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WO2002034784A2 - Production d'antibiotique (ii) - Google Patents

Production d'antibiotique (ii) Download PDF

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Publication number
WO2002034784A2
WO2002034784A2 PCT/GB2001/004709 GB0104709W WO0234784A2 WO 2002034784 A2 WO2002034784 A2 WO 2002034784A2 GB 0104709 W GB0104709 W GB 0104709W WO 0234784 A2 WO0234784 A2 WO 0234784A2
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Prior art keywords
nucleic acid
acid sequence
polypeptide
accb
strain
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WO2002034784A3 (fr
Inventor
Hugo Cesar Gramajo
Eduardo Jose Rodriguez
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Plant Bioscience Ltd
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Plant Bioscience Ltd
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Priority to EP01976514A priority Critical patent/EP1328551A2/fr
Priority to AU2001295782A priority patent/AU2001295782A1/en
Publication of WO2002034784A2 publication Critical patent/WO2002034784A2/fr
Publication of WO2002034784A3 publication Critical patent/WO2002034784A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • Malonyl-CoA is essential as a metabolic substrate in all living organisms studied so far and it also plays a role as a modulator of specific protein activity (for a review see Brownsey et al . , 1997). Malonyl-CoA is a substrate for fatty acid synthase (FAS) (Bloch and Vance, 1977) , for polyketide synthases (PKS) in plants, fungi and bacteria (Hopwood & Sherman, 1990) and for fatty acid chain- elongation systems (Saggerson, et al . , 1992).
  • FAS fatty acid synthase
  • PKS polyketide synthases
  • Biosynthesis of malonyl-CoA occurs in most species through the ATP-dependent carboxylation of acetyl-CoA by an acetyl- CoA carboxylase (ACCase) (Bloch & Vance, 1977; Harwood, 1988).
  • the overall reaction catalyzed by ACCase is a two step process that involves ATP-dependent formation of carboxybiotin followed by transfer of the carboxyl moiety to acetyl-CoA.
  • the importance of this biosynthetic pathway is most directly reflected by the fact that ACCase expression is essential for normal growth of bacteria (Perez, et al . , 1998; Li and Cronan, 1993), yeast (Hasselacher, et al . , 1993) and isolated animal cells in culture (Pizer, et al . , 1996).
  • PCCase purified by Bramwell et al . , (1996) comprises a biotinylated protein of 88 kDa, PccA, and a non-biotinylated component, the carboxyl transferase, of 66 kDa. More recently the inventors have also characterized at both the genetic and biochemical levels, the components of a second PCCase in this bacterium.
  • malonyl-CoA synthesised from malonate by malonyl-CoA synthetase is the major source for fatty acid biosynthesis in the bacterioid R . trifolii .
  • genes with very high identity to MatC and MatB have been recently reported in the S . coellcolor genome project, suggesting that malonyl-CoA could also be synthesized from malonate in this micro-organism.
  • the inventors have identified an essential acyl-CoA carboxylase of S . coellcolor, and provide detailed genetic and biochemical characterization.
  • the enzyme complex contains a unique sub-unit composition and appears to be the main pathway for the biosynthesis of malonyl-CoA, one of the key metabolites in the linkage between primary and secondary metabolism.
  • An alternative pathway for the biosynthesis of malonyl-CoA is also proposed for cultures growing in malonate, and it most probably involves the .ma -B and ma tC homologues of R . trifolii .
  • the acyl-CoA carboxylase seems to be essential for the viability of the micro-organism.
  • AccB was the carboxyl transferase subunit of an acyl-CoA carboxylase .
  • acc-3-5 and accA2 are mainly expressed during vegetative and transition phase of growth, although some expression of these genes also occurred during stationary phase where they should provide the malonyl-CoA subunits for secondary metabolites biosynthesis.
  • accAl is only expressed during the transition phase of growth and its role in the formation of a carboxylase complex involved in providing the substrate for polyketide compounds of S. coellcolor is discussed.
  • the present invention provides a nucleic acid comprising a nucleic acid sequence which encodes an AccB polypeptide and/or an AccE polypeptide, or a nucleic acid sequence complementary thereto.
  • the present invention provides a nucleic acid comprising a nucleic acid sequence which encodes an AccAl and/or AccA2 polypeptide, or a nucleic acid sequence complementary thereto. It is believed that such nucleic acid was not made available to the public before 24 October 1999, when the amino acid sequences of these polypeptides were disclosed in an oral presentation.
  • nucleic acid sequences encoding the AccB, AccE, AccAl and AccA2 polypeptides are given herein. Preferred embodiments of the invention include such sequences. However, it would be a matter of routine for the skilled person to obtain other nucleic acid sequences encoding these polypeptides, e.g. by introducing mutations which do not alter the encoded amino acid sequence, by virtue of the degeneracy of the genetic code, or by introducing mutations which alter the encoded amino acid sequence, within limits as defined below. Moreover, nucleic acids encoding variants of the polypeptides may be obtained e.g. by screening different strains of S. coellcolor or closely related species of Streptomyces using degenerate probes based on the sequences given herein.
  • Preferred nucleic acids of the first and second aspects encode AccB and AccE polypeptides along with an AccAl and/or an AccA2 polypeptide (preferably AccA2) .
  • the nucleic acid sequences encoding Ace polypeptides are preferably in operative association with regulatory sequences, e.g. sequences which enable constitutive or inducible expression in Streptomyces species.
