WO2001021779A2

WO2001021779A2 - Penicillium chrysogenum transcriptional activator blar, a pathway specific regulator of beta-lactam biosynthesis, and uses therof

Info

Publication number: WO2001021779A2
Application number: PCT/EP2000/009031
Authority: WO
Inventors: Marco Alexander Van Den Berg
Original assignee: DSM NV
Current assignee: Koninklijke DSM NV
Priority date: 1999-09-17
Filing date: 2000-09-14
Publication date: 2001-03-29
Anticipated expiration: 2002-03-17
Also published as: AU7288100A; WO2001021779A3

Abstract

The present invention relates to a polynucleotide encoding at least part of a polypeptide having activity of a pathway-specific regulator for the production of beta -lactam (abbreviated BlaR) in a micro-organism. The polynucleotide of the invention is obtainable from a beta -lactam producing micro-organism, preferably from a beta -lactam producing filamentous fungus or bacterium.

Description

REGULATION OF SECONDARY METABOLITE PRODUCTION

The present invention relates to the field of production of anti-infective agents. More specifically, to the field of production of antibiotics by micro-organisms.

The diversity of secondary metabolites synthesised by micro-organisms is enormous. Starting from what mostly are primary metabolites organisms are capable of synthesising a huge range of small molecules via complex multi-step reaction paths. The products often consist of several types of intermediates linked together. The responsible enzymes often are highly specific for each molecule and/or organism. However, in the case of families of closely related compounds (e.g. β- lactams) enzymes with broad substrate specificity are known (e.g. acyltransferase). Recently, detailed knowledge on the genetics behind the production of secondary metabolites is accumulating rapidly. Genes encoding the specialised proteins are mostly found clustered on the genome. These clusters may consist of up to 30 genes, some of which encode huge proteins (e.g. ACV synthetase in penicillin biosynthesis is 425 kDa in size). Expressing these pathways may pose a huge burden on the cell. Obviously, a tight regulation is controlling the expression levels of these pathways in such a way that no energy is wasted. Apart from general regulation principles such as glucose and ammonium repression, several cloned gene-clusters are shown to contain a so-called pathway specific regulator. These genes are imbedded in the gene-cluster and mostly transcribed at a low, but constitutive level. In several cases it was shown that these regulators are positive transcription factors which are essential for transcription of other genes of the cluster, and therefore for the production of the final product. Examples of this type are aflR (aflatoxin production in Aspergilli; Appl. Environ. Microbiol. 60 (1994):2408- 2414), corR (coronatine production in Pseudomonas syringae; J. Bacteriol. 180 (1998):6252-6259), claR (clavulanic acid production in Streptomyces clavuligerus;

Mol. Microbiol. 27 (1998):831-843) and pltR (pyoluteorin production in Pseudomonas fluorescens; J. Bacteriol. 181 (1999):2166-2174). Amplification of ccaR in S. clavuligerus resulted in a twofold to threefold increase in the production of cephamycin and clavulanic acid (Perez-Llarena er a/., J. Bacteriol. 179 (1997): 2053- 2059) These include all types of transcription factors such as binuclear zinc-fingers (e.g. aflR) and LysR-type factors (e.g. claR and pltR).

So far no pathway-specific regulator has been described for /Mactam producing organisms. Although, very complex transcriptional regulation mechanisms were reported (see for example Brakhage Microbiol. Mol. Biol. Rev. 62 (1998):547- 585), these consisted without exception of general transcription factors like CreA, the Hap-complex and PacC.

