery of replicon RK2 into plasmid pUC19 opened with PstI. This cloning step allows conjugation to yield a vector that can be transferred from E. coli to streptomyces.

A map of the vector pOSV 001 is shown in FIG. 17.

Step 2: Vector pOSV002

Insertion of hygromycin markers (Ωhyg cassette), which is selectable in Streptomyces and finally delivers hygromycin-resistant genes, thus allowing complete delivery of BAC along with soil DNA inserts.

Cloning of the hygromycin cassette isolated from pHP45Ωhyg on HindIII-HindIII fragment carrying the hygromycin-resistant gene. This fragment is cloned into the PstI site (position 201) of the vector pOSV001. This PstI site directs the direction of delivery, and therefore the Hygro marker is chosen to be finally delivered during conjugation. The Pst1 and HindIII ends are suitable for generating "smooth ends" after treatment with the Klenow fragment of DNA polymerase. The orientation of the Ωhyg fragment is determined at the end of the construction.

A map of the vector pOSV002 is shown in FIG. 18.

Step 3: Vector pOSV010

XbaI-HindIII fragments isolated from plasmid pOSV002 and containing hygromycin-resistant markers and delivery origin are cloned into plasmid pOSint1 digested with XbaI and HindIII. The orientation of the site is the orientation that ensures that the hygromycin marker is always finally delivered.

The plasmid pOSint1 shown in FIG. 8 was described in Raynal et al. (Raynal A et al., Mol. Microbiol. 1998 28 : 333-42).

This construct allows for integrase expression in E. coli and Streptomyces.

Step 4: Vector pOSV014

The addition of “cassette” eventually allows for plasmids with foreign DNA inserts to be selectable in the final construct.

This "cassette" carries a gene encoding a lambda phage CI repressor and a tetracycline-resistant gene. This gene carries the target sequence of the repressor in the non-coding 5 'region. Inserting the DNA into the HindIII site located in the coding sequence of the CI induces non-production of the repressor and thus induces expression of tetracycline resistance.

This is carried by plasmid pUN99 described in the article: Nilsson et al. (Nucleic Acids Res. 1983, 11 : 8019-30).

The PvuII-HindIII fragment isolated from pOSV010 and containing the sequences int, attP, Hygro and oriT is cloned into the MscI site of pUN99.

A map of the vector pOSV014 is shown in FIG. 19.

Step 5: Vector pOSV 403, and Integration and Conjugation BAC Vectors

The final step, cloned into pBAC11 (shown in FIG. 20), confers final plasmid BAC (Bacterial Artificial Chromosome) characteristics, particularly the ability to accept very large DNA fragments.

The PstI-PstI fragment of vector pOSV014 carrying a set of elements and the previously described function is cloned into plasmid pBAC11 (pBeloBAC11) digested with NotI. Terminals are treated with Klenow enzymes to make them compatible.

A map of the vector pOSV403 is shown in FIG. 21. The schematic of FIG. 21 shows the selected orientation.

Step 6:

Vector pOSV403 containing HindIII and NsiI sites. NsiI sites are rare in Streptomyces and have the advantage of being compatible with PstI. PstI sites, on the other hand, are common in Streptomyces and can be used to perform partial degradation.

Recombinant clones that carry the cloned insert into the CI repressor, thus inactivating the repressor, are tetracycline-resistant. Assuming that BAC is present only at a rate of 1 copy per cell, it is necessary to select recombinant clones at a tetracycline dose, such as a dose of 5 μg / ml, less than the usual dose of 20 μg / ml. Under these conditions, there is no background noise.

It is also possible to use a system developed and marketed by the InVitrogen company, incorporating DNA into the vector inactivates the zyRase inhibitor toxic to E. coli at expression. The fragment is preferably separated from the vector pZErO-2 (http: // www. Invitrogen. Com).

Example 9: S. alboniger (S. algae) in integrated cosmid (pOS700I) and replicable cosmid (pOS700R). alboniger) The creation of the library

1) Construction of the library

To assess the efficacy of the cloning system, the puromycin biosynthetic pathway of Streptomyces alboniger is cloned into two shuttle cosmids pOS700I and pOS700R. The genes of the puromycin biosynthetic pathway are carried by about 15 kb of BamHI DNA fragments.

Genomic DNA of Streptomyces alboniger was isolated. 90% of this DNA has a molecular weight of 20 to 150 kb, measured by pulsed-field electrophoresis.

Both cosmids are digested with the enzyme BamHI (single cloning site).

Partial BamHI degradation conditions of genomic DNA were measured (50 μg of DNA with 12 units of enzyme, 5 min degradation). After checking the size by agarose gel electrophoresis, the partially digested DNA is introduced into the vector. In the linking step, 15 μg genomic DNA + 2 μg integrative vector or replicable vector is used.

Each ligation mixture is used to encapsulate DNA in vitro with the head of bacteriophage lambda. The encapsidation mixture (0.5 mL) is titrated (integration vector pOS7001 = 7.5x10 ⁵ cosmid / ml, replication vector = 5x10 ⁴ cosmid / ml).

The cosmid used to transfect E. coli thus produces a library of about 25,000 ampicillin-resistant clones. DNA from all these clones is isolated and quantified.

To test the library, some clones were purified with DNA and digested with BamHI to select the presence and size of the insert. The clones tested contained 20 to 35 kb of S. alboniger insert.

Identification of Clones Containing 2) -Puromycin Biosynthetic Pathway

Clones that are likely to contain a complete puromycin biosynthetic pathway are identified by hybridization with a probe corresponding to the 1.1 kb pac gene (Lacalle et al., Gene 1989; 79 , 375-80), which is a puromycin-resistant gene.

Libraries made in the integration vector pOS 700I:

Of the 2000 clones analyzed, 9 clones hybridized with the probe and contained about 40 kb of insert.

Libraries built on the replication vector pOS 700R:

Of the 2000 clones analyzed, 12 clones hybridized with the probe; It contains an insert of about 40 kb.

Using data published by Tercero et al. (J Biol. Chem. 1996; 271 , 1579-90), clones containing the entire biosynthetic pathway were identified after hybridization with a suitable probe. Certain integrative and replicable cosmids contain 12,360-base pair fragments after ClaI-EcoRV digestion, leading to the assumption that the insert contains the entire puromycin biosynthetic pathway.

4)-Examination of Puromycin Production by Resistant Clones (Rhone-Poulenc)

a) materials and methods

Strains and Culture Conditions:

Resistant clones are selected to examine the production of puromycin. It contains S. lividans containing inserts (G20) in the integrated vector pOS700I or inserts (G21 and G22) in the replicable vector. Corresponds to the recombinant.

Reference strains are used to ensure that the culture medium used allows for this production. This is S. Alboniger S. lividans, wild type strain ATCC 12461 and containing a complete puromycin cluster cloned into puromycin and plasmid pRCP11 Recombinant strains are generated (Lacalle et al. 1992, the EMBO Journal, 11, 785-792) (G23).

Strains are inoculated in culture medium with the following composition:

Organotechnie bacteriological peptone 5g / final medium ℓ

Springer Yeast Extract 5

Liebig meat extract 5

Prolabo Glucose 15

Prolabo CaCO ₃ (1) 3

Prolabo NaCl 5

Difco Baby Girl 2 1

(1) 3 g of carbonate is mixed with 200 ml of distilled water, separated and sterilized. The addition is carried out after sterilization.

(2) The agar is pre-dissolved in 100 ml of distilled water and then added to other additives in the medium.

Adjust pH to 7.2 before sterilization

Sterilize at 121 ° C for 25 minutes

To maintain culling pressure on clones containing the insert by the marker gene present in the vector, 50 μg / L of hygromycin and 5 μg / L of thiostrepton are added after sterilization (thiostrepton-resistant Gene is carried by plasmid pRCP11).