  • regulatory sequences e.g. sequences which enable constitutive or inducible expression in Streptomyces species.
  • suitable inducible promoter is tipA (inducible by thiostrepton) ; suitable constitutive promoters are ermE and the optimised ermE*.
  • naturally occurring nucleic acid sequences may be in operative association with the regulatory sequences with which they are normally associated, or corresponding regulatory sequences from homologous genes in other strains or species.
  • the nucleic acid sequences may be in operative association with the corresponding regulatory (e.g. promoter) sequences defined herein .
  • the present invention separately provides AccB, AccE, AccAl and AccA2 polypeptides having amino acid sequences encoded or encodable by the respective nucleic acid sequences referred to in the first and second aspects .
  • the present invention provides: vectors containing the nucleic acids of the first and second aspects (preferably vectors, e.g. plasmids, suitable for transforming Streptomyces species for expression therein) and cells, particularly Streptomyces cells, transformed with such vectors. Furthermore, the present invention provides a method of producing a secondary metabolite of a Streptomyces species, the method comprising culturing such transformed Streptomyces cells and extracting the secondary metabolite from the cell culture. The metabolite may be purified and/or formulated as a commercial product according to standard procedures.
  • the invention provides a method of modifying a secondary metabolite-producing strain of a Streptomyces species to increase production of said secondary metabolite, the method comprising modifying said strain to express, or to increase expression of, nucleic acid encoding one or more polypeptides selected from the group consisting of AccB, AccE, AccAl and AccA2.
  • the present invention provides a method of modifying a strain of a Streptomyces species to increase ACCase and/or PCCase activity, the method comprising modifying said strain to express, or to increase expression of, nucleic acid encoding one or more polypeptides selected from the group consisting of AccB, AccE, AccAl and AccA2.
  • the present invention provides a modified strain of a Streptomyces species, produced or producible according to the method of the fifth or sixth aspect. Also provided are cells of said strain, methods of producing secondary metabolites comprising culturing said cells and extracting the secondary metabolite, which may be purified and/or formulated as a commercial product.
  • the invention provides a method of increasing production of a secondary metabolite in cells of a Streptomyces species, the method comprising culturing said cells in the presence of exogenous malonate, preferably at a concentration of at least about 0.1%, more preferably at least about 0.2%, 0.4%, 0.5%, 0.75% or 1%, though higher concentrations may be used.
  • 1% represents Ig per 100 ml of medium.
  • the modification preferably provides for increased expression of nucleic acid encoding more than one of AccB, AccE, AccAl and AccA2, more preferably at least AccB and AccE or at least AccB and either AccAl or AccA2, more preferably AccB, AccE and either AccAl or AccA2.
  • AccAl and AccA2 is preferred.
  • Increased expression of nucleic acid encoding both AccAl and AccA2 (usually in combination with AccB and optionally AccE) is also contemplated.
  • the methods of the fifth and sixth aspects preferably include a step of transforming a Strepto-7.yces cell with a said nucleic acid under the control of a constitutive or inducible promoter, preferably a strong promoter.
  • a constitutive or inducible promoter preferably a strong promoter.
  • the expression of existing said nucleic acid could be increased, e.g. by placing them under the control of a stronger promoter sequence or sequences.
  • Exogenous said nucleic acid can replace existing said nucleic acid in the cell, or can be added without removing or functionally deleting existing said nucleic acid.
  • AccB is intended to include not only a polypeptide having the deduced amino acid sequence encoded by the nucleic acid sequence of Fig. 12 (though this is a preferred embodiment) , but also a polypeptide which is a variant
  • allelic or isoallelic variant e.g. an allelic or isoallelic variant
  • a derivative of said polypeptide having at least about 60% amino acid identity with said polypeptide, preferably at least about 65%, 70% or 75%, especially preferably (in view of the similarity of AccB as disclosed herein to another protein of unconfirmed function) at least about 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% identity.
  • Such a variant or derivative may possess any one or more of the biological properties of the wild-type AccB protein, as disclosed herein, e.g.
  • AccAl, AccA2 and/or AccE complex formation with AccAl, AccA2 and/or AccE (or allosteric regulation by AccE) , ACCase and/or PCCase activity when AccB is co-expressed with AccAl, AccA2 and/or AccE, or increased secondary metabolite production when AccB is overexpressed in Streptomyces species
  • AccE is intended to include not only a polypeptide having the deduced amino acid sequence encoded by the nucleic acid sequence of Fig. 13 (though this is a preferred embodiment) , but also a polypeptide which is a variant (e.g. an allelic or isoallelic variant) or a derivative of said polypeptide, having at least about 40% amino acid identity with said polypeptide, preferably at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity.
  • a variant or derivative may possess any one or more of the biological properties of the wild-type AccE protein, as demonstrated herein, e.g.
  • AccAl complex formation with AccAl, AccA2 and/or AccB (or allosteric regulation of AccB) , ACCase and/or PCCase activity when AccE is co- expressed with AccB, or increased secondary metabolite production when AccE is overexpressed in Streptomyces species (preferably in conjuction with AccB) .
  • AccAl and AccA2 are intended to include not only the polypeptides having the amino acid sequences shown in Fig. 11 (though these are respective preferred embodiments) , but also polypeptides which are variants (e.g. allelic or isoallelic variants) or are derivatives of said polypeptides, having at least about 75% amino acid identity with said polypeptide, preferably at least about 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% identity.
