JPH09503907A - Effective method for producing 7-ADCA via 2- (carboxyethylthio) acetyl-7-ADCA and 3- (carboxymethylthio) propionyl-7-ADCA - Google Patents

Abstract

  (57) [Summary] Using a Penicillium chrysogenum transformant that expresses an expansion enzyme together with acyltransferase, 7-amino via 2- (carboxyethylthio) acetyl-7-ADCA and 3- (carboxymethylthio) propionyl-7-ADCA Provided is an efficient method for producing and recovering desacetoxycephalosporanic acid (7-ADCA).

Description

Detailed Description of the Invention       2- (carboxyethylthio) acetyl-7-ADCA and 3-       Via (carboxymethylthio) propionyl-7-ADCA       Effective manufacturing method of 7-ADCA                   Brief Description of the Field of the Invention and Prior Art   The present invention relates to 7-aminodesacetoxycephalosporanic acid (7-ADCA ) For the production and recovery of   β-lactam antibiotics are the most important antibiotics with a long history of clinical use It comprises a group of compounds. Among this group, the prominent ones are penicillins and cef. It is an arosporin. Each of these compounds is a filamentous fungus Penicillium Lisogenum (Penicillium  Chrysogenum) And Acremonium chrysogenum (Acremonium   Chrysogenum) Is produced naturally.   Penicillium chrysogenum and Acremoni as a result of classical strain improvement techniques Antibiotic production levels in Umm Chrysogenum have increased dramatically over the last few decades I am adding. Knowledge of biosynthetic pathways leading to penicillins and cephalosporins With the increasing number of strains and the advent of recombinant DNA technology, improvements in production strains and New means of derivatization in vivo are becoming available .   Most enzymes associated with β-lactam biosynthesis have been identified and corresponding The gene has been cloned and can be found below. Ingolia and Quee ner's paper (biosynthetic pathways and enzymes) (Med. Res. Rev.,9: 245-264 (1989)), and Aharonowitz, Cohen and Martin (Gene cloning) (Ann, Rev. Mi) crobiol.,46: 461-495 (1992)).   The first two steps in the biosynthesis of penicillin in P. chrysogenum consist of three types of amino acids. Acid, L-5-amino-5-carboxypentanoic acid (L-α-aminoadipic acid) (A), L -Cysteine (C) and L-valine (V) are condensed to give the tripeptide LLD-ACV, followed by Cyclization of the lever peptide to form isopenicillin N. This The compound has a typical β-lactam structure.   The third step is the action of L-5- by the action of the enzyme acyltransferase (AT). By replacing the hydrophilic side chain of amino-5-carboxypentanoic acid with a hydrophobic side chain, You. In the industrial method of penicillin G production, the side chain selected is phenyl vinegar. Acid (PA). European Patent Publication No. 0532341 discloses adipate (5-carboxylate). The use of a feedstock of (cypentanoate) is disclosed. Uptake of this substrate , A penicillin derivative having a 5-carboxypentanoyl side chain, namely 5-carboxyl Leads to cipentanoyl-6-aminopenicillanic acid. This uptake is Due to the fact that spherase has a wide range of proven substrate specificities ( Behrens et al. (J. Biol. Chem.,175: 751-809 (1948)); Cole's paper (Proc ess. Biochem.,1: 334-338 (1966)); Ballio et al.'S paper (Nature,185: 97-99 (1960 ))). Mediated by AT, as disclosed in EP 0448180 The enzymatic exchange reaction that occurs occurs within the microbody of the organelle.   Cephalosporins are much more expensive than penicillins. One of the reasons One is that some cephalosporins (eg cephalexin) have many chemistries. This is because it is made from penicillin by selective conversion. Another reason is However, cephalosporin having D-5-amino-5-carboxypentanoyl side chain It is possible to ferment and produce only Especially important in this regard The starting material cephalosporin C is very good for water at any pH. Long, costly isolation that melts and therefore uses cumbersome and expensive column technology Accompany the method. The cephalosporin C thus obtained may be sometime chemically It has to be converted to cephalosporins for therapeutic use by enzymatic conversion. You.   