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WO1997038107A1 - Enzyme possedant une forte activite d'adipoyl-coenzyme a synthetase et utilisations de celle-ci - Google Patents

Enzyme possedant une forte activite d'adipoyl-coenzyme a synthetase et utilisations de celle-ci Download PDF

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Publication number
WO1997038107A1
WO1997038107A1 PCT/EP1997/001711 EP9701711W WO9738107A1 WO 1997038107 A1 WO1997038107 A1 WO 1997038107A1 EP 9701711 W EP9701711 W EP 9701711W WO 9738107 A1 WO9738107 A1 WO 9738107A1
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WIPO (PCT)
Prior art keywords
coenzyme
enzyme
adipoyl
acid
ligase
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PCT/EP1997/001711
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English (en)
Inventor
Roelof Ary Lans Bovenberg
Derk Jans Hillenga
Philippus Antonius Deen
Diana Esmeralda Pronk-Kraay Veld
Maarten Nieboer
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Gist-Brocades B.V.
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Application filed by Gist-Brocades B.V. filed Critical Gist-Brocades B.V.
Priority to AU25088/97A priority Critical patent/AU2508897A/en
Publication of WO1997038107A1 publication Critical patent/WO1997038107A1/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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates to a novel enzyme which advantageously can be used in the /?-lactam antibiotic biosynthesis. Further the invention relates to a DNA construct encoding said novel enzyme, a recombinant vector or transformation vehicle comprising said DNA construct, and finally a cell comprising said DNA T O construct or recombinant vector. In addition, the invention relates to processes for producing ?-lactam antibiotics.
  • 1 5 ⁇ -Lactam antibiotics constitute the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum, respectively.
  • the third step involves the exchange of the hydrophilic ⁇ -aminoadipic acid side chain derived from L-5-amino-5-carboxypentanoic acid by a hydrophobic side chain, by the action of the enzyme acyltransferase (AT) .
  • AT acyltransferase
  • the enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody, as has been described in EP-A-04481 80.
  • penicillin V and penicillin G produced by adding phenoxyacetic acid or phenylacetic acid, respectively, during fermentation of P. chrysogenum, thereby replacing the side chains of the natural Mactams with phenoxyacetic acid or phenylacetic acid.
  • carboxyiic acid group of the new side chain has to be activated, since the AT enzyme requires the side chain in an activated form.
  • the activated form is a coenzyme A (CoA) derivative, synthesized by a CoA ligase.
  • CoA coenzyme A
  • the gene encoding an acetyl-coenzyme A synthetase of P. chrysogenum has been characterized by Van Hartingsveldt et al. (WO92/07079), Gouka et al. (Appl. Microbiol. Biotechnol., 38, 514-51 9, 1 993) and Martinez-Bianco et al. (Gene, 1 30, p. 265-270, 1 993).
  • the gene codes for a polypeptide of 669 amino acids, corresponding to a molecular weight of approximately 74,000 Dalton.
  • the enzyme not only accepts acetic acid but also phenylacetic acid as a substrate in the synthesis of the corresponding acyl-coenzyme A ester, although the specificity for phenylacetic acid is very low as compared to the specificity for acetic acid.
  • cephalosporin intermediates like 7-aminodeacetoxycephalosporanic acid (7-ADCA) , 7- aminodeacetylcephalosporanic acid (7-ADAC) or 7-aminocephalosporanic acid (7- ACA)
  • P. chrysogenum strains carrying heterologous cephalosporin biosynthetic genes like the expandase gene of S. clavuligerus or the expandase/hydroxylase gene of A. chrysogenum, are used for the production of the corresponding adipoylcephalosporins by feeding adipic acid to these recombinant strains.
  • Subsequent removal of the adipoyl side chain from the adipoylcephalosporins can be achieved by using an amidase (EP 532341 and Crawford etal., Bio/Technology 1 3, p. 58-62, 1 995).
  • the present invention discloses a novel enzyme which is an acyl-coenzyme A synthetase, more specifically an adipoyl-coenzyme A synthetase. According to the present invention, it has been shown that said acyl- coenzyme A synthetase is able to catalyze the formation of adipoyl-coenzyme A with high efficiency.
  • said acyl-coenzyme A synthetase is able to catalyze the formation of other acyl-coenzyme A compounds, for instance adipoyl-coenzyme A derivatives such as carboxymethylthiopropionyl-coe ⁇ zyme A and trans- ⁇ - hydromuconyl-coenzyme A.