  • variants or derivatives may possess any one or more of the biological properties of the wild-type AccAl or AccA2 polypeptides, as disclosed herein, e.g.
  • AccB and/or AccE complex formation with AccB and/or AccE, ACCase and/or PCCase activity when AccAl or AccA2 is co-expressed with AccB and/or AccE, or increased secondary metabolite production when AccB is overexpressed in Streptomyces species (preferably in conjuction with AccB and/or AccE) .
  • a variant or a derivative of a given peptide may have one or more of internal deletions, internal insertions, terminal truncations, terminal additions, or substitutions of one or more amino acids, compared to the given peptide.
  • references to nucleic acid encoding AccAl, AccA2, AccB and/or AccE should be interpreted accordingly.
  • preferred nucleic acids comprise a nucleic acid sequence having at least about 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequence identity with the accB nucleic acid sequence shown in Fig. 12.
  • Other preferred nucleic acids comprise a nucleic acid sequence having at least about 40%, preferably at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequence identity with the accE nucleic acid sequence shown in Fig. 13.
  • nucleic acids comprise a nucleic acid sequence having at least about 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequence identity with the accAl or accA2 nucleic acid sequence shown in Fig. 11.
  • Preferred secondary metabolites are, however, antibiotics, especially Act and Red.
  • Preferred Streptomyces species are the closely related species S . coelicolor, S. violaceoruber r S . lividans and S . parvulus, especially S. coelicolor. Strains of such species are commonly available, e.g. from the ATCC, for example under ATCC deposit numbers 12434 for S. parvulus and 19832 for S . violaceoruber. S . coelicolor A3 (2) and S . lividans 66 are available from the John Innes Culture
  • present invention further provides for the increased production in Streptomyces of acetyl-CoA, since it is thought that when ACCase activity is increased by the methods and means of the present invention, production of malonyl-CoA may become limited by the availability of the substrate acetyl-CoA. It is proposed that increased acetyl-CoA production could then lead to a further increased rate of malonyl-CoA production and hence secondary metabolite production.
  • oils or fatty acids could be used as the carbon source (together with glucose) ; fatty acids are degraded by b- oxidation giving high levels of acetyl-CoA.
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequence with which it is being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the % identity values used herein are generated by WU-BLAST-2 which was obtained from Altschul et al. (1996); http: //blast . wustl/edu/blast/README. html .
  • the HSPS and HSPS2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region, multiplied by 100.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU- BLAST-2 to maximize the alignment score are ignored) .
  • Percent (%) nucleic acid sequence identity is defined as the percentage of nueleotide residues in a candidate sequence that are identical with the nueleotide residues in the sequence under comparison.
  • the identity values used herein were generated by the BLASTN module of WU BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • Streptomyces culturing Streptomyces under conditions suitable for secondary metabolite (e.g. polyketide and/or antibiotic production) and purifying secondary metabolites from Streptomycete cell culture medium are well known, e.g. from Hopwood et al. (1985) and Kieser et al (2000).
  • secondary metabolite e.g. polyketide and/or antibiotic production
  • purifying secondary metabolites from Streptomycete cell culture medium are well known, e.g. from Hopwood et al. (1985) and Kieser et al (2000).
  • active compounds e.g. polyketides, particularly antibiotics
  • Such pharmaceutical formulations may comprise, in addition to the active compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser ox other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, transdermal, transmucosal, intramuscular, intraperitoneal routes.
  • Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington ' s Pharmaceutical Sciences, 18th edition, Mack Publishing
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active compound will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride
  • Formulations suitable for transmucosal administration include liquids, solutions, suspensions, emulsions, suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
  • Ointments are typically prepared from the active compound and a paraffinic or a water-miscible ointment base.
  • Creams are typically prepared from the active compound and an oil-in-water cream base.
  • the aqueous phase of the cream base may include at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more active compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers.
  • Administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, e.g.
  • a pharmaceutical formulation may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Fig. 1 Organization of the genomic region of S . coelicolor M145 chromosome harbouring accB and a ccE genes.
  • A Genetic and physical map of the 6.2 kb insert in pRM08. The secondary structure downstream a ccE represents a rho-independent transcriptional terminator. Fragments I and II were amplified by PCR with the pair of oligos accBup-accBdown and accBEup- accBEdown respectively, uniquely labelled at the 5'- end (*) and used as probes in transcriptional analysis of the accBE operon.
  • B Genetic and physical map of the 6.2 kb insert in pRM08. The secondary structure downstream a ccE represents a rho-independent transcriptional terminator. Fragments I and II were amplified by PCR with the pair of oligos accBup-accBdown and accBEup- accBEdown respectively, uniquely labelled at the 5'- end (*) and used as
  • FIG. 2 Attempted disruption of accB .
  • A Diagram showing the integration of pTR124 through one of the accBE flanking regions and the resolution of the cointegrate by a second event of homologous recombination. The crossed out arrow indicates the impossibility of obtaining the replacement of the wild-type accB by the Hyg R mutant allele.
  • B The integration of a second copy of the accBE genes in the ⁇ C31 att site of T124 (to yield strain T149) allowed the replacement of the wild-type accB by a mutant allele containing the Hyg resistance cassette.
  • FIG. 3 Growth-phase dependent expression and transcription start site of the accBE operon.
  • A SI nuelease mapping of accB, actII-ORF4 and hrdB, using RNA isolated from a liquid time course of S . coelicolor M145.