50 ml of liquid culture medium dispensed into a 250 ml conical flask is inoculated with a 2 ml liquid suspension of spores and mycelium of each strain. Cultures are incubated at 28 ° C. at 220 rpm for 4 days under agitation. 50 ml of the production medium dispensed into 250 ml conical flasks are then inoculated with this 2 ml preculture. The production medium used is an industrial medium (medium RPR 201) optimized for the production of pristinemycin. The culture is incubated at 28 ° C. with 220 rpm. After different incubation times, the conical flasks of each culture are brought to pH 11 and extracted twice with 1 volume dichloromethane. The organic phase is concentrated until dryness under reduced pressure and the extract is taken up in 10 μl of methanol. 100 μl of methanol solution is analyzed in a water-acetonitrile 0.05% TFA V / V gradient system on a C18 column for detection of puromycin by HPLC with a diode-bar detector.

b) results

Comparative HPLC analysis from the cultivation of various strains shows the production of puromycin in the cultivation of wild-type strains in culture for 24 hours or longer. Although production is low, it is also clearly detected in cultures of 48 hours or longer of clone G20 containing cosmid pOS7001 (FIG. 23). Puromycin is also detected in traces in clone G23, which contains a complete operon that encodes a compound in plasmid pRCP11. However, no production was observed in the culture of clones G21 and G22 containing cosmid pOS700R. The results are given in FIG.

c) Conclusion

The results obtained make it possible to demonstrate the efficacy of the cloning system deployed in cosmid pOS700I to express a complete biosynthetic pathway under its own regulatory sequence control in heterologous hosts such as S. lividans. In addition, this data also confirms the screening of libraries obtained on the basis of clones resistant to puromycin because it leads to the identification of recombinants capable of expressing biosynthetic pathways associated with resistance genes among a small number of clones. The absence of puromycin production of other clones may be explained by cloning only a portion of the operon that contains the resistance gene but lacks the specific regulatory, transduction, or transcriptional sequences necessary for the synthesis of the compound.

Example 10 Cloning of Soil DNA into Vectors

1)-Preparation of soil DNA to be cloned

Various DNA fragments need to be purified depending on the application.

Cosmid

The size of the molecule should be between 30 and 40 kb. Now, the DNA extracted from the soil is heterogeneous in size and contains up to 200 or 300 kb of molecules. To homogenize the size, the solution is mechanically disrupted by passing the solution through a 0.4 mm diameter needle. Fragments of size around 30 kb are not affected by repeated passage through the needle and therefore do not need to perform separation based on size, especially since intraparticle packaging automatically removes short inserts.

BAC

DNA production

Soil DNA is separated by pulsed-field electrophoresis (CHEF type) under conditions where 100 to 300 kb fragments are concentrated in a band of about 5 mm. This is obtained by moving for 20 hours at 10 ° C. in a gel containing 1% agarose of low melting point with 0.7% normal agarose or 100 seconds pulsed time.

DNA recovery

Two methods are used, the choice of which depends on the size to be separated, the molecular size of 150 kb or more.

Up to 150 kb

The porosity of the 0.7% agarose gel allows for the release of DNA by electroeluting in the absence of ethidium bromide. This DNA is then treated with a hydrophobic and enlarged orifice pipetting device to avoid mechanical fragmentation of the molecule.

100 to 300 kb

Cut bands containing fragments of 100 to 300 kb in size. For migration, a gel containing a low melting point 1% agarose is used. This property allows the gel to melt at a temperature of 65 ° C. that the DNA can withstand, and can be broken down into agarase (sold by Boehringer) at a temperature of 45 ° C. according to the supplier's prescription.

2)-use of integrative cosmid pOS7001 and replicable cosmid pOS700R

construction of polyA polyT tail

principle

Cosmid vectors open at any cloning site are modified at the 3 ′ end by the addition of repeating polynucleotides. In addition, the DNA to be cloned is modified at the 3 ′ end by the addition of repeating polynucleotides that can be paired with the polynucleotide.

The vector-fragment combination to be cloned is made of this polynucleotide and the cos sequence of the vector allows in vitro packaging of the DNA into lambda phage capsid.

Manufacture of vector

The vector used is self-replicating in E. coli and integrating in Streptomyces.

Selection occurs in ampicillin resistance for E. coli and hygromycin resistance for streptomyces.

The cosmid is open at one of the two possible sites (BamHI or HindIII) and the 3 'end is extended to polyA by terminal transferase under conditions where the enzyme supplier envisions the addition of 50 to 100 nucleotides.

Preparation of DNA to be Inserted

The 3 'end of the DNA is extended to polyT by a terminal transferase under conditions providing an extension comparable to that of the vector. Under the experimental conditions described by the manufacturer, the polyA polyT tail is 30 to 70 bases in length.

Assembly of molecules and in vitro encapsidation

For assembly of molecules, one vector molecule is mixed per inserted DNA molecule. The mass concentration of DNA is 500 μg.ml ⁻¹ .

The mixture is encapsidated and transfection potency depends on the strain used as the receptor and the inserted DNA: 0 with test DNA and strain DH5α, comparable to SURE and DH10B strains; However, the DNA yield at extraction is higher than strain DH10B.

Dephosphorylation

Soil DNA is blunt-ended by removing the protruding 3 'sequence and filling the protruding 5' sequence. This operation is performed with Klenow enzyme, T4 polymerase, 4 nucleotide triphosphate. The cosmid vector is digested with BamHI and then treated with the Klenow enzyme to smooth the ends and dephosphorylation to prevent self closing. After ligation, the mixture is capsidized and transfected as described above.

3)-use of pBAC

principle

Conjugated and integrative plasmid pBAC contains HindIII and NsiI as cloning sites. Insertion of DNA sequences into these sites inactivates lambda phage CI repressors that regulate the expression of tetracycline-resistant genes. Inactivation of the refresher thus makes the cells resistant to antibiotics (5 μg.ml ⁻¹ ). Cloning of these sites is facilitated by modifying the vector and making the DNA to be cloned.

Preparation of the vector. HindⅢ example

Modify the HindIII site so that the vector does not close itself; The original base (A) is reinserted to form a protruding 5 ′ sequence, which is not paired with a partner. This operation is performed by the Klenow enzyme in the presence of dATP.

The success of this task is checked by performing self-linking of the vector before and after treatment with the Klenow enzyme. For the same amount of test DNA, 3000 clones were obtained before treatment and 60 clones after treatment.

Preparation of DNA (Size from 100 to 300 kb)

Granting Smooth Terminals to DNA

DNA is given a blunt end by removing the protruding 3 'sequence and filling the protruding 5' sequence. This operation is performed with Klenow enzyme, T4 polymerase, 4 nucleotide triphosphate.

Preparation of the terminal. HindⅢ example

Adding DNA to the vector is performed by oligonucleotides that recognize the modified HindIII sequence of the vector. Oligonucleotides have a rare restriction site (SwaI; NotI) that allows for subsequent cloning. This technology includes Elledge SJ, Mulligan JT, Ramer SW, Spottswood M, Davis RW. Proc. Natl Acad. Sci. USA 1991 Mar 1; 88 (5): 1731-5.

Two complementary oligonucleotides are used:

Oligo 1: 5'-GCTTATTTAAATATTAATGCGGCCGCCCGGG-3 '(SEQ ID NO: 25)

Oligo 2: 5'-CCCGGGCGGCCGCATTAATATTTAAATA-3 '(SEQ ID NO: 26)

After hybridization, the 5 ′ end is phosphorylated with T4 kinase in the presence of ATP. This phosphorylation step can be omitted by using pre-phosphorylated oligonucleotides.

The linkage of this double stranded adapter with the DNA to be inserted is carried out by T4 ligase at 14 ° C. over 15 hours in the presence of a very large amount of adapter (1000 adapter molecules per DNA molecule to be inserted).

Excess adapter is removed by agarose gel electrophoresis and the molecule is recovered by hydrolysis or electroelutation with agarose.

Vector-DNA Connection

The linking step is carried out at 14 ° C. over 15 hours in an amount of 10 vectors per insertion molecule.

Transformation

The receptor strain is strain DH10B. Transformation is performed by electroporation. To express tetracycline resistance, the transformants are incubated in a medium free of antibiotics for 1 hour at 37 ° C. Clones are selected by incubating overnight in gelated LB medium fed with 5 μg.ml ⁻¹ of tetracycline.