  • adipoyl-coenzyme A derivatives such as carboxymethylthiopropionyl-coe ⁇ zyme A and trans- ⁇ - hydromuconyl-coenzyme A.
  • the enzyme of the invention is at least between 10 and 100 times, preferably 10 3 times and more preferably between 1 0 4 and 10 5 times more active towards adipic acid than towards acetic acid or than towards phenylacetic or phenoxyacetic acid.
  • the enzyme is obtainable from Penici/lium chrysogenum.
  • the novel enzyme can be used in connection with biosynthesis of various Mactam antibiotics.
  • DNA fragment comprising a DNA sequence encoding said novel enzyme exhibiting adipoyl-coenzyme A synthetase activity, and an expression cassette comprising said DNA sequence.
  • Also contemplated according to the invention is a microbial cell comprising said expression cassette or said vector or transformation vehicle.
  • the present invention discloses a novel enzyme which is an acyl-coenzyme A synthetase, more specifically an adipoyl-coenzyme A synthetase.
  • This enzyme will in the following be referred to as "ligase” or "CoA ligase” .
  • ligase or "CoA ligase” .
  • said ligase is able to catalyze the formation of adipoyl-coenzyme A from Mg 2 + , ATP, Co ASH and adipic acid with a high efficiency.
  • said ligase of the invention is able to catalyze the formation of other acyl-coenzyme A compounds than adipoyl-coenzyme A, e.g. the formation of derivatives of adipoyl-coenzyme A, such as carboxymethylthiopropionyl-coenzymeA andtrans- ⁇ -hydromuconyi-coenzymeA, from Mg 2 + , ATP, CoASH and derivatives of adipic acid, such as carboxymethylthiopropionic acid and trans- ⁇ -hydromuconic acid, respectively.
  • the ligase does not show any significant activity towards acetic acid.
  • the CoA ligase of the invention has a high affinity towards adipic acid as a substrate.
  • the ligase of the invention additionally has an affinity towards other important Mactam side chain precursors, such as phenoxyacetic acid and phenylacetic acid, albeit substantially lower than towards adipic acid .
  • the CoA ligase of the invention further has a very low specificity towards acetic acid.
  • the novel enzyme is at least between 1 0 and 1 00 times, preferably 10 3 times and more preferably between 10 4 and 1 0 5 times more active towards adipic acid than towards acetic acid. Further, the enzyme, according to the invention, is at least between 1 0 and
  • the enzyme is unstable in vitro, but addition of ATP, Mg 2 + and ⁇ - mercaptoethanol, dithiothreitol (DTT), ascorbic acid or other reducing agents significantly stabilizes the activity during purification and storage.
  • DTT dithiothreitol
  • enzymes according to the invention include mature proteins or precursor forms thereof and functional fragments thereof which essentially have the activity of the full-length polypeptides.
  • homologues of said enzymes comprise an amino acid sequence exhibiting a degree of identity of at least between 50% and 70%, better between 70% and 80%, even better up to 100%, with the amino acid sequence of the enzyme according to the present invention.
  • the degree of identity may be determined by conventional methods, see for instance: Altshul era/. (Bull. Math. Bio., 48, 603-616, 1 986) and Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA, 89, 10091 5-1091 9, 1 992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 1 0, a gap extension penalty of 1 , and "blosum 62" scoring matrix of Henikoff and Henikoff, supra.
  • the homologue of the enzyme according to the invention may be one encoded by a nucleotide sequence hybridizing with an oligonucleotide probe prepared on the basis of the nucleotide sequence of said enzyme exhibiting ligase activity.
  • Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using standard detection procedures (e.g. PCR technology, Southern blotting) .
  • Homologues of the present polypeptide may have one or more amino acid substitutions, deletions or additions. 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, 95-107, 1 991 ) .
  • conservative substitutions are within the group 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) .
  • 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
  • small amino acids such as glycine, alanine, serine
  • substitutions can be made outside the regions critical to the function of the molecule and still result in an active enzyme.
  • Amino acids essential to the activity of the enzyme of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science, 244, 1 081 -1 085, 1 989) . In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g . ligase 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 photoaffinity labelling. See, for example, de Vos etal. (Science, 5 255, 306-31 2, 1 992), Smith etal. (J. Mol. Biol., 224, 899-904, 1 992) and Wlodaver et al. (FEBS Lett., 309, 59-64, 1 992) .
  • the homologue may be an allelic variant, i.e. an alternative form of a gene that arises through mutation, or an altered enzyme encoded by the mutated gene, but having substantially the same activity as the enzyme of the invention. Hence o mutations can be silent (no change in the encoded enzyme) or may encode enzymes having altered amino acid sequence.