Intermediate 7-ADCA is generally produced by chemical induction of penicillin G. The chemical steps required for the production of 7-ADCA consist of a 5-membered penicillin structure and a 6-membered cephalic ring. Including expanding to the rosporin structure. However, the filamentous bacterium Streptomyces Scrab Ligueras (Streptomyces  clavuligerus) -Derived expandase ) Can perform such ring expansion. Cantwell et al. (Proc. R. Soc . Lond. B.,248: 283-289 (1992)); and European Patent Publication No. 0532341 and European Patent When introducing to P. chrysogenum, as described in Kyushu No. 0540210 , The penicillin ring structure can be converted to a cephalosporin ring structure. This expansion The enzyme is well characterized both biochemically and functionally (European patent publication). No. 0366354), and its corresponding gene.cefBoth physical markers of the E gene (European Patent Publication No. 0233715), DNA sequence andcefP. Chrysogen by E Studies on the transformation of Nam have become clear.   Another source of suitable ring-spreading enzymes is the filamentous bacterium Nocardia lactamdura. (Nocardia  lactamdurans) (Formerly Streptomyces lactamdurans (Streptomyces   lactamdurans)). Biochemical properties of this enzyme and gene D Both NA sequences have been identified (respectively by Cortes et al. (J. Gen. Mic robiol.,133: 3165-3174 (1987)); and Coque et al. (Mol. Gen. Genet.,236 : 453-458 (1993))).   More specifically, European Patent Publication No. 0532341 was found in P. chrysogenum. Feedstock 5-carboxypenta used as a substrate for acyltransferase Using the P. chrysogenum extended enzyme in combination with Noil side chains in vivo It is shown that. This is the formation of 5-carboxypentanoyl-6-APA. Which is converted by the expanding enzyme introduced into the P. chrysogenum strain, This results in carboxypentanoyl-7-ADCA. Finally, 5-carboxype Removal of the tannoyl side chain is shown, giving 7-ADCA as the final product. You. A patent application, European Patent Publication No. 0540210, describes a similar method of making 7-ACA. The method is described, and the 3-methyl side chain of the ADCA ring is added to 3-acetoxy of ACA. It includes the step of converting to a methyl side chain. However, the aforementioned patent application is It does not show an efficient and cheap effective method, which is, first of all, the acyltran. At the same time as the expression of the spherase enzyme, the problem of the timely expression of the expansion enzyme in the cell Because it has not been clarified.   In contrast, the present invention shows that the expansion enzyme and the acyltransferase are co-expressed. The present invention provides an effective method for producing 7-ADCA.   In addition, the use of a novel side chain precursor, 3'-carboxymethylthiopropionic acid, The present invention shows use. This precursor has been described by P. chrysogenum in the corresponding pen Very effectively incorporated into syrin, which is expanded by the subsequent continuous activity of the expansion enzyme. Is done.   Furthermore, until now, the 7-ADCA derivative from the fermentation broth was de-acylated before deacylation. No effective method of recovery has been identified. The present invention is a 7-ADCA derivative An effective solvent extraction method for recovering   The present invention provides a substantially effective overall process for producing 7-ADCA. , Which is not disclosed or proposed in the prior art. It includes a reaction step.   Also, by using the invention and those similar to EP 0540210. Thus, 7-ACA can be produced in all effective steps of this method.                             Brief description of the drawings     Figure 1: Functional map of plasmid pMcTNE.     Figure 2: Restriction map of plasmid pMcTSE.     Figure 3: Restriction map of plasmid pMcTNde.     Figure 4: Restriction map of plasmid pGNETA.     Figure 5: Restriction map of plasmid pGSETA.     Figure 6: Restriction map of plasmid pANETA.     Figure 7: Restriction map of plasmid pASETA.     Figure 8: Nocardia lactam du, similar to the sequence of PCR product 1 (top). LancecefDNA sequence of E (Coque et al., Supra) (bottom).                           