Example 11 Clone-to-Clone Conjugation between E. coli and Streptomyces

containing pPM803 E. coli strain S17.1 and Streptomyces lividans Junction between TK21

Introduction

It is possible to carry out the conjugation between E. coli and Streptomyces (Mazodier et al, 1989). Application of this method by the development of the so-called drop technique of mixing 10 μl of E. coli culture containing a recombinant vector with a drop of receptor S. lividans results in all libraries constructed from E. coli at the end of the work. During introduction into the lividans, it consists of performing clone to clone transformation. Bulk transformation actually needs to induce proliferation of Streptomyces transgenic clones so that the E. coli library can be fully expressed in S. lividans. This method is also easy for automation.

Preliminary examination

Conjugation of E. coli strain S17.1 with S. lividans TK21 with vector pOSV303.

Under these conditions, 6 × 10 in 20 μl final volume⁶E. coli cells are 2 × 10⁶Pre-germinated Mixed with S. libidans spores.

Development of the method

It is known that DNA extracted from certain actinomysheets is modified and consequently cannot be introduced into certain strains of E. coli without limitation. E. coli strain DH10B that accepts this DNA cannot deliver plasmids with only oriT to streptomyces and it is necessary to prepare such plasmids. Derivatives of RP4 will be introduced into the plasmid by integration into the chromosome, which may provide all the functions necessary for the delivery of recombinant clones containing the oriT of origin of delivery.

Example 12 Construction of Cosmid Library in E. coli and Streptomyces Lividans: Cloning of Soil DNA

The aim is to produce a library of large environmental DNA with the purpose of accessing metabolic genes of unknown bacteria (or other organisms) without knowing the prior steps of microbial culturing, and how to culture under standard laboratory conditions.

The process described DNA extracted and purified from E. coli-S. Libidans shuttle cosmid pOS7001 and soil bacterial fractions is used to generate DNA libraries in E. coli. This final method makes it possible to obtain a high purity average size 40 kb of DNA. In addition, alternative strategies based on using terminal transferases that add polynucleotide tails to the 3 'end of the DNA and the vector are employed to avoid partial degradation of the DNA extracted from cloning.

5 μg of DNA was extracted from 60 mg of “St-Andreshore” soil according to the protocol of Example 3 and treated with terminal transferase (Pharmacia) to extend the 3 ′ end with repeating polynucleotide (polyT) ( Example 10).

Integral cosmid pOS700I is prepared according to protocol B1, Orsay. After standard purification steps in the presence of phenol / chloroform, the DNA and the vector are assembled by mixing one molecule of vector and one molecule of inserted DNA. The mixture is then encapsidated in the head of the lambda bacteriophage (Amersham kit), which contributes to transfection of E. coli DH10B. The transfected cells are then seeded in LB agar medium in which ampicillin is present to select antibiotic resistant recombinants.

A library of about 5000 ampicillin-resistant E. coli clones was obtained. Each clone is inoculated in LB or TB medium + ampicillin in microplate wells (96 wells) and stored at -80 ° C.

Site sequences into which soil fragments were generated during construction of the library were inserted into the vector pOS7001 were analyzed. To this end, 17 cosmids of the library are purified and sequenced with a primer, seq.5 'CCGCGAATTCTCATGTTTGACCG 3', which hybridizes between the BamHI site and the HindIII cloning site in the vector.

The sequence obtained allows one to assess that the length of the homopolymer tail at the junction is very variable, from 13 to 60 poly-dA / dT. Beyond the tails, the resulting soil fragments have a G + C% of 53-70%. This high percentage is unexpected, but similar results have already been reported in raw material preparations of soil DNA (Chatzinotas A. et al., 1998).

Analyze microbial and metabolic abundance using a strategy of "pooling" 48 or 96 clones. Cosmid DNA extracted from these "pools" of clones is used to perform PCR or hybrid experiments.

Example 13: Diversity of 16S ribosomal DNA in cloned DNA

A) Materials and Methods

Cosmids in the library are extracted by alkaline dissolution from pools of clones and purified on a cesium chloride gradient for the purpose of taking bands of cosmid DNA in supercoil form and removing E. coli chromosomal DNA that may interfere with the study.

After linearizing the cosmid under the action of S1 nuclease (50 units, for 30 minutes at 37 ° C.), the 16S rDNA sequence contained in the pool of clones was subjected to universal primer 63f (5 ′) as defined by Marchesi et al under standard amplification conditions. Amplified using -CAGGCCTAACACATGCAAGTC-3 ') and 1387r (5'-GGGCGGWGTGTACAAGGC-3'). Amplification products of about 1.5 kb are purified using Qiaquik gel extraction kit (Qiagen) and cloned directly into vector pCRII (invitrogen) in E. coli TOP10 according to the manufacturer's instructions. Inserts are amplified using M13 forward and M13 reverse primers specific for the cloning site of the vector pCRII. Amplification products of expected size (about 1.7 kb) are analyzed by Restriction Fragment Length Polymorphism (RFLP) using CfoI, MspI and BstUI (0.1 units) to select clones to be sequenced. The restriction profiles obtained are separated on 2.5% Metaphore agarose gel (FMC products) containing 0.4 mg of ethidium bromide per ml.

16S rDNA sequences are determined directly using PCR products purified with a "Qiaquick gel extraction" kit with the aid of sequencing primers as defined by Normand (1995). Lineage by comparing this sequence to the prokaryotic 16S rDNA contrasted in the Ribosome Database Project (RDP) database (version 7.0 (Maidak et al.1999)) using the SIMILARITY MATCH program which allows to obtain similar values for the database sequence. Analysis results are obtained.

b) results

To measure phylogenetic diversity shown in the library, 47 sequences of 16S rRNA were isolated from 288 clones and almost completely sequenced. The results are given in Table 7.

Sequencing by interrogation of the database revealed that most sequences (> 61%) had a percent similarity of 95% or less with the identified bacterial species (Table 7). 28 of the 47 sequences analyzed have non-cultured bacteria as their nearest neighbors, the sequences of which are obtained directly from DNA extracted from the environment. Most of these sequences also have very low similarities% (88-95%), so 17 out of 28 sequences differ by at least 5% relative to their nearest neighbors.

Of the sequences that can be classified as phylogenetic, most of the sequences belong to proteobacteria subclass a (18 sequences with% similarity between 89 and 99%). The second group of sequences is represented by the proteobacteria subclass g and includes 9 sequences having% similarity ranging from 84 to 99%. The groups of b-proteobacteria and d-proteobacteria comprising the sequences 1, 4, 3 and 5 respectively are Permicute with low G + C% and high G + C%. Of the major bacterial lineages defined, only one sequence cannot be classified: sequence a22.1 (19), its nearest neighbor, Aerothermobacter marianas (89% similarity), which itself is marine It is a strain isolated from the environment and is not currently classified. Finally, the six sequences can be classified into groups of Ashidobacterium / Holophaga. This group has the special characteristics represented by only two cultured bacteria, Acidobacterium capsulatum and Holophaga foetida, in which the entire group contains only 16S rRNA genes in environmental samples (primarily soil). ), Which is detected by amplification and cloning using DNA extracted (Ludwig et al., (1997)). The low similarity between the different sequences that make up this group makes it possible to predict large heterogeneity and diversity within these groups.

The set of results is shown in Table 7.

These results suggest that the sequences containing the cosmid library are derived not only from phylogenetic diversification but from all microorganisms that have not been isolated at all.

The sequencing results of the amplified DNA allow the characterized sequences to establish phylogeny of organisms present in new soil samples.

The phylogeny shown in FIG. 7 is sequenced by MASE software (Faulner and Jurak, 1988), calibrated by Kimura 2-parameter method (1980), and of the Naver joining algorithm (Saitou and Nei, 1987). Generated with the help. Phylogenetic analysis was performed using the ribosomal database project (RDP) database (version 7.0, SIMILARITY-MATCH program, Maidak et al., 1999) and BLAST 2.0 software (Atschul et al, 1997) to clone 16S rDNA sequences cloned from soil DNA libraries. Allows comparison with the prokaryotic 16S rDNA sequence in the GenBank base.

Example 14 Gene Screening of Libraries for Assessing Metabolic Richness

In order to characterize the library obtained in terms of metabolic diversity and to identify clones containing inserts carrying genes that may be involved in biosynthetic pathways, gene screening techniques based on PCR methods detect and identify type I PKS genes. Has been developed in accordance with the present invention.