  • the homologue of the present enzyme may also be a species homologue, i.e. an enzyme with a similar activity derived from another species.
  • homologues of the present enzyme are those which are 5 immunologically cross-reactive with antibodies raised against the enzyme of the invention.
  • the ligase is obtainable from bacteria or fungi.
  • the ligase is obtainable from the filamentous fungi Penicillium chrysogenum, Aspergillus nidulans or Acremonium chrysogenum. More preferably, 0 the enzyme is obtainable from Penicillium chrysogenum.
  • the present invention surprisingly shows that ligases partially purified from cell-free extracts obtained from P. chrysogenum fermentations which are fed by different side chain precursors display an acyl-coenzyme A synthetase activity with a different specificity towards different side chain precursors.
  • the CoA ligase of the invention specifically accumulates within the cell when performing a fermentation in the presence of an inducing substance, i.e. the relevant side chain precursor, which is adipic acid, or an analogue thereof.
  • the expression of the ligase of the invention is specifically increased in respect to other CoA ligases with a different substrate specificity.
  • the enzyme can be purified from P. chrysogenum biomass using conventional protein purification techniques.
  • the enzyme can be purified from cell extracts of P. chrysogenum by precipitation with ammonium sulphate and by elution from columns packed with 5 various gels (e.g. Phenylsepharose, Cibacron blue and Sephacryl ® S-200) .
  • various gels e.g. Phenylsepharose, Cibacron blue and Sephacryl ® S-200
  • partial amino acid sequences of the protein from its N-terminus and/or from internal fragments.
  • partial amino acid sequences can be directly determined from protein or peptide bands separated by electrophoresis of a protein or peptide preparation on a denaturing SDS gel (Matsudaira, Methods Enzymol. 1 82, 602-61 3, 1 990).
  • the thus-obtained amino acid sequence data subsequently enable the skilled person to construct oligonucleotide probes to be used for cloning of the corresponding gene or cDNA sequence from an appropriate library.
  • PCR technology can be used to facilitate cloning .
  • a DNA fragment comprising a nucleotide sequence encoding the ligase of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1 989).
  • the DNA fragment comprising the gene or cDNA encoding the ligase of the invention may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683, 202 or Saiki et al. (Science, 239, 487-491 , 1 988) .
  • the DNA fragment comprising the DNA sequence encoding the ligase of the invention is derivable from a filamentous fungus belonging to genus of Aspergillus, Penicillium or Acremonium, preferably from a strain of P. chrysogenum, A. chrysogenum, or A. nidulans, more preferably from a strain of P. chrysogenum.
  • the present invention relates to an expression cassette comprising said DNA fragment containing a gene or cDNA encoding said enzyme exhibiting ligase activity.
  • the DNA sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA.
  • operably linked indicates that the segments 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 the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., EMBO J ., 4, 2093-2099, 1 985) or the rp/A promoter.
  • ADH3 promoter McKnight et al., EMBO J ., 4, 2093-2099, 1 985
  • rp/A promoter examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A.
  • niger ac ⁇ stable ⁇ -amylase A. niger ox A. awamori glucoamylase (gluA), Rhizomucor miehei Wpase, A. oryzae alkaline protease, A. oryzae triose 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. Preferred are the A.
  • niger glucoamylase or P. chrysogenum promoters It is often advantageous to use identical or similar promoters to regulate two or more of the biosynthetic genes in order to obtain a synchronized production of the intermediates involved in the Mactam antibiotic synthesis. If the production of intermediates is not synchronized an accumulation of intermediates (bottle neck) might arise. Consequently the production of Mactam antibiotic may be retained .
  • the promoter of said ligase gene is replaced by the promoter from another gene involved in the biosynthesis of ⁇ - lactams.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters.
  • the DNA fragment encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
  • a targeting signal or a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • the targeting signals or secretory signal sequence are joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory sequences are commonly positioned 5' to the DNA sequence encoding the enzyme, whereas the targeting signal sequences are commonly positioned 3' to the DNA sequence.
  • the targeting signal or secretory signal sequences may be a sequence natively associated with the gene or may be from a gene encoding another protein having the desired signal sequence.
  • the targeting sequence is preferably a targeting signal capable of directing the ligase activity to the desired location within the cell (e.g. to the microbodies) .
  • a peroxisomal targeting signal for the use in filamentous fungi, an example of a peroxisomal targeting signal
  • microbodies in e.g. Neurospora crassa, is described by Keller etal.