To increase the efficiency of a production process it is necessary to increase the flux of the product wanted. Most approaches focus on the structural genes which encode the enzymes for the actual conversions. This has been the case also with penicillin production. Several papers describe increased penicillin titres of wild type strains transformed with the genes of the penicillin biosynthetic cluster (Veenstra et al. J. Biotechnol. 17 (1991 ):81-90; Fernandez-Canon and Penalva, Mol. Gen. Genet. 246 (1995):110-118; Kennedy and Turner Mol. Gen. Genet. 253 (1996): 189-197; Van den Berg et al. Anth. Leeuwenhoek Int. J. 75 (1999): 155-161 ). Moreover, industrial penicillin production strains of Penicillium chrysogenum were shown to contain an increased number of gene-clusters, accounting for the increased production rate (Barredo et al., Curr. Genet. 16 (1989):453-459; Newbert et al. J. Industr. Microbiol. Biotechnol. 19 (1997): 18-27). However, these clusters are still controlled by the complex general regulation mechanisms. Moreover, the promoter regions of the penicillin biosynthetic genes of P. chrysogenum strains are shown to be identical in low and high producing strains (Newbert et al. supra). Therefore, the regulation will be de facto the same. A possible pathway-specific regulator will still be transcribed at a low level. The number of binding sites in the promoter regions of all genes in all clusters will simply outnumber the amount of active pathway regulator. Therefore, the organism is not capable of fully exploit the increased genetic capability.

The invention described below provides a system were increased numbers of gene clusters are anticipated by a sufficient amount of pathway specific transcription factor. Therefore, it provides a pathway-specific regulator for /Mactam antibiotics. This transcription factor blaR was cloned and shown to be located on the amplified region as described by Barredo et al. (supra) and Newbert et al. (supra), downstream of the penDE gene. It is located also on the fragment used by Turner et al. (Bio/Technology 8 (1990):39-41 ) which was used to confer non-producing organisms like Neurospora crassa and A. niger to penicillin production. Thereby, showing that the fragment they used contained all the genetic information needed. Here, we show that this also includes the cluster specific transcription factor. This is in contradiction to Newbert et al. (supra), who claimed that there were no other transcripts found in the amplified region. Otherwise, Fierro et al. (abstracts GIM

Jerusalem (1998)) described three unidentified mRNA transcripts downstream of the penDE gene.

The present invention relates to a polynucleotide encoding at least part of a polypeptide having activity of a pathway-specific regulator for the production of β- lactam (abbreviated BlaR) in a micro-organism. The polynucleotide of the invention is obtainable from a Mactam producing micro-organism, preferably from a Mactam producing filamentous fungus or bacterium.

The DNA fragment(s) according to the present invention comprising a nucleotide sequence encoding a BlaR polypeptide may suitably be of genomic or of cDNA origin, for instance obtained by preparing a genomic or a cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridisation using at least part of the novel nucleotide sequence as disclosed herein as a probe applying standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

The DNA fragment(s) of the invention comprising the gene or cDNA encoding a BlaR polypeptide may also be prepared by polymerase chain reaction using specific primers advantageously primer derived from the nucleotide sequence as disclosed herein. To this end use can be made, for instance of the method described in US 4,683,202 or by Saiki et al. (Science, 239 (1988): 487-491 ).

In another embodiment of the invention, nucleotide sequences encoding a BlaR polypeptide from other Mactam producing micro-organisms, such as filamentous fungi like A. chrysogenum or A. nidulans, or Actinomycetes like Streptomyces clavuligerus, Nocardia lactamdurans, Flavobacterium sp. are obtained by screening genomic or cDNA libraries prepared from the respective organisms with probes derived from the P. chrysogenum nucleotide sequence as disclosed herein. The present invention further encompasses nucleotide sequences which are substantially homologous to a blaR coding sequence as obtained from a /Mactam producing micro-organism. Preferably, the present invention encompasses nucleotide sequences which are substantially homologous to a blaR coding sequence as obtained from P. chrysogenum, A. chrysogenum, A. nidulans or S. clavuligerus.

Substantially homologous sequences include nucleotide sequences encoding a BlaR polypeptide which may have one or more amino acid substitutions, deletions or additions as compared to their natural counterparts. These changes are preferably of a minor nature (that is conservative amino acid substitutions that do not adversely affect the folding or activity of the protein), small deletions (typically of one to about 30 amino acids), small amino- or carboxyl-terminal extensions (such as an amino-terminal methionine residue), a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (such as a poly-histidine tract, an antigenic epitope or a binding domain). See in general Ford et al. (Protein Expression and Purification, 2 (1991), 95-107,).