1. Bacteria strains, plasmids and culture conditions

S. Ko elementary color (S.coelicolor) ATCC101478, S. memorizing Pacific Enschede (S.ambofaciens) NRRL2420, S. lactase buns lance (S.lactamandurans) ATCC27382, S. remote suspension (S.rimosus) ATCC109610, B. sub B. Subtilis ATCC6633 and B. licheni formis THE1856 (collection RPR) are used as DNA sources for PCR experiments. S. lividans TK24 is the host strain used for shuttle cosmid POS1700.

For the preparation of suspensions of genomic DNA, spores and protoplasts and transformation of S. lividans, follow the standard protocol described in Hopwood et al. (1986). E. coli Top 10 (INVITROGEN) is used as a host for cloning PCR products and E. coli Sure (STRATAGENE) is used as a host for shuttle cosmid pOS700I. Under E. coli culture conditions, preparation of plasmids, digestion of DNA, agarose gel electrophoresis is performed according to standard procedures (Sambrook et al., 1996).

2. PCR primers:

Primer pairs a1-a2 and b1-b2 are defined by the N.Bamas-Jacques team and their use is optimized for screening soil libraries for genetic investigations encoding DNA and PKSI from pure strains.

Table 8

PCR primers homologous to PKSI used to screen libraries

a1 (+) 5'CCSCAGSAGCGCSTSTTSCTSGA3 ' a2 (-) 5'GTSCCSGTSCCGTGSGTSTCSA3 ' b1 5'CCSCAGSAGCGCSTSCTSCTSGA3 ' b2 5'GTSCCSGTSCCGTGSGCCTCSA3 '

Amplification Condition:

For the study of PKSI from pure strain DNA, the amplification mixture was: 50 μl final volume, 50-150 ng genomic DNA, 200 μM dNTP, final 5 mM MgCl ₂ , 7% DMSO, 1 × Appligene buffer , 0.4 μM of each primer and 2.5 U of Appligene Taq polymerase. Amplification conditions used are: in the first cycle, as described by K. Seow et al., 1997, denature for 2 minutes at 95 ° C., hybridization for 1 minute at 65 ° C., elongation for 1 minute at 72 ° C., This is followed by 30 cycles in which the temperature is reduced to 58 ° C. The final extension step is carried out at 72 ° C. for 10 minutes.

To study PKS I from the DNA of the library, PCR conditions are the same for a1-a2 pairs using 100-500 ng cosmid extracted from a pool of 48 clones.

For b1-b2 primer pairs, 500 ng cosmid derived from 96 clones is used. The amplification mixture contains 200 μM of dNTP, final 2.5 mM MgCl ₂ , 7% DMSO, 1 × Quiagen buffer, 0.4 μM of each primer and 2.5 U hot-start Taq polymerase (Quiagen). Amplification conditions used are: 15 cycles of denaturation at 95 ° C., 30 cycles: 1 minute denaturation at 95 ° C. + 1 minute hybridization at 65 ° C. and hybridization at 62 ° C., 1 minute at 72 ° C. in other cycles. Elongation, final extension step at 72 ° C. for 10 minutes.

Identification of positive clones from pools of 48 or 96 clones is performed using replicas of the corresponding parent microplates on solid medium or using other standard replication methods.

3) subcloning and sequencing

PCR products of the identified clones are sequenced according to the following protocol:

Fragments are purified on agarose gel (gel extraction kit (Quiagen)) and cloned into E. coli TOP 10 (invitrogen) using TOPO TA cloning kit (invitrogen). The plasmid DNA of the subclone is extracted by alkaline lysis on Biorobot (Qiagen) and dialyzed for 2 hours on a 0.025 μm VS membrane (Millipore). Samples are sequenced on an ABI 377 96 sequencer (Perkin Elmer) with "universal" and "reverse" M13 primers.

4) Result

Definition and Identification of PCR Primers

The two highly conserved regions of actinomycete type I PKS that contain the active site of the enzyme are targeted to amplify the same gene with degenerate primers. These two regions correspond to the sequences PQQR (L) (L) LE and VE (A) HGTGT, respectively.

Primers (Table 8) are tested with DNA from strains that produce or do not produce macrolides: Streptomyces coelicolor, which produces spiramycin, Streptomyces ambofaciens, Saccharopolis, which produces erythromycin. Fora Eritrea. Regardless of the primer used, bands representing fragments of about 700 pb or corresponding to the length of the fragments expected are obtained with all strains.

These results demonstrate the specificity of the silent genes in primers a and b or S. coelicolor for the PKS I gene of the producing strain.

The sequencing of PCR products obtained with a1-a2 primer pairs was previously described as belonging to the biosynthetic pathway of the macrolide precursors of plantenolide, spiramycin, from S. ambofaciens strains (European Patent Application No. EP 0). 791 656) allows the identification of the KS gene, and two sequences not described, Stramb 9 and Stramb 12 (see sequence listing).

Regarding S. erythrrea, the screening method is identical to the sequence of the KS gene of Module 1 (accession number M63677) previously published in Genebank, and the 6-deoxyerythronolide B synthetase 1 ( To enable identification of the sequence of KS (Sacery 17) encoding DEBSI). Other sequences unrelated to the erythromycin biosynthetic pathway have been identified and are sequences of SEQ ID NO: 32.

conclusion

Methods have been developed for analyzing the presence of genes encoding type I PKS by PCR from different microorganisms. The highly conserved structure of the type I keto-synthase domain allows for the generation of PCR methods based on the use of GC-biased degenerate primers for the selection of codons.

This approach shows the possibility of identifying genes or clusters involved in the biosynthetic pathway of type I polyketides. Cloning of these genes allows for the creation of collections that can be used to prepare polyketide hybrids. The same principle can be applied to other antibiotic classes.

The results obtained here also show the presence of genes that can belong to silent clusters (SEQ ID NOs: 30-32).

The presence of silent clusters has already been documented in S. lividans and its expression is triggered by specific or pleiotropic regulators (Horinouchi et al .; Umeyama et al. 1996). These results imply that the detection of genes belonging to the so-called silent pathway encodes an active enzyme that, together with other specific enzymes of the pathway, can direct the enzymatic steps necessary for the synthesis of secondary metabolites.

Screening of Libraries

Screening is performed using primer pairs identified in productivity strains under conditions as described in the Materials and Methods section.

In the presence of a1-a2 primer pairs, the size of the PCR product obtained from cosmid DNA extracted from the pool of 48 or 96 clones is about 700 kb, which is expected result.

The intensity of the bands obtained varies but there is only one amplification band for the target DNA of each pool.

Under these conditions, eight groups of target DNA corresponding to nine positive clones were detected after decoupling.

Screening performed with the second primer pair b1-b2 results in less specific amplification results because a large number of accessory bands are observed near the 700 bp band. Nevertheless, nine groups of target DNA corresponding to 14 positive clones starting with these positive clones were detected after decoupling, and the DNA is extracted for the sequencing and transformation steps of S. lividans.

Cosmid Analysis

Digestion of the cosmid identified by PCR with the enzyme DraI, which recognizes the AT-rich site, did not affect fragments larger than 23 kb (FIG. 22). This suggests that the PCR method preferentially targets soil DNA containing high G + C%. This result is the result of the degeneracy of the primers used GC-biased for the selection of codons. As expected in the case of cosmid, the insert is larger than 23 kb, except in one case (clone a9B12), which may reflect a certain level of instability of the cosmid. In addition, of all the clones selected, only two, GS.F1 and GS.G11, showed the same restriction profile, indicating low levels of abundance in the library.

Selected cosmids are delivered to Streptomyces lividans by transfection of the protoplasts in the presence of PEG 1000. Transformation efficacy ranges from 30 to 1000 transformants per μg cosmid DNA used.

Sequencing and Phylogenetic Analysis of Soil PKSI Gene

PCR methods developed on pure strains are used as described on the cosmid of the library and thus 24 clones are identified.

About 700 bp of PCR product obtained from the DNA of two pools (48 clones) and 8 unique clones is purified on agarose gel and then cloned and sequenced. This allows for the identification of 11 sequences.

The arrangement of inferred protein sequences of soil PKS I (FIG. 24) with other PKS I present in different microorganisms shows the presence of highly conserved regions corresponding to active site consensus regions of beta-ketoacyl synthase.