A brief description of the sequence listing   SEQ ID NOS: 1 to 13: Streptomyces crabligeras and Nocardia lac Tom DurancecefUsed to construct a P. chrysogenum expression cassette for the E gene Oligonucleotides.   SEQ ID NO: 14: Nocardia lactamduranscefDNA sequence of E (described above Coque et al.).                               Summary of the Invention   Therefore, the present invention provides a 7-aminodesacetoxy cephalosporpoie comprising the following steps: A method for producing and recovering lanic acid (7-ADCA) is provided:   a) Under the transcriptional and translational control of the filamentous fungal expression signal, the enzyme is used to Transforming the N. chrysogenum strain;   b) fermenting the strain in a culture medium and expanding in situ, 2- (carboxyethylen (Luthio) acetyl- and 3- (carboxymethylthio) propionyl-7-ADCA 2- (carboxyethylthio) acetyl- and 3- (carboxyme Tylthio) propionyl-6-aminopenicillanic acid (2- (carboxyethylthio) Acetyl- and 3- (carboxymethylthio) propionyl-6-APA) are obtained. 3'-Carboxymethylthiopropionic acid or a salt thereof suitable for Adding an ester to the culture medium;   c) 2- (carboxyethylthio) acetyl- and 3- (carboxyl) from fermentation broth Recovering (cimethylthio) propionyl-7-ADCA;   d) the 2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) E) deacylating propionyl-7-ADCA; and   e) A step of collecting crystalline 7-ADCA.   Step (e) is preferably a filtration step.   The expression of the expansion enzyme gene is regulated by transcription and translation of each regulatory element of AT-gene. It is preferred that it underlies and is concomitant with expression of the AT gene.   2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propyi Onyl-7-ADCA was dissolved in an organic solvent immiscible with water at a pH below 4.5. By extracting the Roth filtrate and back-extracting it with water at pH 4-10, the fermentation medium It is preferable to collect them from   Furthermore, the AT-gene of P. chrysogenum or A. nidulans (A.nidulans)g pd An expansion enzyme that is functionally linked to the transcriptional and translational regulatory elements of the A gene Recombinant DNA vector containing the encoding DNA, and transformed therewith Host cells are provided.                             Detailed Description of the Invention   The present invention provides a functional P. chrysogenum that expands the penicillin ring structure in vivo. The use of gene constructs and 7-ami, a key intermediate in cephalosporin biosynthesis Forms a derivative of nodesacetoxycephalosporanic acid, 7-ADCA For the combination of the use of novel substrates for biosynthetic enzymes. This derivative is It has a chemical composition to enable effective solvent extraction, resulting in cheap and attractive Provide a recovery method.   In principle, P. chrysogenum transformation is based on PEG-Ca mediated protoplasts. Various DN such as capture of water, electroporation and particle gun technology This can be achieved by the A transfer method and selection of transformants. For example Van de Gene transfer and vector development of n Hondel filamentous fungus (Applied Molecular G enetics of Fungi (edited by Peberdy, Laten, Ogden, Bennet, Cambridge University Press (1991))). Application of primary and non-primary selectable markers has been demonstrated ( Van den Hondel paper mentioned above). Same species (from P. chrysogenum) and different species (P. chrysogenum) Both selectable markers of origin (from Genum) are known.   In the selection of transformants, the presence or absence of vector sequences that are homologous or heterologous Whether it is physically associated with non-selectable DNA in the absence of Regardless, applying different transformant selectable markers is well known. You.   