Examples of conservative substitutions are substitutions within the respective groups of

♦ basic amino acids (such as arginine, lysine, histidine), ♦ acidic amino acids (such as glutamic acid and aspartic acid),

♦ polar amino acids (such as glutamine and asparagine),

♦ hydrophobic amino acids (such as leucine, isoleucine, valine),

♦ aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and

♦ small amino acids (such as glycine, alanine, serine, threonine, methionine).

It will be apparent to persons skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still have to result in an active polypeptide. A test can be applied to determine the /Mactam regulatory activity of the polypeptide. For example an in vitro gel shift assay can be employed to show that the BlaR protein binds to the promoter of the penicillin biosynthetic gene, pcb C, encoding isopenicillin N synthase.

Amino acids essential to the activity of the BlaR polypeptide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science, 244 (1989): 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e. BlaR activity) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photo-affinity labelling. See, for example, de Vos et al. (Science, 255 (1992): 306- 312), Smith et al. (J. Mol. Biol., 224 (1992): 899-904) and Wlodaver et al. (FEBS Lett, 309 (1992): 59-64). The present invention further comprises a novel polypeptide capable of acting as a pathway-specific regulator for the production of a /Mactam compound in a micro-organism, characterised by an amino acid sequence comprising the sequence of SEQ ID NO 2 or a sequence showing substantial homology thereto. The polypeptide preferably contains a CX₂CX₆CX₁₆CX₂CX₆C zinc-finger, which is an active DNA binding site. The amino acids beside this region determine the specificity of BlaR as a /Mactam transcriptional regulator.

Nucleotide and amino acid sequences which are substantially homologous may show at least 70% homology, preferably at least 80% homology, more preferably at least 90% homology and more preferred at least 95% homology. In a further aspect, the present invention relates to an expression cassette comprising said nucleotide sequence encoding said polypeptide exhibiting BlaR activity.

In the expression cassette, the DNA sequence of the invention encoding a BlaR polypeptide is operably linked to at least one regulatory sequence, e.g. a sequence required for transcription of the DNA and/or a terminator. The term,

"operably linked" indicates that the sequences are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.

The promoter may be any DNA sequence which shows transcriptional activity in a host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Suitable host organisms include fungi, yeasts or bacteria. A preferred host organism is a filamentous fungus, especially a β- lactam producing fungus. An overview of fungal promoters can be found in, for instance, Applied Molecular Genetics of filamentous fungi (Kinghom, Turner (eds.), Blackie, Glasgow, UK, 1992). Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., EMBO J., 4 (1985): 2093-2099,) or the tpif promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable a-amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae those phosphate isomerase, A. nidulans acetamidase, P. chrysogenum ACV synthetase, P. chrysogenum isopenicillin N synthase, P. chrysogenum acyltransferase, P. chrysogenum phosphoglycerate kinase, P. chrysogenum gene Y, P. chrysogenum sulphate permease (sutB), Saccharomyces cerevisiae GAPDH. Preferred are the A. niger glucoamylase or P. chrysogenum promoters.

In an embodiment of the invention, the native promoter of said blaR gene is replaced by a promoter which is differently regulated than said native promoter. In another embodiment of the invention the native promoter of said blaR gene is replaced by the promoter from another gene involved in the biosynthesis of /Mactams.

The DNA sequence of the invention encoding a BlaR polypeptide may also, if necessary, be operably connected to a suitable terminator.