Analysis of the sequences obtained by the “codon preference” method (Gribskov et al., 1984; Bibb et al., 1984) reveals the presence of a strong bias in the use of G + C-rich codons in a single reading frame. Proteins inferred according to this reading frame show a strong similarity with the known type I KS (Blast program). In particular, the similarity between the KS sequence from the soil and that of the erythromycin cluster is about 53%.

After decoupling of the pool and identification of specific clones, the sequence of PCR products obtained from this clone is identical to that of the pool, which confirms the authenticity of the method used.

Sequencing the clone's PCR products allows for the probable identification of different 3 KSI genes. One of these sequences (SEQ ID NO: 34) has 98.7% similarity with the sequence of the other pool, suggesting that it encodes the same enzyme. The other two sequences are different but strongly homologous.

Cloning and identification in soil DNA libraries of biosynthetic pathways of secondary metabolites containing genes encoding type I KS were described for the first time.

The high G + C% in the soil sequence suggests that they are derived from a genome with it similar to the codon usage of actinomycetes.

Although the data available in the literature is limited, it is known that genes encoding type I PKS vary widely because of the physical composition, size, and number of modules contained in each gene.

The presence of some domains derived from monoclonals is affirmation that they belong to asymmetric polyketide clusters. In one case, two clones appear to form a contiguum because they share the same sequence for the KS domain.

The size of the gene region involved in PKSI synthesis ranges from a few kb for penicillin to about 120 kb for rapamycin. The size of the cosmid insert is therefore not sufficient for the expression of most complex clusters.

Genes encoding PKS I have been described as repeatable as PKS II and can regulate the synthesis of aromatic polyketides (Jae-Hyuk et al. 1995). Research of soil PKSI clusters may provide additional novelty in this area.

5. Identification of 6 Genes Encoding Polyketide Synthetase

While continuing library screening of cosmids according to the protocol described in this example, we found that cosmid clones containing 34071-bp inserts with several open reading frames encoding polypeptides of the polyketide synthase type Was identified.

More specifically, cosmids are thus identified by screening libraries containing 6 open reading frames encoding polyketide synthase polypeptides or polypeptides in very close relation, non-ribosome synthase peptides. A detailed map of this cosmid is shown in FIG. 36.

The complete nucleotide sequence of the cosmid constitutes the sequence of SEQ ID NO: 113 in the sequence listing. The DNA insert contained in the sequence of SEQ ID NO: 113 constitutes the complementary nucleotide sequence (-backbone) of the nucleotide sequence encoding various polyketide synthase.

The nucleotide sequence of the DNA insert included in the cosmid in FIG. 36 and comprising an open reading frame encoding the polyketase synthase polypeptide (+ strand) is shown in FIG. 37 approximately, SEQ ID 114 in the sequence listing. Constitutes a sequence of.

Further, detailed maps of the various open reading frames contained in the DNA insert of this cosmid are also shown in FIG. 37.

Features of the nucleotide sequence comprising the open reading frame contained in the DNA insert of this cosmid are detailed below.

ORF1 sequence

The orf1 sequence is partially open reading frame 4615 nucleotides in length. This sequence constitutes the sequence of SEQ ID NO: 115, starting at the nucleotide of position 1 of the sequence of SEQ ID NO: 114 and ending at the nucleotide of position 4615.

The sequence of SEQ ID NO: 115 encodes a 1537-amino acid ORF1 polypeptide, which polypeptide constitutes a sequence of SEQ ID NO: 121.

The polypeptide of SEQ ID NO: 121 sequence is associated with a non-ribosome synthetase peptide. This polypeptide has 37% amino acid identity with Anabaena sp.90 synthetase peptide referred to by the accession number "emb CACO1604.1" in the Genbank database.

ORF2 sequence

The orf2 nucleotide sequence is 8301 nucleotides in length and constitutes the sequence of SEQ ID NO: 116, begins at the nucleotide at position 4633 of the sequence of SEQ ID NO: 114, and ends at the nucleotide at position 12933.

The ORF2 sequence encodes a 2766-amino acid ORF2 peptide, which constitutes the sequence of SEQ ID NO: 122.

The polypeptide of sequence SEQ ID NO: 122 shows 41% amino acid sequence identity with the MtaD sequence of Stigmatella aurantiaca , referenced from Genbank database under access number "gb AAF 19812.1".

ORF2 polypeptides constitute a polyketide synthetase.

ORF3 sequence

The orf3 nucleotide sequence is 5292 nucleotides in length and constitutes the sequence of SEQ ID NO: 117. The sequence of SEQ ID NO: 117 corresponds to the sequence starting at the nucleotide at position 12936 of SEQ ID NO: 114 sequence and ending at the nucleotide at position 18227.

The nucleotide sequence of SEQ ID NO: 117 encodes a 1763-amino acid ORF3 polyketase synthase polypeptide, which constitutes the sequence of SEQ ID NO: 123 according to the present invention.

The ORF3 polypeptide of SEQ ID NO: 123 shows 42% amino acid identity with the MtaB sequence of Stigmatella Orantiaca, referred to by access number "gb AAF 19810.1" from the Genbank database.

ORF4 sequence

The orf4 nucleotide sequence is 6462 nucleotides in length and constitutes the sequence of SEQ ID NO: 118 according to the present invention.

The nucleotide sequence of SEQ ID NO: 118 corresponds to the sequence starting at the nucleotide at position 18224 of the nucleotide sequence of SEQ ID NO: 114 and ending at the nucleotide at position 24685.

The nucleotide sequence of SEQ ID NO: 118 encodes a 2153-amino acid ORF4 polyketase synthase polypeptide, which constitutes the sequence of SEQ ID NO: 124 according to the present invention.

The ORF4 polypeptide of SEQ ID NO: 124 has 46% amino acid sequence identity with the epoD sequence of Sorangium cellulosum referenced by the accession number "gb AAF 62883.1" from the Genbank database.

ORF5 sequence

The orf5 nucleotide sequence is 5088 nucleotides in length and constitutes the sequence of SEQ ID NO: 119 according to the invention.

The sequence of SEQ ID NO: 119 corresponds to the sequence starting at the nucleotide of position 24682 of the nucleotide sequence of SEQ ID NO: 114 and ending at the nucleotide of position 29769.

The nucleotide sequence of SEQ ID NO: 119 encodes a 1695-amino acid ORF5 polyketase synthase polypeptide, which constitutes the sequence of SEQ ID NO: 125 according to the present invention.

The ORF5 polyketide synthetase polypeptide of SEQ ID NO: 125 sequence has 43% amino acid identity with the epoD sequence of Sorangium cellulose, referred to by the accession number "gb AAF 62883.1" from the Genbank database.

ORF6 sequence

The orf6 nucleotide sequence is 4306 nucleotides in length and constitutes the sequence of SEQ ID NO: 120 in accordance with the present invention. The nucleotide sequence of SEQ ID NO: 120 corresponds to the sequence starting at the nucleotide at position 29766 of SEQ ID NO: 114 and ending at the nucleotide at position 34071.

The sequence of SEQ ID NO: 120 contains a partially open reading frame encoding a 1434-amino acid ORF6 polypeptide of the polyketide synthase type, which constitutes the sequence of SEQ ID NO: 126 according to the present invention.

The polypeptide of SEQ ID NO: 126 has 43% amino acid identity with the epoD sequence of Sorangium cellulose, referred to by the accession number "gb AAF 62883.1" from the Genbank database.

Example 15 Construction of Shuttle Vector of Integrative BAC Type in Streptomyces

Construction of Integrative and Conjugated BAC Type Shuttle Vectors in Streptomyces

15.1 Construction of the vector pMBD-1

Vector BAC pMBD-1 is obtained according to the following steps.

Step 1: Vector pOSVO10 is digested with enzymes PsTI and BstZ171 to yield 6.3-kb nucleotide fragments.

Step 2: Vector pDNR-1 is digested with enzymes PsTI and PvuII to yield 4 145-kb nucleotide fragments.

Step 3: The 6.3-kb nucleotide fragment derived from vector pOSV017 is fused by linking with the 4.15-kb fragment derived from vector pDNR-1, resulting in vector pMBD-1 as shown in FIG. 30.

15.2 Construction of the vector pMBD-2

Vector pMBD-2 is a BAC type vector containing a "Φc31int-Ωhyg" integration box.