Allogeneic selection markers were used to select for transformants of P. chrysogenum and It is preferable to limit the amount of heterologous DNA introduced into Sogenum. Main selection marker Most preferably, the marker is, for example, A. nidulans or Of other filamentous fungiamdIt can be selectively removed from transformants such as the S gene. (European patent application No. 94201896.1). Selectable markers for P. chrysogenum transformants These favorable features of the product are very useful in the process and method of product registration. , The reason is that antibiotic resistance markers are not included in the process or transmitted to the environment. It will not happen.   In the most preferred embodiment, it can be selectively removed from the strainadmS selection Marker allows for iterative steps of transformation using the same dominant selection over and over again To A P. chrysogenum strain expressing this novel expansion enzyme provides a selectable marker. The characteristic of not having is for rapid development of high-producing strains in the improvement plan of industrial strains. Very important.   Ring-expansion reactions mediated by expansion enzymes include Introduced into P. chrysogenum, such as the Wisconsin 54-1255 strain, It is expressed by the method of. This ring expansion reaction also resulted in improved β-lactam yield. It is also carried out with these mutants. In that case, the medium conditions should be adjusted to obtain effective growth. It will be clear that some improvement is needed.   Further,cefThe E gene is preferably the acyl transfer gene of P. chrysogenum. Transcriptional and translational regulation of regulatory elements of each filamentous fungal gene derived from the proteinase (AT) gene Placed below, therefore expression in the optimal time frame It becomes possible to synchronize with the action of the enzyme itself. These measurements are based on the penicillin molecule Is related to the effectiveness of the ring expansion reaction of.   In addition to the synchronized expression of the genes encoding the expansion enzymes and acyltransferases, In the microbody (acyl transferase is intracellularly localized), It is an inexpensive manufacturing method that co-localization of luciferase and a part of the expansion enzyme in the cell Useful for law development. These preferred embodiments are 7-ADCA for registrars. Significantly contributed to reducing the amount of unacceptable penicillin by-products in the final product. Will give.   In summary, the present invention shows that the activity of the expanding enzyme introduced into P. chrysogenum is synchronized. Expression can contribute to the ring expansion of the penicillin ring. Is shown.   In accordance with the present invention, the β-lactam intermediate, 2- (carboxyethylthio) acetate. Cyl- and 3- (carboxymethylthio) propionyl-7-ADCA are 3-carboxyl. To add ruboxymethylthiopropionic acid or salts or esters thereof Manufactured in P. chrysogenum. Suitable salts are, for example, those Natriu. Are sodium salts or potassium salts. 2- (carboxyethylthio) acetyl- and 3- (carboxyl) Ruboxymethylthio) propionyl-7-ADCA is a simple compound such as Efficient recovery from the medium by solvent extraction:   The broth is filtered and a water immiscible organic solvent is added to the filtrate. Adjust the pH The cephalosporins are extracted from the water layer. Its pH range is less than 4.5 It is necessary that it is between 4 and 1, and more preferably between 2 and 1. This The method separates cephalosporins from many other impurities present in the fermentation broth. Let go. Make a concentrated solution of cephalosporin using a small volume of organic solvent , Thereby reducing the volumetric flow rate. The second possibility is at pH 4 or below To extract the entire broth. The broth is treated with an organic solvent immiscible with water to It is preferable to extract in the range of 1.   It is possible to use any solvent that does not interfere with the cephalosporin molecule. it can. Suitable solvents include, for example, butyl acetate, ethyl acetate, methyl isobutane. Examples include alcohols such as chill ketone and butanol. 1-butanol or iso Preference is given to using butanol.   After this, the cephalosporins are treated with water in a pH range of 4-10, preferably 6-9. Back extract. Also, the final volume is dramatically reduced. The temperature range is from 0 to 50 ℃ And preferably at room temperature.   