The expression cassette comprising the DNA sequence encoding said polypeptide exhibiting BlaR activity may be incorporated in a recombinant vector or transformation vehicle. The vector into which the expression cassette of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The recombinant vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or one which confers resistance to a drug. Examples of the latter are phleomycin or hygromycin. For filamentous fungi, additional selectable markers include amdS, pyrG, argB, niaD, facA, and sC (Applied Molecular Genetics of Filamentous fungi (ibid.), Biotechnology of Filamentous fungi, Finkelstein, Ball (eds.), Butterworth-Heinemann, Boston, 1992). It is also possible to introduce the expression cassette comprising the DNA sequence of the invention encoding a BlaR polypeptide into a host cell on a DNA fragment separate from the vector comprising a selectable marker, by a so-called co-transformation process. Another aspect of the present invention relates to improvement of the production of a Mactam compound by an organism producing said Mactam compound, by transforming said /Mactam producing organism with an expression cassette comprising the DNA sequence of the invention and selecting transformed cells with an increased production of said /Mactam compound as compared to non- transformed cells. The increase in the /Mactam level of said transformed strain is advantageously due to an increase of the regulatory activity of BlaR.

Still another aspect of the invention concerns the production of /Mactam compounds via fermentation of a /Mactam-producing strain transformed with a an expression cassette comprising the DNA sequence of the invention and, optionally, isolation of the /Mactam compound. In one embodiment of the invention, an increased BlaR activity is used in combination with modifications in biosynthetic routes leading to the production of β- lactam compounds.

In another embodiment of the invention the expression of said regulatory activity is synchronised to the expression of other genes belonging to the /Mactam biosynthetic pathway. Said genes may e.g. be the pαbAB, pcbC and/or penDE genes.

The above mentioned Mactam compound for instance is a penicillin, a cephalosporin or a cephamycin. Preferably said /Mactam compound is a penicillin or a cephalosporin or another compound containing a penam, a cephem, a clavam, a carbapenem or a monobactam nucleus. Advantageously the Mactam compound may be an intermediate for the production of semisynthetic antibiotics. Description of the Figures

FIG. 1 : Schematic representation of the 1016 bp PvuW-Eaή DNA sequence including the blaR ORF.

FIG. 2: The plasmid plPBWA, containing the blaR expression cassette under control of the pcbC promoter

FIG. 3: The plasmid pGPBWA, containing the blaR expression cassette under control of the gpdA promoter

FIG. 4: The plasmid pdelta-jb/aP, with the blaR disruption cassette

EXAMPLE 1 CLONING OF BLAR

A 1016 base pair PvuW-Eaή fragment of the penicillin biosynthetic gene cluster (Barredo et al., supra) was cloned, after Klenow fill-in, into the Smal site of pBluescript. This clone maps to the 8 kb EcoRI-SamHI region on the right site of the penicillin biosynthetic gene cluster as described by Barredo et al. (supra).

Sequencing revealed a single Open Reading Frame of 556 nucleotides, encoding a Zinc-finger protein of 154 amino acids (FIG. 1 and SEQ. ID NO 1 ). A putative intron of 52 base pairs was found at position 880 to position 931.

EXAMPLE 2

CLONING THE CDNA OF BLAR

A cDNA library from RNA isolated from a high producing P. chrysogenum strain (e.g. Panlabs P2) was used to isolate the blaR cDNA. Using the primers (5¹- ccggtcaaaatgccatcgataatttctctg-3' (primer #1 ) and

5'-caatggcgtaagtcgacctatcccaaggagtagcaagg-3' (primer #2); SEQ ID NO 5 and 6, respectively) and Supertaq® polymerase a specific 500 bp fragment was amplified. The library was amplified in E. coli and plated out in 100 pools of approximately 1000 colonies. Using the same primers as above pools which contain the blaR insert were isolated. Finally, one pool was selected and plated out on agar plates. The colonies were transferred to nylon filters and subsequently lysed and hybridised to the 500 bp fragment. A single positive colony was isolated and sequenced (SEQ ID 3). The putative intron was shown to be spliced out from this sequence.