  • Exp, Trans and Stat indicate the exponential, transition and stationary phase of growth, respectively.
  • B The nueleotide sequences of both strands from the accB promoter region are shown. The arrow indicates the most likely transcription start point for the a ccBE promoter, as determined by SI nuelease mapping. The potential -10 and -35 regions for the a ccBEp are underlined.
  • C SI nuelease mapping of the accB accE intergenic region using a 563 nt probe. FLP represents the full-length RNA-protected fragment that is 13 nt shorter than the probe.
  • Fig. 4 Growth-phase dependent expression of accA2 and accAl .
  • SI nuelease mapping of accA2 (A) and accAl (B) using RNA isolated from a liquid time course of S. coelicolor M145.
  • FIG. 5 Mapping of the a ccA2 and accAl transcription start point.
  • A High resolution SI nuelease mapping of the 5 ' end of the accA2 transcript.
  • SI RNA-protected products of the SI nuelease protection assay.
  • Lanes labelled A, C, G and T indicate a dideoxy sequencing ladder using the same oligonueleotide that was used to make the SI probe (accA2down) .
  • B High resolution SI nuelease mapping of the 5' end of the accAl transcript.
  • SI RNA-protected products of the SI nuelease protection assay.
  • Lanes labelled T, G, C and A indicate a dideoxy sequencing ladder using the same oligonueleotide that was used to make the SI probe (accAldown) .
  • C Sequence of the accA2 and a ccAl upstream regions, indicating the most likely transcription start points for the promoters of each of the accAl and accA2 genes (bent arrows) .
  • the potential -10 and -35 sequences for the accAl and accA2 promoters are underlined.
  • the potential ribosomal binding sites (rbs) are highlighted with bold letters.
  • the 16 nt direct repeats (DR) found upstream of the transcription start point of accAlpl are indicated with straight arrows.
  • FIG. 6 Construction and analysis of the accBE conditional mutant.
  • A Diagram showing the integration of pIJ8600 in strain M86 and the expected organisation of the Campbell integration of pTR94 in M94. Restriction sites: B. BamHI; N, ⁇ fotI; Nd, Ndel ; S, Sacl ; Sp, Sphl ; Xb, X-al.
  • B Hybridisation analysis of Southern blot of Sad-digested D ⁇ As from M145, M86 and M94. The probe was the internal Nde ⁇ -Xbal fragment of accB shown in A (see Fig. 10) .
  • Fig. 7 Expression of the acyl-CoA components in M86 and M94.
  • A. SDS-PAGE of cell-free extracts of S. coelicolor M86 and M94 strains grown in YEME medium containing 10 ⁇ g/ml Am with or without the addition of 5 ⁇ g/ml Th.
  • B. A duplicate of the SDS-PAGE gel shown in A was subjected to Western blotting and stained for biotinylated proteins by using alkaline phosphatase- streptavidin conjugate.
  • Fig. 8A Growth curves of M145, M86 and M94 strains. 10 8 spores of strains M86 and M94 were inoculated in YEME medium containing 10 ⁇ g of Am or 10 ⁇ g/ml Am and 5 ⁇ g/ml of Th . 10 8 spores of M145 were inoculated in YEME. The growth was followed by measuring OD 45 o n m-
  • Fig. 8B Actinorhodin production in M94 and M145 in cultures grown in the presence of 5 ⁇ g of Th.
  • Fig. 9 Morphological and physiological differentiation of M86 and M94 in the presence of Th. Spores of M86 and M94 were spread in R2 or R5 medium containing lO ⁇ g/ml Am. A drop containing 1 ⁇ g of Th was spotted in the centre of each plate. The picture shows the results obtained after the incubation of the plate at 30°C for 48h.
  • Fig. 10 The sequence of the amplification product obtained from accB using primers TC16 and TC17. ⁇ /del (CATATG) and X-al (TCTAGA) sites introduced into the accB by the primers are shown in bold. The 1 kb ⁇ /del- Xbal fragment was cloned into pIJ8600.
  • Fig. 11 A. Amino acid sequences and B. Nucleic acid sequences of accAl and accA2.
  • Fig. 12 A. Amino acid sequence and B. Nucleic acid sequence of accB .
  • Fig. 13 A. Amino acid sequence and B. Nucleic acid sequence of a ccE .
  • Fig. 14 Plasmid map for the construction of an expression vector for accA, accB and accE .
  • pccB of S. coeli color (Rodriguez and Gramajo, 1999) was used as an heterologous probe in Southern blot experiments.
  • a Ba-nHI digest of S. coelicolor DNA was probed with pccB and washed under low stringent conditions, a second, low hybridising, band was readily detected (data not shown) .
  • the target sequence was cloned from a size-enriched library as a 2.5 kb Bai ⁇ Hl fragment and sequenced as described in Experimental Procedures (below) . The sequence revealed the presence of an incomplete ORF with high homology to pccB. The complete gene was finally cloned as a 6 kb Sstl fragment yielding pRM08 (Fig.
  • the sequence also revealed the presence of a small ORF, designated a ccE, whose start codon is only 17 bp downstream of the termination codon of accB .
  • a 17 nt inverted repeat which could function as a factor- independent bidirectional transcriptional terminator (reviewed in Lewin, 1994), separates accE from three convergent ORFs with homology to putative proteins of M. tuberculosis with unknown functions.