The cephalosporin aqueous solution thus obtained was treated with an appropriate enzyme to give 2- (cal Boxyethylthio) -acetyl- and 3- (carboxymethylthio) propionyl side Stripping off yields the desired 7-ADCA.   It is preferred that the enzyme can be used repeatedly using an immobilized enzyme. like that Methods for making particles and immobilizing enzymes are broadly disclosed in EP 0222462. Have been. The pH of the aqueous solution has a value in the range of pH 4 to pH 9, for example. Minimize the degradation reaction of farosporin and optimize the desired conversion by the enzyme . Therefore, maintain the enzyme at an appropriate level of pH while adding the enzyme to the aqueous cephalosporin solution. In addition, at this time, the pH is adjusted by adding an inorganic base such as potassium hydroxide solution. Or by the use of a cation exchange resin. When the reaction is complete Then, the immobilized enzyme is recovered by filtration. Another possibility is a fixed bed or flow The use of immobilized enzyme in a moving bed column, or the use of enzyme in solution for membrane fractionation. The product is removed by separation. After that, the reaction mixture is not mixed with water. Acidify in the presence of some organic solvents.   Suitable enzymes include, for example, those derived from Pseudomonas SY77 microorganisms, , 177, 178 and 179 have mutations at one or more positions. Also, other An enzyme derived from Pseudomonas microorganism may be used, and as the microorganism, Pseudomonas SE83, Pseudomonas SY77 Nos. 62, 177, 178 and 179 It is preferable that the site corresponding to the position has a mutation at one or more sites.   After adjusting the pH to about 0.1-1.5, separate the layers and adjust the pH of the aqueous layer to 2-5 . The crystalline 7-ADCA is then filtered.   Deacylation can also be done chemically, as is known in the art, eg For example, add phosphorus pentachloride at a temperature below 10 ° C, then isobutanol below ambient temperature. Via the formation of imino chloride side chains.   The following examples are provided for purposes of illustration and are not intended to be limiting. Yes.                                 Example                                 Example 1 Streptomyces and Nocardia in Penicillium chrysogenumcefE Gene expression a. General gene cloning and gene transformation methods   Conventional techniques used in gene cloning methods are used in this application. These techniques include synthetic enzyme chain reaction (PCR), synthetic oligonucleotide synthesis. , Nucleotide sequence analysis of DNA, enzymatic ligation and cleavage of DNA, Escherichia A coli (belowE. FIG. coli) Vector subcloning, transformation and transformation Body selection, DNA isolation and purification, Southern blot analysis and³²For P-labeled probe DNA specification by Random primer method³²Includes P label. this All of these techniques are well known in the art and have already been described in many references. Have been. For example, Sambrook et al. (Molecular cloning, experimental procedures, goals De Spring Harbor, U.S.A (1989)), Innes et al. (PCR protocol , Guide to Methods and Uses, Academic Publishing (1990)), and McPherson et al. (PCR, Practical Approach, IRL Publishing (1991)).   Common methods used in transformation of filamentous fungi and selection of transformants include Preparation of protoplasts, conditions for DNA transfer and protoplast regeneration, transformants Purification and specification. All of these methods are well known in the art and include: Described in detail below: Finkelstein and Ball (edit), Biochemistry of filamentous fungi, Technology and Methods, Butterworth-Heinemann (1992); Bennett and Lasure (edit), Shin Genetic engineering in fungi, Academic Publishing (1991); Turner's paper, Puhler (edit) ) Biochemistry, Second Edition, Revised, VCH (1992).   