Φ c31 is a broad host spectral lysogenic phage with well distributed attachment sites (attP). The Φ c31 int fragment is the smallest fragment of actinophage Φ c31 that can induce the integration of plasmids into the chromosome of Streptomyces lividans.

Ω hyg is a derivative of Ω interposon that can confer hygromycin resistance in E. coli and S. libidans.

BAC vectors containing a φc31 integrated system have been described in Sosio et al. (2000) and PCT patent application No. 99/6734 of December 29, 1999.

Vector BAC pMBD-2 is constructed according to the following steps.

Step 1: Construction of Φ c31 in Ω hyg integrative box in E. coli multicopy plasmid.

The φc31 int fragment is first amplified from plasmid pOJ436 using the following primer pairs.

Primer EVΦc31I (SEQ ID NO: 109) (allowing the EcoRV site to be introduced into the 5 'end of the Φc31 sequence) and primer BII Φc31F (SEQ ID NO: 110) (allowing the BgLII site to be introduced into the 3' end of the Φc31 sequence ).

Ωhyg fragments are obtained by digestion using the BamHI enzyme of plasmid pHP45 Ωhyg described by Blondelet-Rouault (1997).

The φc31 int-Ωhyg integrative box is then cloned into vector pMCS5 digested with enzymes BgIII and EcoRV.

Step 2: Construction of the Vector pMBD-2

The bacterial artificial chromosome pBAce3.6 described by Frengen et al. (1999) is degraded with the enzyme NheI and then treated with Eco polymerase.

The vector pMCS5 φc31 int-Ωhyg is then digested with the enzymes SnaBI and EcoRV to recover the integrated box.

A detailed map of the vector pMBD2 is shown in FIG. 31.

15.3 Construction of the vector pMBD-3

Vector pMBD-3 is a BAC type of integration (Φc31 int) and conjugation (ori T) vectors and includes the selection marker Ωhyg.

A map of the vector pMBD-3 and its construction method are shown in FIG. 31.

Vector pMBD-3 is obtained by amplifying the Ori T gene starting with plasmid pOJ436 using primer pairs of SEQ ID NO: 111 and SEQ ID NO: 112 having pacI restriction sites.

Nucleotide fragments amplified using primers SEQ ID NO: 111 and SEQ ID NO: 112 are cloned into vector pMBD2, previously digested with PacI enzyme. An outline of the construction of the vector pMBD-3 is shown in FIG. 31.

15.4 Construction of the vector pMBD-4

A detailed map of the vector pMBD-4 is shown in FIG. 32.

Vector pMBD-4 is obtained by cloning the φc31 int-Ωhyg integrity box into the vector pCYTAC2.

15.5 Construction of the vector pMBD-5

The construction scheme of the vector pMBD-5 is shown in FIG. 33.

Vector pMBD-5 is constructed by recombining a nucleotide fragment contained between two loxP sites of vector pMBD-1 illustrated in FIG. 33 and a loxP site contained in a BAC vector named pBTP3, and a detailed map of plasmid pBTP3 is shown in FIG. 34. It is shown in.

15.6 Construction of the vector pMBD-6

Vector pMBD-6 is constructed by recombining the nucleotide fragment contained between two loxP sites of vector pMBD-1 into the loxP site of the BAC pBeloBac11 vector.

Table 1

The location of the sampling site and the characteristics of the soil used in the various experiments.

Perform microbial counts directly by staining with acridine orange before and after crushing the soil.

number origin Texture The following amount (%) sand octopus clay Organic matter (g / kg dry soil) pH Total cell count ^a (× 10 ⁹ / g dry weight of soil) Number of cells after grinding ^a (× 10 ⁹ / g dry weight of soil) One Australia Sandy clay 62 22 6 49.7 5.8 6.5 (0.9) 2.9 (1.3) 2 Peyrat le Chateau, France Sandy clay 61 26 13 48.2 4.9 7.3 (0.6) 5.4 (0.8) 3 St-Andre coast, France Sand mix 50 41 9 40.6 5.6 10.0 (0.7) 7.5 (1.4) 4 Chazay d'Azergue, France Clay sand mixture 34 47 19 13.9 5.8 7.8 (1.1) 4.2 (0.6) 5 Guadeloupe, France clay 27 26 47 17.0 4.8 1.4 (0.4) 0.5 (0.1) 6 Dombes, France Clay sand mixture 20 67 13 30.3 4.3 7.5 (0.5) 5.6 (0.9) ^a n = 3; The parentheses are the standard deviation

TABLE 2

Primers and Probes Used for PCR Amplification and Dot-Blot Hybridization

Primer or probe Target ^a) Sequence (5 'to 3' direction) Reference number FGPS431 Probe Universal (1392-1406) ACGGGCGGTGTGT (A / G) C Amann et al., 1995 FGPS122 Primer Bacteria (6-27) GGAGAGTTTGATCATGGCTCAG Amann et al., 1995 FGPS350 Primer Streptosporangium (616-635) CCTGGAGTTAAGCCCCCAAGC germinal FGPS643 probe Streptosporangium (122-142) GTGAGTAACCTGCCCC (T / C) GACT germinal R499 Primer Bacillus anthracis TTAATTCACTTGCAACTGATGGG Patra et al., 1996 R500 Primer Bacillus anthracis AACGATAGCTCCTACATTTGGAG Patra et al., 1996 C501 probe Bacillus anthracis TTGCTGATACGGTATAGAACCTGGC Patra et al., 1996 FGPS516 Primer S. libidans 0S48.3 TCCAGATCCTTGACCCGCAG germinal FGPS517 Primer S. libidans 0S48.3 CACGACATTGCACTCCACCG germinal FGPS518 Probe S. libidans 0s48.3 CCGTGAGCCGGATCAG germinal ^A) The positions on the E. coli 16S rRNA genes are given in parentheses. For B. anthracis and S. lividans, primers and probes target chromosomal sequences specific to each organism. This sequence is not located in the 16S rRNA gene. This cassette containing the target region of S. lividans has been described by Clerc-Bardin et al. (Private).

TABLE 3

Amount of DNA extracted from different soils after lysis treatment according to protocols 1-5 (μg DNA / dry soil weight in g ± standard deviation) ^a

Soil 1, 2, 3 and 6; n = 3; Soil 4: n = 1

Soil Dissolution Protocol No. ^b Number and origin One 2 3 4a 4b 5a 5b Australia 17 +/- 2 52 +/- 2 32 +/- 5 16 +/- 3 33 +/- 2 59 +/- 1 27 +/- 0 2.peyrat 29 +/- 2 58 +/- 1 40 +/- 2 29 +/- 2 18 + / 3 56 +/- 1 15 +/- 1 3.St-Andre Beach 36 +/- 7 60 +/- 6 148 +/- 10 94 +/- 7 38 +/- 6 73 +/- 5 47 +/- 6 4.Chazay 9 16 Not detected 32 15 15 70 6.Dombes 4 +/- 2 26 +/- 3 43 +/- 1 61 +/- 66 +/- 1 160 +/- 7 102 +/- 5 ^a Quantitative analysis by dot-blotting with universal probe FGPS431 and phosphorescence imaging (Table 2) ^b 1: non-treated; 2: dry-grinding (G) of soil; 3: chromium + Ultra-turrax homogenization (H); 4a: G + H + microtip sonication (MT); 4b: G + H + Cup Horn sonication (CH); 5a: Chromium + H + NT + chemistry / enzyme lysis. See FIG. 1. ^c ND = unmeasured

Table 4

Primers and Probes Used to Molecular Characterization of DNA Extracted from Soil

Target (primer or probe) Sequence (5'-3 ') Position ^a FGPS 612 Yu Bacteria (Primer) C (C / T) AACT (T / C / A) CGTGCCAGCAGCC 506-525 FGPS 669 Yu Bacteria (Primer) GACGTC (A / G) TCCCC (A / C) CCTTCCTC 1174-1194 FGPS 618 Oil Bacteria ATGG (T / C) TGTCGTCAGCTCG 1056-1073 FGPS 614 a-proteobacteria (probes) GTGTAGAGGTGAAATTCGTAG 683-703 FGPS 615 b-proteobacteria (probes) CGGTGGATGATGTGGATT 939-956 FGPS 616 g-proteobacteria (probes) AGGTTAAAACTCAAATGA 900-917 FGPS 621 Low GC% Gram + (Probe) ATACGTAGGTGGCAAGCG 532-549 FGPS 617 Actinomysheet (probe) GCCGGGGTCAACTCGGAGG 1159-1149 FGPS 680 Streptomycin (probe) TGAGTCCCCA (A / C / T) C (T / A) CCCCG 1132-1149 FGPS 619 Streptosporangium (probe) GCTTGGGGCTTAACTCCAGG 609-628 ^a : location on E. coli 16S rRNA gene

Table 5

Extraction efficacy and amount of DNA extracted from Nycodenz gradient bacteria cells.