EXAMPLE 3

OVERPRODUCTION OF BLAR

The complete cDNA of blaR was PCR-amplified with High Fidelity Polymerase (Boehringer Mannhein) using the oiigonucleotides:

5'-ccttccgccgaaaccggtattaatgccaaaggaaatttctctgcgg-3' (primer #3); and 5'-gggtcaatggcgttaatgcatctatcccaaggagtagcaaggagg-3' (primer #4), SEQ ID NO 7 and 8, respectively. Sequence analysis was used to ensure the introduction of Ase\ and Λ/s/^'l restriction sites for further cloning procedures. The fragment was cloned under control of the gpdA or pcbC promoter. Therefore, vector plSEWA (supra) was digested with Nde\ and Λ/s/^'l to insert the blaR fragment. This yielded plasmid plPBWA (FIG 2). To insert the constitutive gpdA promoter from Aspergillus nidulans plasmid plSEWA was digested with EcoRI and Λ/s/^'l . The gpdA promoter was amplified from A. nidulans using the oiigonucleotides 5'-cacacttgtcgaattcgagtcctg-3' (primer #5; SEQ ID NO 9) and

5'-ggattgaggcatatggatgtctgctcaagcggg-3' (primer #6; SEQ ID NO 10), introducing EcoRI and Λ/cfel restriction sites. This fragment was inserted in the digested vector together with the blaR fragment, yielding pGPBWA (FIG. 3).

Both expression cassettes were isolated by digesting the plasmids plPBWA and pGPBWA with Λ/o l and transformed to Penicillium chrysogenum using co- transformation with amdS as described in EP-2922. Positive transformants were identified by colony PCR and screened for an increased mRNA level of the blaR transcript and a subsequent increased penicillin titre.

Claims

1. An isolated polynucleotide comprising a polynucleotide fragment encoding at least part of a pathway specific regulator for the production of /Mactam in a micro-organism.

2. An isolated polynucleotide according to claim 1 comprising a polynucleotide fragment encoding at least part of the polypeptide of SEQ ID NO 2.

3. An isolated polynucleotide according to claim 1 comprising at least part of a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO. 1 , a nucleotide sequence which is essentially homologous to SEQ ID NO. 1 and the complement of any of the foregoing nucleotide sequences.

4. An isolated polynucleotide according to claim 1 wherein the micro-organism is a filamentous fungus.

5. An isolated polynucleotide according to claim 4 wherein the filamentous fungus is selected from the group consisting of Penicillium chrysogenum, Acremonium chrysogenum and Aspergillus nidulans.

6. An isolated polynucleotide according to claim 1 wherein the micro-organism is a bacterium.

7. An isolated polynucleotide according to claim 6, wherein the bacterium is an Actinomycete.

8. An isolated polynucleotide according to claim 7 wherein the Actinomycete is selected from the group consisting of Streptomyces clavuligerus, Nocardia lactamdurans, Flavobacterium sp.

9. A recombinant DNA comprising a polynucleotide according to claim 1-8.

10. An expression cassette comprising a polynucleotide according to any of the claims 1-8 operably linked to at least one regulatory sequence.

5

11. A vector or transformation vehicle comprising the expression cassette according to claim 10.

12. A transformed host comprising the expression cassette according to claim 10 o or the vector or transformation vehicle according to claim 11.

13. The transformed host according to claim 12 which is a Mactam producing micro-organism.

5 14. A method to improve the production of a Mactam compound in a Mactam producing organism by transforming said /Mactam-producing organism with an expression cassette according to claim 10 or the vector or transformation vehicle according to claim 11 and selecting transformed cells with an increased production of said Mactam compound as compared to non-transformed cells. 0

15. A method for the production of a /Mactam compound comprising fermenting the transformed host according to claim 13 and, optionally, isolating said β- lactam compound from the fermentation medium.

5 16. A polypeptide capable of acting as a pathway-specific regulator for the production of a /Mactam compound in a micro-organism, characterised by an amino acid sequence comprising at least part of the sequence of SEQ ID NO 2 or a sequence showing substantial homology thereto.