Gene cloning and transformation for Penicillium chrysogenum A more detailed application of the technique is described by Bennett and Lasure (supra) and Finkelstein and Details are described in Ball (supra). -Synthetic DNA oligonucleotides should be commercially available DN according to the manufacturer's instructions. A synthesizer (Biosystem, CA, U.S.A.) is used for synthesis. -PCR is a commercially available automatic PCR device (perkin Elmer, U.S.A. and Ultma DNA polymerase (Perkin Elma Company). -hGC PCR protocol (Dutton et al., Nucleic Acids Res.,twenty one, (No. 1 2): 2953-2954 (1993)), N. Lactam Durance and S. Crab Righellas. Of chromosomal DNAcefThe E coding region could be amplified. -Restriction enzymes and other DNA modifying enzymes are manufactured by BRL (MD, U.S.A.) Use according to the instruction manual. A) get from. -All other chemicals used are of analytical grade and obtained from various suppliers. -DNA nucleotide sequence analysis based on detection of sequence-specific fluorescent labels, DN Follow the manufacturer's instructions using an A-sequence automatic analyzer (Biosystem). I will carry out. b. Microbial culture   Streptomyces crabrigellas ATCC 27064 is trypsinized broth Proliferate in (Difco). The chromosomal DNA of this strain iscefIsolation of E gene (Kovacevic et al., J. Bacteriol.,: 754-760 (1989)).   Nocardia Lactam Durance ATCC 27382 is also trypsinized Proliferate in Ross (Difco). The chromosomal DNA of this strain iscefSingle E gene Used for separation (Coque et al., Supra).   Penicillium chrysogenum wisconsin 54-1255 (ATCC 28089) is In YPD complete medium (YPD; yeast extract 1%, peptone 2%, glucose 2%) Grows in. The chromosomal DNA of this strain iscefRequired for E gene expressionpenD Used to isolate the 5'and 3'regulatory regions of the E gene. Penicillium chrysogenum ATC C 28089 alsocefIt is also used as a host for E gene transformation experiments. Another strain of Penicillium chrysogenum, a variant of Wisconsin 54-1255 strain Strains containing variants and having improved β-lactam yields are also suitable. Use According to the selectable marker of the transformant used,pyrG,niaD orfacA remains P. chrysogenum strains containing mutations in the gene may be used. These mutant strains It can be obtained by methods well known in the art (Cantoral, Bio / Techno. l. ，5: 494-497 (1987); Gouka et al., J. Biotechn.,20: 189-200 (1991); and And Gouka et al., Appl. Microbiol. Biotechnol., 514-519 (1993)).   The P. chrysogenum culture for protoplast formation used in transformation is also YP D medium.   The procedure for protoplast formation and regeneration depends on the specific Penicillium chryso It may be slightly different depending on the Genum strain and the transformant selection method applied. This is well known in the art.   E. FIG. coli WK6 (Zell and Fritz ,, EMBO J.,6: 1809-1815 (1987)), XLI-Blue ( Stratagene) and HB101 (Boyer and Roulland-Dussoix, J. Mol. Biol.,41: 459 (1969); Bolivar and Backman, Messages Enzymol.,68: 2 040 (1979)) is the standardE. FIG. coliThe culture medium (Sambrook, cited above) was used to preserve Incubate. c.cefConstruction of E expression cassette   Table I lists the cefE expression cassettes and the name used for these plasmids. The name is also shown.   S. Club RiguerascefE gene (Kovacevic, supra);N. lactamde New LancecefE gene (above Coque article); A. NidulansgpdA gene (Punt et al., Gene69: 49-57 (1988)); P. chrysogenumpenDE gene (Ba rredo et al., Gene83: 291-300 (1989); Diez et al., Mol. Gen. Genet.2 18 : 572-576 (1989)) nucleotide sequence has been published and , Have designed the synthetic oligonucleotides listed in Table II. c1.E. FIG. coli  cefConstruction of E expression plasmids pMCTSE and pMCTNE PCR, 1: N. lactam durancecefE   In the first PCR, the chromosomal DNA of N. lactamdurans and oligonucleosides N. lactam durance using chid 1 and 2cefThe reading frame of E is 5'- End-specificNdeUnique to I restriction site and 3'-endXba0.9k including I site Obtained as the PCR product of b. PCR, 2: S. Crab RiguerascefE   In the second PCR, chromosomal DNA and oligonucleotides of S. crabrigellas S. Club Rigueras using 3 and 4cefThe reading frame of E is at the 5'-end characteristicNdeUnique to I restriction site and 3'-endXba0.9 kb, which also contains I site Was obtained as a PCR product of   E. FIG. coliInsidecefE gene was expressed and PCR was performed by DNA sequence analysis. To characterize the products, PCR products 1 and 2 were used as derivatives of pMC-5 (Stanssens Et al., Nucleic Acids Res.17: 4441 (1989)), the vector pMCTNde I learned. The plasmid pMCTNde contains the RBS site andNdeI cloning site Upstream oftacBy inserting the fragment encoding the promoter Derived from pMC5-8 (European patent application No. 0351029) (Fig. 3).   