  • the putative AccE polypeptide has a deduced molecular mass of 7.07 kDa and no significant homology to this polypeptide was found in a search of sequences deposited in the GenBank database.
  • a ccB mutant was constructed by gene replacement (Fig. 2A) .
  • a Hyg-resistant cassette was cloned in the unique BaraHI site present in the coding sequence of accB, contained in pTR80.
  • pIJ2925 After an intermediate construction in pIJ2925, a Bgill fragment containing the mutated allele was finally cloned in the conjugative vector pSET151.
  • the resulting plasmid, pTR124 was cloned into the E . coli donor strain ET12567/pUZ8002 and transferred by conjugation into M145. Exconjugants were selected for Th R Hyg R for a simple crossover event.
  • One of the exconj ugants, named T124 was taken through four rounds of non-selective growth
  • Example 3 Heterologous expression of accB, accE and in vitro reconstitution of an acyl-CoA carboxylase complex .
  • ACCase activity cannot be assayed directly by carboxylation of acetyl-CoA (Polakis et al . , 1972); therefore, the acyl- CoA carboxylase activity measured in these crude extracts exclusively represents the activity of the heterologous complexes reconstituted in vitro .
  • hrdB was expressed constantly throughout growth (Buttner, M.J., 1990), while actII-ORF4 had a peak of expression during transition phase that shut off in stationary phase (Gramajo, et al . , 1993).
  • RNA-protected fragments found for accB corresponded to a transcription start site 1 bp upstream, or in the adenine, of the most likely translation start site of accB.
  • Upstream of the transcription initiation site we found a putative -10 and -35 promoter regions with a high consensus sequences of promoters recognised by the vegetative ⁇ h dB (Strohl, 1991) (Fig. 3B) .
  • a new 563 bp probe was obtained by PCR.
  • a 5' oligo corresponding to a sequence within the coding region of accB and a 3'oligo corresponding to a sequence within accE.
  • the full- length RNA-protected fragment was easily differentiated from the probe-probe re-annealing due to the addition of a 13 nt tail to the 5'oligonucleotide (Experimental Procedures) .
  • the results obtained in this experiment clearly showed that accB and accE were part of the same transcript, confirming that these two genes form a single- copy operon (Fig. 3C) .
  • the expression of accBE during the different growth phases as detected with this new probe followed the same profile as the expression observed with the probe used for accB.
  • accA2 and accAl mRNA present throughout growth were also studied by SI protection experiments (Fig. 4) .
  • the probe used for accA2 was a 766 bp DNA fragment generated by PCR and uniquely labelled on the 5' end of the oligo corresponding to the sequence within accA2. This experiment showed the existence of three mRNA-protected fragments.
  • the growth phase-dependent expression of two of them, accA2pl and acc-2p2 resemble very much that of the accBE operon.
  • TP exponential and transition phase
  • Fig. 4A the transcription shuts down when the cultures reach stationary phase
  • SI nuelease protection of a ccAl mRNA was performed by using a 563 bp PCR product, uniquely labelled at the 5' end of the downstream oligo, corresponding to a sequence within accAl .
  • the expression of this gene occurs from at least three different putative promoters, and all of them showed a clear burst of expression during the first hours of the TP, which rapidly shut down during late TP.
  • This pattern of transcription resembled very much the one observed for the third RNA-protected band found for a ccA2.
  • the transcription starts sites for the accA2pl and p2 were mapped by high resolution SI mapping (Fig. 5A and B) .
  • Fig. 5C The transcription start points and the putative -10 and -35 promoter regions of these two promoters are shown in Fig. 5C.
  • a certain degree of homology was found between the -10 consensus sequence of acc ⁇ 2pl and p2 and the promoters recognised by the vegetative ⁇ 3 (Strohl, 1992) .
  • High resolution SI mapping of accAl revealed that the transcription start point of the most abundant mRNA species starts 88 bp upstream of the GTG initiation codon of AccAl and the putative -10 regions resemble, in some extent, the consensus sequences of promoters recognised by ⁇ ⁇ rds .
  • accBE genes are essential in the presence of malonate
  • coelicolor M145 was able to grow, indicating that MatC and MatB could be the proteins involved in the transport and activation of malonate to malonyl-CoA, and suggesting that a decarboxylase that could convert malonyl- into acetyl-CoA should also be present in this bacterium, to allow the use of malonate as a carbon and energy source.
  • Example 6 Construction of a strain with the accBE operon under the control of a fcipA promoter
  • the accBE operon which encodes the earboxyl-transferase and a previously unidentified ⁇ sub- unit of an acyl-CoA carboxylase, is essential for the viability of S . coelicolor A3 (2).
  • S . coelicolor A3 (2) In order to regulate the expression of this operon and study its effect on the physiology of this microorganism, we constructed a conditional mutant strain where the expression of the a ccBE operon was under the control of the thiostrepton-inducible ti A promoter (Murakami, et al . , 1989).
  • M94 One of these exconjugants, designated M94, was purified in SFM medium for further analysis. Integration of pTR94 could only take place by Campbell recombination through the accBE homologous sequences, and this event should leave a complete copy of the accBE operon under the tipA promoter (Fig. 6A) . To confirm that this event had occurred in M94, we performed Southern blot experiments of DNA samples prepared from strains M145, M94 and M86. The last strain (M86) was obtained by integration of pIJ8600 in the ⁇ C31 att site of the chromosome and used as the best isogenic control for M94 (Fig. 6A) . As shown in Fig.