Effect of soil sample culture in 6% yeast extract solution prior to density gradient phase dispersion and centrifugation.

Extracted Bacteria Extracted DNA Total microorganisms ^a bacteria / dry soil g Cultivable microflora ^b cfu / g dry soil Cultivable Actinomycete ^c cfu / g dry soil Direct lysis ^d DNA ng / dry soil g Soluble ^{d, e} DNA ng / dry soil g on agarose block Not incubated Soil suspension 1.3 × 10 ⁹ (± 0.1) 6.9 × 10 ⁶ (± 0.2) 8.6 × 10 ⁶ (± 1.2) Cell extraction 1.9 × 10 ⁸ (± 0.2) 4.1 × 10 ⁶ (± 1.5) 2.5 × 10 ⁶ (± 0.7) 333 (± 35) 221 (± 70) Extraction efficacy 15% 59% 38% Cultured from 6% Yeast Extract Soil suspension 1.2 × 10 ⁹ (± 0.1) 7.6 × 10 ⁷ (± 1.1) 6.6 × 10 ⁷ (± 0.4) Cell extraction 1.6 × 10 ⁸ (± 0.3) 5.3 × 10 ⁶ (± 1.4) 3.7 × 10 ⁶ (± 0.7) 344 (± 30) 341 (± 67) Extraction efficacy 13% 7% 5% a: stained with acridine orange and counted microscopically b: counting on 10% Trypcase-Soja solid medium c: 6% yeast extract-0.05% concentrated in SDS solution at 40 ° C. for 20 min and then on HV agar solid medium Counting: The amount of extracted DNA is measured on an electrophoretic gel for the calibration range of calf thymus DNA e: Quantification is performed after digestion of agarose by action of beta-agarase

Table 6

Characterization of the extracted DNA as a function of Gram + proteobacterial subclasses a, b, and g with actinomycete having a low G + C%; Hybridization Signals with Prokaryotic Probes That Act as 100% References

a-proteobacteria b-proteobacteria g-proteobacteria Low G + C% Gram + Actinomy Sheet Streptomycin Direct extraction ^a 7.7% (± 1.4) 5.3% (± 0.5) 3.3% (± 0.9) 3.1% (± 1.7) 14.7% (± 0.6) 0.8% (± 0.1) Indirect extraction Melt + CsCl 10.9% (± 1.4) 6.4% (± 1.4) 14.3% (± 1.4) 7.9% (± 1.4) 8.5% (± 1.4) 3.0% (± 1.4) Block melting 2.9% (± 1.4) 5.4% (± 1.4) 11.1% (± 1.4) 8.0% (± 1.4) 11.3% (± 1.4) 2.6% (± 1.4) Block dissolution + YE culture 6.3% (± 1.4) 7.5% (± 1.4) 17.0% (± 1.4) 18.1% (± 1.4) 19.4% (± 1.4) 4.6% (± 1.4) a: Grinding in a centrifugal force tungsten bead mill (extraction protocol described in Froststegard et al.) YE: 6% yeast extraction solution

TABLE 7

Diversity of 16S rDNA Sequences in Cosmid Library

Table 9 : Sequence

Reference

Claims (81)