  • the expression cassette comprising a DNA fragment containing the gene or cDNA encoding said enzyme exhibiting ligase 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.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • 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, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene, 40, 1 25-1 30, 1 985), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, phleomycin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces pombe TPI gene described by P.R. Russell, Gene, 40, 1 25-1 30, 1 985
  • a drug e.g. ampicillin, kanamycin, tetracyclin, phleomycin, chloramphenicol, neomycin,
  • 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, 1 992) .
  • such improved processes can be provided if the production of ?-lactam antibiotics of interest takes place in the presence of increased ligase activity.
  • such improved process of producing a /9-lactam antibiotic comprises i) fermentation of a microorganism capable of producing said /3-lactam antibiotic, and ii) recovering said Mactam antibiotic in substantially pure form, wherein said fermentation takes place in the presence of increased ligase activity, in comparison to the ligase activity present when fermenting with the original microorganism alone and/or under the original fermenting conditions.
  • An increased ligase activity is defined as an enhanced conversion of the carboxylic acid in question towards the corresponding acyl-coenzyme A ester, in comparison to the unmodified original microorganism and/or the original fermentation conditions.
  • Said original microorganism, capable of producing Mactams, lacks or only has a relatively low ligase activity and/or said original fermentation conditions ⁇ o do not give any or result in only a relatively low ligase expression .
  • the increased expression of ligase activity may be accomplished by any suitable way.
  • the ligase activity may be increased by modulation of the physical conditions of the 15 fermentation process, such as temperature and pH.
  • Another possibility is subjecting the microorganism to compounds or agents, leading to an increased expression of ligase.
  • the nature of said compound or agent depends on e.g . the promoter used for initiating the expression of the ligase.
  • increased expression of ligase 20 activity can be achieved.
  • said ligase activity or increased ligase activity is obtained by modifying said microorganism.
  • the introduction of said expression cassette or recombinant vector into a host cell may the performed according to, for instance, Applied Molecular Genetics of Filamentous fungi (ibid. ) or Biotechnology of Filamentous fungi (ibid. ) .
  • the ligase activity or increased ligase activity is 30 obtained by random or site specific mutagenesis of said microorganism.
  • a modification leading to increased ligase activity may be obtained by amino acid substitutions, deletions or additions of the ligase enzyme.
  • the DNA fragment encoding the ligase of the invention may thereby be derived from a species which is similar to or different from the microorganism in
  • the DNA fragment encoding the ligase of the invention is used to delete its genomic counterpart. This may be important when the ligase is not expressed at the proper location inside the cell. Upon deletion of the gene, it is possible to introduce the DNA fragment encoding the ligase of the invention provided with the appropriate targeting signals.
  • the DNA fragment encoding the ligase of the invention is used to delete a gene encoding an isozyme of said ligase, i.e. a mitochondrial enzyme.
  • new biosynthetic routes may be desirable in an organism.
  • the above mentioned recipient or host microorganism is from the group comprising Penicillium, Cephalosporium, Aspergillus, Nocardia, Streptomyces, Bacillus, Cerospora, Microspora, other Eubacteria , other Actinomycetes or filamentous fungi.
  • said microorganism belongs to species from the group comprising Penicillium chrysogenum, Penicillium notatum, Acremonium chrysogenum, Aspergillus nidulans, Nocardia lactamdurans and Streptomyces clavu/igerus.
  • the expression of said ligase activity is synchronized to the expression of other genes belonging to the Mactam biosynthetic pathway.
  • Said genes may e.g. be the pcbA , pcbC and/or penDE genes.
  • the above mentioned /Mactam antibiotic is selected from the group compris ⁇ ing penicillins, cephalosporins, cephamycins.
  • said antibiotic is a penicillin or a cephalosporin, more preferably an adipoyl-penicillin or an adipoyl-cephalosporin, such as adipoyl-7-ADCA, adipoyl-7- ADAC or adipoyl-7-ACA.
  • the culturing of said microorganism takes place under conditions inducing expression of the DNA sequence encoding the ligase of the invention, thus resulting in an increased production of the coenzyme A thioester of the acid corresponding to the side chain in the desired ⁇ -lactam antibiotic.
  • this enables an increased flux through the step of the biosynthesis of the Mactam in which the side chain is connected to the Mactam nucleus and results in a reduction of the intermediate 6-APA and byproducts like 8-OH penicillic acid .
  • Said inducing culture conditions may be dependent on the promoter which is involved in directing expression of the ligase of the invention.