PCR products 1 and 2NdeI andXbaCut with I,NdeI-Xba I was ligated into the cut vector pMCTNde. Using the ligation mixture,E. FIG. coli  WK6 was transformed. Transformants develop resistance to chloramphenicol Selected more. Plasmid DNA was isolated using these transformants. ThecefInsertion of the E expression cassette The expected occurrence was first analyzed. Ultimately, the Lasmids are analyzed by an automatic DNA sequence analyzer.   S. crabligeras in the plasmid pMCTSE (Fig. 2)cefDN of E reading frame The A sequence was 100% identical to the known sequence (Kovacevic, supra).   N. lactam durancecefD of all clones analyzed, including the open reading frame of E The NA sequence (Fig. 8) was different from the known sequence (said Coque article).   Induced known N. lactam durancecefThe amino acid sequence of the E gene is It has a proline at position 41 of the no acid (see SEQ ID NO: 14). This proline is It disappears in the clone obtained by PCR1. This plasmid is called pMCTNE (Fig. 1). c2. P. chrysogenumcefConstruction of E expression plasmid   PCR, 3:gpdA promoter   In this third PCR, pAN7-1 plasmid DNA (Punt et al., Gene,56: 1 17-124 (1987)), which was used by A. Nidulans.gpdRegulation of A promoter BelowE. FIG. coli  hphContains genes and oligonucleotides 5 and 6 ,thisgpdA promoter is unique to the 5'-endEcoRI restriction site and 3'- End-specificNdeObtained as a 0.9 kb PCR product containing the I site.   PCR, 4: AT promoter   In the fourth PCR, chromosomal DNA of P. chrysogenum and oligonucleotide 7 And 8 were used to obtain a 1.5 kb AT promoter fragment, which was The fragment also has a unique 5'-end.EcoUnique to RI restriction site and 3'-end ofNdeIt had an I site.   PCR, 5: AT terminator and N. lactam durancecefE gene 3'-end   In the fifth PCR, the chromosomal DNA of P. chrysogenum and the oligonucleotides respectively With octides 9 and 10, 11 and 10penDE (AT) terminator I got the area. Therefore, these PCR products are With or withoutcefThis signal contains the 3'-terminal sequence of the E gene. Is the C-terminal amino acid sequence ARL (Muller et al., Biochimica et Biophysica.  Acta1116: 210-213 (1992)).   The oligonucleotides are unique to the 5'-end of the PCR product.BspEI department A position unique to the 3'-end of the PCR product.SpeTo introduce the I site Designed.   PCR, 6: AT terminator and S. crabrigellascef3'-end of E gene end   In the sixth PCR, the chromosomal DNA of P. chrysogenum and the oligonucleotides respectively Using octides 12 and 10, 13 and 10penDE (AT) terminator I got the area. Therefore, these PCR products are S. club Rigueras, with or withoutcefIncluding the 3'-terminal sequence of the E gene This signal is due to the C-terminal amino acid sequence SKL (De Hoop et al., Biochem. . J.286: 657-669 (1992)).   The oligonucleotide is unique to the 5'-end of the PCR product.BglI site Introduced at the 3'-end of the PCR productSpeDesigned to introduce I site Have been.   In P. chrysogenumcefTo express the E gene, thegpdA Promo From the plasmids pMCTNE and pMCTSE. ofcefLigated to the E fragment. These ligated fragments are It was cloned by placing it in pBluescript II KS.   PCR3EcoRI andNdeI cut. pMCTNE and pMCTSE areNde I andXbaI cut. Agarose gel electrophoresis of restriction fragments Separated. 0.9kbcefPurification of E-coding fragment from agarose gel did.EcoRI-NdeThe I promoter fragment isNdeI-XbaIcef With E fragmentEcoRI-XbaVector pBluescr cleaved from I bound in ipt II KS. The following plasmids were thus obtained: pGSE and pGNE. .   In P. chrysogenumcefIn order to obtain the optimal expression of the E gene, the above-mentioned penicill The AT termination signal sequence was cloned downstream of the cefE gene of the Um expression plasmid. Selected to train.   