  • a Sa d digested DNA from M145 and M86 lights up a unique hybridisation band of 5.94 kb that contains the accBE operon.
  • DNA from M94 instead, lights up two hybridising bands corresponding to the expected sizes for the integration of pTR94 in the accBE operon (Fig. 6A and B) .
  • Fig. 7A shows a 60 kDa protein that is only induced in cultures of M94 grown in the presence of Th; the size of this protein corresponded to the molecular mass of AccB. We were not able to detect an inducible band corresponding to AccE.
  • AccAl or AccA2 The levels of the biotinylated components (AccAl or AccA2) of the acyl-CoA carboxylase, in each of the cell-free extracts, were analysed by a modified Western Blotting procedure (Fig. 7B) . As shown in this figure the levels of AccAl and/or AccA2 were not modified by presence of Th . However, cell free-extracts of M94 do contain a slightly higher amount of the 65 kDa protein compared to M86.
  • ACCase and PCCase activities were assayed in cell-free extracts of M94 and M86.
  • the levels of both enzyme activities were similar in cell-free extracts prepared from cultures of M86 grown in the presence or in the absence of Th (Table 3) .
  • Cell-free extracts prepared from induced cultures of M94 show instead a remarkable increase in both ACCase (11.5 fold) and PCCase (3.5 fold) activities, compared with the levels found in non-induced cultures of the same strain or in M86.
  • Example 8 Influence of the acyl-CoA carboxylase levels in the physiological properties of M94
  • Fig 8A Growth curves (Fig 8A) were determined for the conditional mutant M94 and for M86 by inoculating a spore suspension in YEME medium supplemented with 10 ⁇ g of Am, with or without the addition of 5 ⁇ g of Th .
  • YEME medium without the addition of any antibiotic was used for M145.
  • M94 supplemented with the inducer (Th) showed a growth rate during exponential phase very similar to M145, judged from the slope of the curves.
  • Actinorhodin and undecylprodigiosin were also quantitated throughout growth.
  • Table 4 shows that antibiotic production was only detected in cultures of M94 grown in the presence of 1 or 5 ⁇ g of Th. No antibiotic production was observed in cultures of M145 or M94 without Th, at least until after 60 h of growth. No antibiotic production was detected in M86.
  • Fig. 8B shows the stimulatory effect on actinorhodin production in M94 compared to M145 in cultures grown in the presence of 5 ⁇ g of Th.
  • Example 9 Co-expression of accA, accB and accE in S . coelicolor
  • the Ndel-Xbal fragment of pTR154 (Fig. 14) is introduced into pIJ8600 and then transformed into S. coelicolor M145 (Fig. 14). Transformants are selected with apra yein and thiostrepton . Overexpression of the three components a ccA2, accB and accE results in increased ACCase activity and antibiotic production compared to the wild type M145 strain.
  • accB, accE and the BC-BCCP- (biotin carboxylase- and biotin carboxylase carrier protein-) encoding genes accAl and accA2 in E. coli allowed in vi tro studies to be performed in order to understand the role of the corresponding encoded proteins as components of a previously uncharacterized acyl-CoA carboxylase.
  • the reconstitution, by mixing cell-free extracts of E. coli containing AccB and AccAl (or AccA2), of an active enzyme with the ability to carboxylate either acetyl- or propionyl-CoA clearly established that AccB was the carboxyl transferase component of an acyl-CoA carboxylase complex.
  • Malonyl-CoA is an essential component of all living organisms, since it is the main elongation unit for fatty acid biosynthesis (Brownsey et al . , 1997). This primary metabolite is synthesised in most species through the carboxylation of acetyl-CoA by an ACCase (Bloch and Vance, 1977) . If this was also the case for S. coelicolor and, if AccB was the component of an essential acyl-CoA carboxylase, mutation of this gene should be lethal for the micro-organism. Replacement of the wild-type accB for the Hyg R mutant allele prove to be unsuccessful, and it only occurred when a second copy of the accBE genes was present in the chromosome (Fig. 2B) .
  • accB and accA2 throughout growth also support this interpretation, since both genes are principally transcribed during exponential and transition phase.
  • ACCase and PCCase activities also showed the highest and constant levels of activities during exponential and transition phase while in stationary phase the activities were low but readily measurable.
  • a putative pathway for the utilization of this substrate could involve the R . trifolii MatC and MatB homologues which are found in the genome of S. coelicolor .
  • the biochemical characterization of MatB in R . trifolii demonstrated that this protein is a malonyl-CoA synthetase, which catalyzes the formation of malonyl-CoA directly from malonate and CoA.
  • MatC instead, has not been characterized biochemically but computer analysis indicate that it is a transmembrane protein that could function as a dicarboxylate (malonate for example) carrier (An and Kim, 1998). If these enzymes were part of the pathway that allows S.
  • Transformants were selected on media supplemented with the appropiate antibiotics : ampicillin (Ap) 100 ⁇ g/ml; apramaycin (Am) 100 ⁇ g/ml; chloramphenicol (Cm) 25 ⁇ g/ml or kanamycin (Km) 30 ⁇ g/ml.
  • Strain BL21(DE3) is an E. coli B strain [F ⁇ ompT (r B ⁇ m B " ) (DE3) ] lysogenized with 1DE3, a prophage that expresses the T7 RNA polymerase downstream of the IPTG-inducible IacUV5 promoter (Studier & Moffat,
  • ET12567/pUZ8002 (MacNeil et al (1992)/Paget et al (1999)) was used for E. coli - S. coelicolor conjugation experiments (Bierman, 1992) .