  • the invention permits the production of the ⁇ -lactam antibiotics of interest, without the production of a significant amount of waste products. Consequently this makes the processes more efficient, due to more energy available to synthesize the products of interest. Further the waste-products may interfere adversely with the biosynthetic pathway.
  • the yield of the Mactam antibiotic of interest is, at any time in the fermentation process, significantly elevated in comparison to fermentation processes with microorganisms lacking or having low activity of said ligase.
  • the yield may be an overall yield or the yield over a certain period of time.
  • ligase activity or just an increase of said ligase activity, in microorganisms lacking or having low ligase activity can make non-enzymatic removal of side-chains of /Mactam antibiotic intermediates superfluous in the production of e.g. penicillins and cephalosporins.
  • an adipoyl-penicillin or an adipoyl- cephalosporin such as adipoyl-7-ADCA or adipoyl-7-ACA is produced entirely, by enzymatic means, in vivo, by inducing the host microorganism to express an increased ligase activity, according to the invention.
  • an acyltransferase e.g. the acyl-coenzyme A:isopenicillin N acyltransferase from P. chrysogenum
  • enzymes able to transform penicillins into cephalosporins e.g. expandase from S. c/avuligerus
  • Acetyl-coenzyme A synthetase (Sigma) Phenoxyacetic acid coenzyme A ester (M.J.Alonso efa/., J . Antibiot., 41 , p. 1 074- 1084, 1 988)
  • Adipic acid coenzyme A ester was prepared as follows: Adipoyl chloride (0.1 4 ml; Aldrich) was added to a well-stirred and ice-water cooled suspension of coenzyme A - Na salt (400 mg; 5.1 52 x 10 "4 mmol; Sigma) in acetone
  • Solution A 10 //I of 0.25 M MgCI 2 , 50 /vl of 0.1 M ATP in 50 mM Tris/HCI, pH 8.0, 50 ⁇ of 20 mM CoASH in 50 mM Tris/HCI, pH 8.0 and 30 ⁇ of 0.2 M phenoxyacetic acid or phenylacetic acid or adipic acid or hexanoic acid in 50 mM Tris/HCI, pH 8.0 mixed and equilibrated at 25°C for 5 minutes.
  • Solution B 100 ⁇ of 0.25 M MgCI 2 , 500 ⁇ of 0.1 M ATP in 50 mM Tris/HCI, 500 l of 20 mM CoASH in 50 mM Tris/HCI, and 300 ⁇ of 0.067M potassium acetate in 50 mM Tris/HCI.
  • the reaction was started by addition of 100 ⁇ of enzyme solution to Solution A containing phenylacetic or phenoxyacetic acid and CoASH. After incubation for 30 minutes at 25°C the reaction was stopped by addition of 240 ⁇ l of 0.5 % trifluoroacetic acid. The precipitate formed was removed by centrifugation and the amount of phenoxyacetic acid coenzyme A ester (or phenylacetic acid coenzyme A ester) formed was analyzed by HPLC, eluent 1 5% (v/v) acetonitrile in 25 mM sodium phosphate buffer, pH 6.5.
  • Phenyl- and phenoxyacetic acid coenzyme A esters were quantitated relative to a standard.
  • the reaction was started by addition of 1 volume of enzyme to 1 .4 volumes of solution A containing adipic acid, adjusted to pH 8.5.
  • a typical incubation mixture consisted in 250 ⁇ enzyme and 350 ⁇ l solution A. Incubation was at 30° C. At regular time intervals samples were withdrawn from the incubation mixture and mixed with an equal part of 0.5 % trifluoroacetic acid . The precipitate formed was removed by centrifugation. The amount of adipoyl-coenzyme A ester formed was analyzed by HPLC, eluent 5% acetonitrile in 25 mM sodium phosphate buffer pH 6.5.
  • Retention time for adipoyl-coenzyme A ester 1 1 .4 minutes.
  • Adipoyl-coenzyme A ester was used as reference. Formation of adipoyl-coenzyme A derivatives was also analyzed in this assay.
  • the reaction was started by addition of 1 volume of enzyme to 1 .4 volumes of solution A containing hexanoic acid, adjusted to pH 8.5.
  • a typical incubation mixture consisted in 250 ⁇ l enzyme and 350 ⁇ l solution A. Incubation was at 30°C. At regular time intervals samples were withdrawn from the incubation mixture and mixed with an equal part of 0.5% trifluoroacetic acid. The precipitate formed was removed by centrifugation. The amount of hexanoyi-coenzyme A ester formed was analyzed by HPLC, eluent 1 5 % acetonitrile in 25 mM sodium phosphate buffer pH 6.5. Retention time for hexanoyi-coenzyme A ester: 20.4 minutes. Direct assay for acetyl-coenzvme A synthetase activity
  • Solution B was mixed and pH was adjusted to 8.0 with 4 M KOH. 40 ⁇ l of acetic acid-1 - 14 C, sodium salt, (41 .8 mCi/mmol, 1 .0 mCi/ml) was added and the mixture was equilibrated at 35 °C.