I (a) pulverizing the pre-dried soil sample to obtain fines, which are then suspended in a liquid buffer medium; (b) extracting the nucleic acid present in the particulates; (c) passing the nucleic acid-containing solution onto the molecular sieve, and then recovering the elution fraction concentrated in the nucleic acid, passing the elution fraction concentrated in the nucleic acid on an anion exchange chromatography support, and then eluting fraction containing the purified nucleic acid. Method of producing a collection of nucleic acids from the soil-containing sample, comprising the steps of recovering the sequence. II (i) the environmental sample is dispersed in a liquid medium to form a suspension and then the suspension is homogenized by gentle stirring; (ii) separating the organism and other inorganic and / or organic components of the homogeneous suspension obtained in step (i) by centrifugation on a density gradient; (iii) dissolving the organism isolated in step (ii) and extracting the nucleic acid; and (iv) purifying the nucleic acid on a cesium chloride gradient. The method of claim 1, wherein after step I- (a) A further step of sonicating the particulates suspended in the liquid buffer is carried out. The method of claim 1, wherein after step I- (a) Sonicating the particulate suspended in liquid buffer; Incubating the suspension at 37 ° C. in the presence of lysozyme and acromopeptidase after sonication; Adding SDS; A further step of recovering the nucleic acid is carried out. The method of claim 1, wherein after step I- (a) Homogenizing the microparticles using a strong mixing (vortex) step followed by a simple stirring step; Freezing and then thawing the homogeneous suspension; Sonicating the suspension after thawing; Incubating the suspension at 37 ° C. in the presence of lysozyme and acromopeptidase after sonication; A further step of adding SDS is carried out. The method of any one of claims 1 to 5, wherein the nucleic acid is a DNA molecule. A method for producing a recombinant vector collection, characterized in that the nucleic acid obtained by the method according to any one of claims 1 to 6 is inserted into a cloning and / or expression vector. 8. The method of claim 7, wherein the nucleic acid is isolated as a function of its size prior to insertion into the cloning and / or expression vector. 8. The method of claim 7, wherein the average size of the nucleic acid is substantially uniform by physical disruption prior to insertion into the cloning and / or expression vector. 8. The method of claim 7, wherein the cloning and / or expression vector is plasmid type. 8. The method of claim 7, wherein the cloning and / or expression vector is cosmid. 12. The method of claim 11, wherein the cosmid is replicable in E. coli and integrative in Streptomyces. 13. The method of claim 12, wherein the cosmid is pOS7001. The method of claim 1, wherein said cosmid is conjugative and integrative in Streptomyces. The method of claim 14, wherein the cosmid is selected from cosmids pOSV303, pOSV306, and pOSV307. 12. The method of claim 11, wherein the cosmid is replicable in both E. coli and Streptomyces. The method of claim 16, wherein the cosmid pOS 700R is used. 12. The method of claim 11, wherein said cosmid is replicable in E. coli and Streptomyces and conjugated in Streptomyces. 8. The method of claim 7, wherein the coloning and / or expression vector is of type BAC. 20. The method of claim 19, which is a BAC vector that is integrative and conjugative in Streptomyces. The method of claim 20, wherein the vector is selected from BAC vectors pOSV403, pMBD-1, pMBD-2, pMBD-3, pMBD-4, pMBD-5, and pMBD-6. Insertion of nucleic acids into cloning and / or expression vectors Using a suitable restriction endonuclease to open the cloning and / or expression vector at the selected cloning site; Adding a first homopolymeric nucleic acid at the free 3 ′ end of the open vector; Adding a second homopolymer nucleic acid whose sequence is complementary to the first homopolymer nucleic acid to the free 3 ′ end of the nucleic acid from the collection to be inserted into the vector; Hybridizing the first and second homopolymer nucleic acids of mutually complementary sequences to assemble the nucleic acids of the vector and the nucleic acids of the collection; -Cloning the vector by ligation, characterized in that the method of producing a recombinant cloning and / or expression vector. The method of claim 22, The first homopolymer nucleic acid is a poly (A) or poly (T) sequence; The second homopolymer nucleic acid is a poly (T) or poly (A) sequence. Method according to claim 22 or 23, characterized in that the size of the nucleic acid to be inserted is at least 100 kilobases, preferably at least 200 kilobases. 25. The method of claim 22, wherein the nucleic acid to be inserted is contained in the nucleic acid collection obtained by the method according to any one of claims 1 to 6. Insertion of nucleic acids into cloning and / or expression vectors Creating a blunt end on the nucleic acid end of the collection by removing the overhang 3 'sequence and filling the overhang 5' end; Opening cloning and / or expression vectors at selected cloning sites using appropriate restriction endonucleases; Creating a blunt end at the nucleic acid terminus of the vector by removing the overhang 3 'sequence, filling the overhang 5' sequence and then dephosphorylation of the 5 'end; Adding complementary oligonucleotide adapters; -Inserting the collection of nucleic acids into the vector by linkage, the method of producing a recombinant cloning and / or expression vector. 27. The method of claim 26, wherein the nucleic acid to be inserted is at least 100 kilobases, preferably at least 200 kilobases. 28. The method of producing a recombinant vector according to claim 26 or 27, wherein the nucleic acid to be inserted is contained in the nucleic acid collection obtained by the method according to any one of claims 1 to 6. 29. The nucleic acid according to any one of claims 22 to 28, wherein the nucleic acid is inserted as it is obtained without treatment with one or more restriction endonucleases prior to insertion into the cloning and / or expression vector. Way. A nucleic acid collection consisting of nucleic acids obtained by the method of any one of claims 1 to 6. A nucleic acid characterized in that it is contained in the nucleic acid collection according to claim 30. 32. A nucleic acid according to claim 31 comprising at least one operon, or a nucleotide sequence encoding a portion of the operon. 33. A nucleic acid according to claim 32 wherein the operon encodes all or part of the metabolic pathway. 34. The nucleic acid according to claim 33 wherein the metabolic pathway is a polyketide synthesis pathway. 35. The nucleic acid according to claim 34 selected from polynucleotides comprising the sequences of SEQ ID NOs: 30-44 and SEQ ID NOs: 115-120. 32. A nucleic acid according to claim 31 comprising all of the nucleotide sequences encoding the polypeptide. 37. A nucleic acid according to any of claims 31 to 36, characterized in that it is of prokaryotic origin. 38. A nucleic acid according to claim 37 which is derived from a bacterium or a virus. 37. A nucleic acid according to any one of claims 31 to 33 and 36 which is of eukaryotic origin. 40. The nucleic acid according to claim 39 which originates in fungi, yeasts, plants or animals. a) a vector comprising a nucleic acid according to any one of claims 35 to 40; b) a vector obtained according to the method of any one of claims 22 to 25 and 29; c) a recombinant vector, characterized in that it is selected from the vectors obtained according to the method of any one of claims 26-29. A vector characterized by a cosmid pOS7001. A vector characterized by a cosmid pOSV303. A vector characterized by cosmid pOSV306. A vector characterized by a cosmid pOSV307. A vector characterized by a cosmid pOS700R. A vector characterized by a BAC vector pOSV403. A vector characterized in that the vector pMBD-1. A vector characterized in that the vector pMBD-2. A vector characterized by a vector pMBD-3. A vector characterized by a vector pMBD-4. A vector pMBD-5. A vector characterized by a vector pMBD-6. A collection of recombinant vectors obtained according to the method of any one of claims 7 to 21, 25 and 28. Recombinant cloning and / or expression vector, characterized in that it is contained in a collection of recombinant vectors according to claim 54. A recombinant host cell comprising the nucleic acid according to any one of claims 31 to 40 or the recombinant vector according to claim 55. 59. The recombinant host cell of claim 56, wherein the recombinant host cell is a prokaryote or eukaryote. 59. The recombinant host cell of claim 57, wherein the recombinant host cell is a bacterium. 59. The recombinant host cell of claim 58, wherein the bacterium is a bacterium selected from E. coli and Streptomyces. 59. The recombinant host cell according to claim 58, which is a yeast or filamentous fungus. Construction of the Collection A collection of recombinant host cells, wherein each host cell comprises nucleic acids from the nucleic acid collection according to claim 30. 59. A recombinant host cell collection, wherein each of the constituent host cells of the collection comprises a recombinant vector according to claim 41 or 55. Placing the recombinant host cell collection in contact with a pair of primers that hybridize with a given nucleotide sequence or hybridize with a nucleotide sequence that is structurally similar to a given nucleotide sequence; Performing at least three amplification cycles; Detecting any nucleic acid that has been amplified, 63. A method of detecting a nucleic acid in a recombinant host cell collection according to claim 61 or 62 wherein a given nucleotide sequence, or nucleotide sequence, is structurally similar to a given nucleotide sequence. Placing the recombinant host cell collection in contact with a probe that hybridizes with a given nucleotide sequence or hybridizes with a nucleotide sequence that is structurally similar to a given nucleotide sequence; Detecting a hybrid possibly formed between the probe and the nucleic acid included in the vector of the collection, 63. A method for detecting a nucleic acid in a collection of recombinant host cells according to claims 61 or 62, wherein a given nucleotide sequence, or nucleotide sequence, is structurally similar to a given nucleotide sequence. Incubating the collection of recombinant host cells in a suitable culture medium; Detecting the compound of interest in the culture supernatant or in one or more cell lysates of the cultured recombinant host cell, 63. A method for identifying the production of a compound of interest by one or more recombinant host cells in a collection of recombinant host cells according to claim 61 or 62. Incubating the collection of recombinant host cells in a suitable culture medium; Detecting the compound of interest in the culture supernatant or in one or more cell lysates of the cultured recombinant host cell; Selecting a recombinant host cell which produces the compound of interest, A method for screening recombinant host cells for producing the compound in a collection of recombinant host cells according to claim 61 or 62. Culturing the selected recombinant host cells according to the method of claim 66; -Recovering and optionally purifying the compound produced by the recombinant host cell. A compound according to claim 67 which is obtained according to the method of claim 67. 69. The compound of claim 68, wherein said compound is polyketide. A polyketide, characterized in that it is produced by expressing at least one nucleotide sequence comprising a sequence selected from SEQ ID NO: 30 to 44 and SEQ ID NO: 115 to 120. 70. A composition comprising the polyketide according to claim 69 or 70. A pharmaceutical composition comprising a pharmaceutically active amount of a polyketide according to claim 69 or 70 together with a pharmaceutically acceptable vehicle. The nucleic acid of the nucleic acid collection to be tested is placed in contact with an oligonucleotide pair that hybridizes with any sequence of bacterial 16S ribosomal DNA; At least three amplification cycles, The amplified nucleic acid is detected using an oligonucleotide probe or a plurality of oligonucleotide probes each hybridizing specifically with sequences of 16S ribosomal DNA, each of which is common to a bacterial system, throat, subclass or genus Optionally, for nucleic acids of known sequence constituting the calibration range, using a probe or a plurality of probes, comparing the results of the preceding detection step with the detection results, the most Specifically a method for detecting the diversity of nucleic acid collections originating from environmental samples, preferably soil samples. 74. The method of claim 73, wherein the primer pair hybridizing with any sequence of bacterial 16S ribosomal DNA consists of primers FGPS 612 (SEQ ID NO: 12) and primers FGPS 669 (SEQ ID NO: 13). 74. The method of claim 73, wherein the primer pair hybridizing with any sequence of bacterial 16S ribosomal DNA consists of primers 63 f (SEQ ID NO: 22) and primer 1387 r (SEQ ID NO: 23). A nucleic acid comprising a 16S rDNA nucleotide sequence selected from sequences having at least 99% nucleotide identity with the sequences of SEQ ID NOs: 60-106. A recombinant host cell comprising a nucleic acid encoding a type I polyketide synthetase comprising a nucleotide sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120 Generate; Incubating the recombinant host cell in a suitable culture medium; Recovering type I polyketide synthetase from the culture supernatant or cell lysate, and optionally purifying the type I polyketide synthase. A polyketide synthetase comprising an amino acid sequence selected from SEQ ID NOs: 45 to 59 and SEQ ID NOs: 121 to 126. An antibody against a polyketide synthase according to claim 78. a) placing the antibody according to claim 79 in contact with the sample to be tested; b) a method for detecting a type I polyketide synthetase or peptide fragment of the enzyme, comprising detecting an antigen / antibody complex that is possibly formed. a) the antibody according to claim 79; b) optionally a Type I polyketide synthase detection kit in the sample, comprising reagents for detection of the antigen / antibody complexes possibly formed.