  • media were overlayed with thiostrepton (Th) (300 ⁇ g per plate), hygromycin (Hyg) (1 mg per plate) or apramycin (Am) (1 mg per plate) .
  • Thiostrepton Thiostrepton
  • Hyg hygromycin
  • Am apramycin
  • S. coelicolor M145 was grown at 30°C in shake flasks in YEME medium for 24-48 h. When necessary, 10 mg Am ml "1 or 5 mg Th ml "1 were added to the medium. Mycelia were harvested by centrifugation at 5000 x g for 10 min at 4 °C, washed in 100 mM potassium phosphate buffer pH 8 containing 0.1 mM DTT, 1 mM EDTA, 1 mM PMSF and 10% glycerol (buffer A) and resuspended in 1 ml of the same buffer.
  • E. coli strain BL21(DE3) harbouring the appropriate plas ids were grown at 37°C in shake flasks in LB medium in the presence of 25 ⁇ g Cm ml "1 or 100 ⁇ g Ap ml "1 for plasmid maintenance.
  • biotinylated proteins 10 ⁇ M d-biotin was supplemented to the medium. Overnight cultures were diluted 1:10 in fresh medium and grown to A 60 o 0.4-0.5 before the addition of IPTG to a final concentration of 0.1 mM.
  • the reaction mixture contained 100 mM potassium phosphate pH 8.0, 300 ⁇ g BSA, 3 mM ATP, 5 mM MgCl 2 , 50 mM NaH 14 C0 3 [specific activity 200 ⁇ Ci mmol "1 (740 kBq mmol "1 ) ] , 1 mM substrate (acetyl-CoA or propionyl-CoA) and 100 ⁇ g cell-free protein extract in a total reaction volume of 100 ⁇ l .
  • the reaction was initiated by the addition of NaH 14 C0 3 , allowed to proceed at 30 °C for 15 min and stopped with 200 ⁇ l 6 M HCl. The contents of the tubes were then evaporated to dryness at 95 °C.
  • the reaction mixture contained 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1 mM MgCl 2 , 6% glycerol, 25 ⁇ M of each of the four dNTPs, 2.5 U Taq DNApolymerase, 20 pmol of each primer and 50 ng of S. coelicolor chromosomal DNA in a final volume of 100 ⁇ l .
  • Samples were subjected to 30 cycles of denaturation (95°C, 30 s), annealing (65°C, 30 s) and extension (72°C, 1 min) .
  • a 1 kb PCR fragment was used as a 32 P-labelled probe to screen a size-enriched library.
  • a 2.7 kb BamRI fragment containing an incomplete a ccB gene was cloned in Bam I- cleaved pBluescript SK(+), yielding pTR62.
  • the synthetic oligonueleotide TC16 ( 5 -
  • TATTCTAGACATATGACCGTTTTGGATGAGG used to introduce an Ndel site at the translational start codon of the S. coelicolor accB gene
  • TC17 5' -ACCTCTAGACAACGCTCGTGGACC, used to introduce an Xbal site in the accB coding sequence
  • the reaction mixture was the same as the one indicated above. Samples were subjected to 30 or 35 cycles of denaturation (95°C, 30 s) , annealing (65°C, 30 s) and extension (72°C, 1 min) .
  • the 1 kb PCR product was digested with Ndel and Xbal (these sites were introduced in the 5 ' ends of the oligos TC16 and TC17 and are shown in bold in Fig. 10) and cloned in Xbal- cleaved pBluescript SK(-f) in E. coli DH5 ⁇ , yielding pTR82.
  • This plasmid was digested with Bst-SII and Sacl, ligated with a B5t.£H-SacI fragment cleaved from pRM08 and introduced by transformation into E. col i DH5 ⁇ , yielding pTR87.
  • Ndel-Xbal fragment from the plasmid pTR82 was cloned in ⁇ del- ial-cleaved pIJ8600 (Sun et al (1999)), yielding pTR93.
  • pTR93 In order to place the chromosomal copy of accBE operon under the tip- promoter we removed from pTR93 a Hindll l fragment containing the int gene and att of ⁇ C31, yielding pTR94.
  • Plasmid pTR94 was transformed into strain ET12567/pUZ8002 and transferred by conjugation to S. coelicolor M145 (Hopwood et al (1985)).
  • Ndel-Sacl fragment from the plasmid pTR87 was cloned in Ndel-Sa cI-cleaved pET22b(+) ( ovagen) (pTR88), thus placing the accBE operon under the control of the powerful T7 promoter and ribosome-binding sequences.
  • ATGAATTCTATGCATCGGGTCAGCGCCAGCTG were used to amplify the a ccE gene of S. coelicolor.
  • the reaction mixture was the same as the one indicated above.
  • Samples were subjected to 35 cycles of denaturation (95°C, 30 s) , annealing (65°C, 30 s) and extension (72 C C, 30 s) .
  • the PCR product was cloned using pGEM-T easy vector (Promega) in E. coli DH5 ⁇ , yielding pTR106.
  • a -Vdel- ⁇ -coRI fragment from the plasmid pTR106 was cloned in -Vdel-BcoRI-cleaved pET22 (b) ( ⁇ ovagen) yielding the plasmid pTR107, thus placing the accE gene under the control of the powerful T7 promoter and ribosome- binding sequences.