  • the seed stage was initiated by adding 2 * 10 8 spores to 50mL/500mL flask of medium composed of (g/L) : glucose, 30; (NH 4 ) 2 S0 4 , 10; KH 2 P0 4 , 10; trace element solution I (MgS0 4 .7H 2 0, 25; FeS0 4 .7H 2 0, 10; CuS0 4 .5H 2 0, 0.5; ZnSO 4 .7H 2 0, 2; Na 2 S0 4 , 50; MnS0 4 .H 2 0, 2; CaCI 2 .2H 2 0, 5), 10 (mL/L) (pH before sterilization 6.5).
  • medium composed of (g/L) : glucose, 30; (NH 4 ) 2 S0 4 , 10; KH 2 P0 4 , 10; trace element solution I (MgS0 4 .7H 2 0, 25; FeS0 4 .7H 2 0, 10; CuS0 4 .5H 2 0, 0.5; ZnSO 4
  • the seed culture is incubated for 48-72 hours at 25-30 °C and subsequently used to inoculate 10-20 volumes of a production medium containing (g/L) lactose, 80; maltose, 20; CaS0 4 , 4; urea, 3; MgS0 4 .7H 2 0, 2; KH 2 P0 4 , 7; NaCI, 0.5; NH 4 ) 2 S0 4 , 6; FeS0 4 .7H 2 0, 0.1 ; trace element solution II (CuS0 4 .5H 2 0, 0.5; ZnS0 4 .7H 2 0, 2; MnS0 4 .H 2 0, 2; Na 2 S0 4 , 50), 10 (mL/L); phenylacetic acid or adipic acid, 2.5% (w/v) (pH before sterilization 5.5-6.0) . The incubation is then continued for another 96-1 20 hours.
  • a production medium containing (g/L) lactose, 80; mal
  • Phenyl-Sepharose CL 4B column 1 50 ml
  • Pharmacia LKB PhastSystem Pharmacia Biotech
  • Solutions Solution A 50 mM Tris/HCI, pH 8.5; 35 % ammonium sulphate, 4 mM EDTA,
  • Buffers Buffer A 50 mM Tris/HCI, pH 8.5, 1 .36 M ammonium sulphate, 4 mM EDTA,
  • Buffer B 50 mM Tris/HCI, pH 8.5, 0.68 M ammonium sulphate, 4 mM EDTA,
  • the cells from a 6 days old culture of P. chrysogenum (Example 1 ) were isolated by filtration and quickly washed with 5 volumes of 0.9% sodium chloride. The cells were frozen in liquid nitrogen and homogenized in a mortar. The enzyme was extracted by suspension in Solution A. Cell debris was removed by centrifugation. The extract was brought to 50 % ammonium sulphate saturation and the precipitate was removed by centrifugation. The enzyme could then be precipitated by addition of ammonium sulphate to 70 % saturation. After centrifugation the pellet was dissolved in Solution B. Phenyl-Sepharose ® CL 4B column chromatoqraphy
  • acyl-CoA synthetases from the P. chrysogenum Wisconsin 54-1255 strain cultured with either phenylacetic acid (PA) or adipic acid (AD) (Example 2), resulted in 2 different acyl-CoA synthetase preparates.
  • a third acyl-CoA synthetase preparate was obtained by purification from cells obtained from a phenoxyacetic acid (POA) fermentation (see International patent application WO 96/1 0085) .
  • the obtained partially purified acyl-coenzyme A synthetases were incubated with phenoxyacetic acid, phenylacetic acid, adipic acid, hexanoic acid and acetic acid, using the assay conditions given in the section Experimental.
  • the substrate specificities which were obtained are depicted in Table 1 (relative activities are given) .
  • the enzyme preparation from a cell-free extract obtained by fermentation of P. chrysogenum in the presence of adipic acid displays a large increase in specificity towards adipic acid relative to the specificity towards other precursors.
  • Example 3 an enzyme preparation obtained from mycelium grown in the presence of adipate had activity towards other sidechain precursors as well.
  • An enzyme preparation from adipate-induced mycelium was subjected to further purification, as described below.
  • Buffer A 50 mM Tris/HCI pH 8.5, 1 .36 M ammonium sulfate, 5 mM MgCI 2 , 5 mM ATP, 4 mM EDTA, CompleteTM protease inhibitor ( 1 tablet /
  • Buffer B 50 mM Tris/HCI pH 8.5, 0.68 ammonium sulfate, 5 mM MgCI 2 ,
  • the enzyme was extracted by suspension of lyophilized mycelium in buffer A (1 g / 20 ml) for 45 min on ice. Cell debris was removed by centrifugation (1 3.000 r.p.m. GSA-rotor, 30 min) . The extract was brought to 45 % ammoniumsulfate saturation, precipitated on ice for 45 min, and the precipitate was removed bij centrifugation ( 1 3.000 r.p.m. GSA-rotor, 30 min) . The enzyme could then be precipitated bij addition of ammonium sulfate to 65 % saturation, precipitation on ice for 45 min, and collection of the precipitate by centrifugation ( 1 3.000 r.p.m. GSA-rotor, 30 min).
  • PD-10 buffer 1 0 mM Tris/HCI pH 8.5, 4 mM EDTA, 5 mM MgCI 2 , 20% glycerol,
  • Triton X-100 20 % Triton X-100 was made in PD-10 buffer. 20% Triton X-100 solution was added dropwise to the protein solution, to a final concentration of 1 % Triton X-100 and 1 0 mg/ml protein. Triton X-1 00 extraction was carried out for 1 hr on ice.
  • Buffers Buffer A 5 mM potassium phosphate buffer pH 7.8, 5 Mm MgCI 2 , 4 mM EDTA, 10% glycerol, 5 mM ascorbic acid, 5 mM ATP, CompleteTM
  • Buffer B 200 mM potassium phosphate buffer pH 7.8, 5 Mm MgCI 2 , 4 mM EDTA, 1 0% glycerol, 5 mM ascorbic acid, 5 mM ATP, CompleteTM
  • Buffer A 1 0 mM Tris/HCI pH 7.8, 2 mM MgCI 2 , 20% glycerol, 5 mM ATP,
  • Fractions of 2 ml were collected. Fractions were analyzed for adipoylCoA synthetase activity. Activity was measured in fractions 40-56 ml, showing a peak at 48 ml.
  • a partially purified protein fraction containing adipoyl-CoA synthetase activity was subjected to chromatography on a native gel (Methods in Molecular Biology, Volume 32, p. 1 7-22, Basic Protein and Peptide protocols. J.M.Walker) . Two 7.5 % gels were prepared:
  • Electrophoresis buffer was chilled before use.
  • the Mini-PROTEAN II Electrophoresis System was placed on icewater during electroforesis. A stirrer bar was placed in the System to ensure proper mixing of the chilled water. Electrophoresis was performed at 30 mA until the bromophenol blue reached the bottom of the gel (approximately 2 hr) .
  • the gel was allowed to equilibrate in a buffering solution (see below) .
  • a buffering solution see below
  • an activity staining was carried out, based on the formation of pyrophosphate from ATP during the acivation of adipate. The formation of pyrophosphate results in precipitation with Ca 2 + , appearing as a white band in the gel.
  • the following mixture for activity-staining was prepared in degassed 50 mM Tris/HCI, pH 8.5: 4 mM CoA, 5 mM ATP, 1 5 mM MgCI 2 , 1 0 mM CaCI 2 . Incubation took place for 1 0 min. bij gently shaking (the gel was completely covered with the mixture) . Then, adipate was added to a final concentration of 10 mM, and the gel was further incubated without shaking.
  • the precipitate occurred within 30 min of incubation (no precipitate was visible without adipate) .
  • Adipoyl-CoA ligase activity was clearly present as a single band.
  • the protein had an apparent mobility between 46 and 66 kD, based on the mobility of the marker proteins (Rainbow Marker, Amersham) on a native gel.
  • the gel fragment can be cut out to isolate the enzyme expressing the adipoyl ⁇ CoA synthetase activity and/or can be used for partial amino acid sequence determination.
  • CMTP carboxymethylthiopropionate
  • adipoyl-CoA ligase has a substrate specificity which includes S-derivatives of adipate (CMTP) and other mono- and dicarboxylic acids (saturated or unsaturated) .