  • Plasmid pIJ8600 was digested with Bgill and EcoRI and the fragment containing ori T RK2, ori pUC18, attP site, int ⁇ C31 and aac (3) IV (Am R cassette) genes was ligated with a linker containing the following enzymes (Mike Butler personal comunication) : Bgill , Asel, EcoRI , Bgill , Ndel , Kpnl , Xba l , Ps tl , Hindll l , Bamtil , Ss tl , Notl and -ScoRI, yielding pTR141.
  • a 4.0 kb Kpnl fragment containing the complete accBE operon from pRM08 was cloned into pz-I- cleaved pTR141, yielding pTR149.
  • the amplified DNA was then cloned into pGEM-T (Promega) , to give pTR99.
  • pGEM-T Promega
  • To introduce a Nsil site upstream of the RBS of accA2 we amplified this gene using the oligo N-accA2 (5' ATG AAT TCA TGC ATG AGG GAG CCT CAA TCG 3') / for the 5' end and the oligo C-accA2 (5' AGA TCT AGA TCA GTC CTT GAT CTC GC 3') containing a Xbal and a BcoRI site, for the 3' end of the gene.
  • the amplified DNA was cloned in pGEM-T to give pTR112.
  • the sequence of the Sphl original fragment was performed from plasmids DNA constructed by subcloning Apal DNA fragments from pRM08 into pSKBluescribe SK(+). Synthetic oligonucleotides were used to complete the sequence.
  • the nueleotide sequence of the accBE region was determined by dideoxy sequencing (Sanger et al . , 1977) using the Pro ega TaqTrack sequencing kit and double-stranded DNA templates.
  • the complete sequence of the 1C2 cosmid, that includes the Sphl fragment harbouring a ccBE, is available from the S. coelicolox genome sequencing project.
  • RNA for each SI nuelease reaction, 30 ⁇ g of RNA were hybridized in NaTCA buffer (Murray, 1986) ; solid NaTCA (Aldrich) was dissolved to 3M in 50mM PIPES (pH 7.0), 5mM EDTA, to about 0.002 pmol (approximately 10 4 cpm) of the following probes.
  • the synthetic oligonueleotide 5'- GCTTTGAGGACCTTGGCGATG (accA2down) corresponding to the sequence within the coding region of accA2 was uniquely labelled at the 5' end of the oligonueleotide with [ 32 P]- ATP using T4 polynucleotide kinase.
  • the labelled oligo was then used in the PCR reaction with the unlabelled oligonueleotide (accA2up) 5' -GAAGTACAGGCCGAAGACCAC, which corresponds to a region upstream of the accA2 promoter region, to generate a 766 bp probe.
  • the accAldown oligo was later used in the PCR reaction with the unlabelled oligonueleotide (accAlup) 5'- CCGATATCAGCCCCTGATGAC, which corresponds to a region upstream of the a ccAl promoter to generate a 563 bp probe.
  • a ccB the synthetic oligonueleotide (accBdown) 5'- CGTCAGCTTGCCCTTGGCGTG, corresponding to the region within the coding region of a ccB, was uniquely labelled with [ 3 ⁇ P] ⁇ ATP using T4 polynucleotide kinase at the 5' end of the oligonueleotide. accBdown was then used in the PCR reaction with the unlabelled oligonueleotide (accBup) 5'- CTACGCTCCGGGTGAGCGAAC, which corresponds to a region upstream of the accB promoter, to generate a 483 bp probe.
  • accBEdown oligo was then used in the PCR reaction with the unlabelled oligonueleotide (accBEup) 5'- GAGGAACTGGTACGCGCGGGCG(GTACAAGCAAGCT) , which corresponds to a region within the coding region of accB (bracketed oligonucleotides are a tail added to the probe to differentiate probe reannealing from fully protected DNA- RNA complexes), to generate a 563 bp probe. Subsequent steps were as described by Strauch et al . (1991).
  • Plasmids pBluescript SK(+) Phagemid vector (Ap R lacZ ' ) Stratagene pGEM-T Easy For cloning PCR products Promega pIJ2925 pUCIS derivative (Ap R lac ) Janssen & Bibb (1993) pSET151 For the conjugal transfer of DNA from E. coli to Bierman et al. (1992) Streptomyces spp. (Ap R Th R lac ⁇ A pET22b(+) Phagemid vector (Ap R lacZ') for expression of Novagen recombinant proteins under control of strong T7 transcription and translation signals pUZ8002 RK2 derivative with defective oriT (Km R ) Pagt etal.

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Abstract

L'invention concerne les séquences d'acide nucléique et d'acide aminé des sous-unités α1, α2, β et ε de l'acétyl-CoA carboxylase (ACCase) provenant d'un Streptomyces coelicolor. Cette structure de sous-unités diffère de celle des acyl-carboxylases connues. L'invention concerne également des substances et des procédés pour augmenter l'activité de l'ACCase et pour produire des métabolites secondaires (tels que des polycétides et surtout des antibiotiques) en provoquant l'expression dans un Streptomyces d'un tel acide nucléique. L'invention a encore trait à des procédés pour augmenter l'activité de l'ACCase et produire des métabolites secondaires (tels que des polycétides et surtout des antibiotiques) en mettant en culture un Streptomyces en présence d'un malonate exogène.
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