  • CMTP S-derivatives of adipate
  • saturated or unsaturated S-derivatives of adipate
  • Table 3 Substrate specificity of adipoyl-CoA synthetase

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Abstract

La présente invention se rapporte à une nouvelle enzyme qui est une adipoyl-coenzyme A synthétase, de même qu'à un fragment d'ADN codant cette nouvelle enzyme, à une cassette d'expression, à un vecteur de recombinaison ou à un véhicule de transformation comprenant ce fragment d'ADN, ainsi qu'enfin à une cellule comprenant la cassette d'expression ou le vecteur de recombinaison. En outre, l'invention concerne l'utilisation de cette nouvelle enzyme dans des procédés de production d'antibiotiques β-lactames.
PCT/EP1997/001711 1996-04-04 1997-04-04 Enzyme possedant une forte activite d'adipoyl-coenzyme a synthetase et utilisations de celle-ci WO1997038107A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071579A3 (fr) * 1999-05-21 2001-03-01 Dsm Nv Regulation de l'organite de l'homeostasie dans une production de cellule
EP2392649A2 (fr) 2008-08-05 2011-12-07 DSM IP Assets B.V. Adipoyl-7-ADCA produisant des souches
CN114686547A (zh) * 2020-12-30 2022-07-01 中国医学科学院药物研究所 一种以双醋瑞因为供体的酶促合成乙酰辅酶a的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005158A1 (fr) * 1991-09-11 1993-03-18 Merck & Co., Inc. Nouveau procede biochimique pour la preparation de 7-adca
WO1993008287A1 (fr) * 1991-10-15 1993-04-29 Merck & Co., Inc. Nouveaux procedes biologiques de preparation de 7-aca et de 7-adac
WO1995004148A1 (fr) * 1993-07-30 1995-02-09 Gist-Brocades B.V. Procede de production efficace de 7-adca par l'intermediaire du 2-(carboxyethylthio)acetyl-7-adca et du 3-(carboxymethylthio)propionyl-7-adca
WO1996010085A1 (fr) * 1994-09-28 1996-04-04 Gist-Brocades B.V. PROCEDE DE PRODUCTION D'ANTIBIOTIQUES A BASE DE β-LACTAMES A L'AIDE DE MICRO-ORGANISMES A ACTIVITE LIGASE ACCRUE

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005158A1 (fr) * 1991-09-11 1993-03-18 Merck & Co., Inc. Nouveau procede biochimique pour la preparation de 7-adca
WO1993008287A1 (fr) * 1991-10-15 1993-04-29 Merck & Co., Inc. Nouveaux procedes biologiques de preparation de 7-aca et de 7-adac
WO1995004148A1 (fr) * 1993-07-30 1995-02-09 Gist-Brocades B.V. Procede de production efficace de 7-adca par l'intermediaire du 2-(carboxyethylthio)acetyl-7-adca et du 3-(carboxymethylthio)propionyl-7-adca
WO1996010085A1 (fr) * 1994-09-28 1996-04-04 Gist-Brocades B.V. PROCEDE DE PRODUCTION D'ANTIBIOTIQUES A BASE DE β-LACTAMES A L'AIDE DE MICRO-ORGANISMES A ACTIVITE LIGASE ACCRUE

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CRAWFORD, L., ET AL.: "PRODUCTION OF CEPHALOSPORIN INTERMEDIATES BY FEEDING ADIPIC ACID TO RECOMBINANT PENICILLIUM CHRYSOGENUM STRIANS EXPRESSING RING EXPANSION ACTIVITY", BIO/TECHNOLOGY, vol. 13, January 1995 (1995-01-01), pages 58 - 62, XP002037169 *
MARTINEZ-BLANCO, H., ET AL.: "ISOLATION AND CHARACTERIZATION OF THE ACETYL-CoA SYNTHETASE FROM PENICILLIUM CHRYSOGENUM", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 8, 15 March 1992 (1992-03-15), pages 5474 - 5481, XP002016601 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000071579A3 (fr) * 1999-05-21 2001-03-01 Dsm Nv Regulation de l'organite de l'homeostasie dans une production de cellule
EP2392649A2 (fr) 2008-08-05 2011-12-07 DSM IP Assets B.V. Adipoyl-7-ADCA produisant des souches
EP2392649A3 (fr) * 2008-08-05 2012-01-11 DSM IP Assets B.V. Adipoyl-7-ADCA produisant des souches
CN114686547A (zh) * 2020-12-30 2022-07-01 中国医学科学院药物研究所 一种以双醋瑞因为供体的酶促合成乙酰辅酶a的方法
CN114686547B (zh) * 2020-12-30 2024-05-14 中国医学科学院药物研究所 一种以双醋瑞因为供体的酶促合成乙酰辅酶a的方法

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