pNETA-pGNEWA   PCR5 productBspEI andSpeCut with I,BspEI andSpe I was ligated into the cut vector pGNE. Using the ligation mixture,E. FIG. coli HB1 01 was transformed. Transformants were selected by their resistance to ampicillin. Plasmids isolated from these transformants were subjected to restriction fragment analysis and subsequent analysis. Was characterized by DNA sequence analysis. As a result, the following plasmid was obtained: pGNEWA And pGNETA (Fig. 4).   pGSETA-pGSEWA   PCR6 productBglI andSpeCut with I,BglI andSpeI It was ligated into the cut vector pGSE. Using the ligation mixture,E. FIG. coli HB101 Transformed. Transformants were selected by their resistance to ampicillin. this The plasmids isolated from the transformants were subjected to restriction fragment analysis and then D Characterized by NA sequence analysis. As a result, the following plasmid was obtained: pGSEWA and pGSETA (Fig. 5).   pANETA, pANEWA and pASETA and pASEWA   Plasmids pGNETA, pGNEWA, pGSETA and pGSEWAEcoRI andNdeI Cut. The restriction fragments were separated by agarose gel electrophoresis and , A 4.5 kb fragment was purified.   PCR4 productEcoRI andNdeCut with I and purify the above fragment Connected with the event.E. FIG. coli After transformation with the ligation mixture into HB101, the Transformants were selected from ampicillin resistance.   The transformants are propagated, their plasmids isolated, restriction fragment analysis and It was characterized by final DNA sequence analysis. As a result, the desired structure, namely pA NETA (Fig. 6), pANEWA, pASETA (Fig. 7) and pASEWA were obtained. d. Transformation of P. chrysogenum   Ca-PEG mediated protoplast transform ation procedure) is used.   Cantoralno paper (see above), Gouka et al. Paper (see J. Biotechn. Above) and According to the method described in the article by Gouka et al. (See Appl. Microbiol. Biotechnol. Above). Whole plasmid or purifiedcefE expression cassette (E. FIG. coliVector sequence Respectively) as selection markers usingpyrG,niaD,facA as well asamdS (Beri et al., Curr. Genet.11 : 639-641 (1987)) gene The P. chrysogenum strain was transformed.   E. FIG. coliA purified form of the homologous form without the vector sequencepyrG,niaD as well asfacBy using the A selection marker, a form without a bacterial resistance gene A quality-converted P. chrysogenum strain was obtained.   European Patent Application No. 94201896.1 describes recombinant strains that do not contain a selectable marker gene. It describes how to obtain it. This method uses A. HurranceamdIt was used successfully with P. chrysogenum transformants containing the S gene.   So the only element that is heterogeneous is the 0.9kbcefE-code area, optionally , 0.9kbgpdA promoter region. e. Analysis of transformants   The P. chrysogenum transformant is purified by repeatedly culturing it in a selective medium. single Using stable colonies, make agar slopes to allow sporulationcefE-expressing cassette The transformant containing the recombinant was screened. From transformants on agar plates The fresh mycelium fragment obtained was boiled andcefE inheritance Enough template DN to efficiently screen hundreds of transformants with offspring I got A (Seth's paper, Fungal Genetic Society, Asilomar (1991), Fungal Gene News Letter summary,38: 55). From this, the efficiency of transformation was evaluated.   Selection of transformants was also performed using a bioassay. Select transformants Were grown on agar containing side chain precursors.E. FIG. coli ESS2231 is on the agar surface Used as an indicator bacterium, this surface also contains bactopenase. And distinguish penicillin and cephalosporin production by methods well known in the art. This is described, for example, in the paper by Guttierez et al., Mol. Gen. Genet.225 : 56-64 (1991)).   Spores were used to inoculate P. chrysogenum culture medium as described in section d. 72 After culturing for a time (under 25 ° C.), chromosomal DNA was isolated from the mycelium. The DNA ToEcoI orPstA 6 bp recognition sequence such as I was cut with a restriction enzyme. .   The DNA fragments are separated by agarose gel electrophoresis and Copy on a lean nylon membrane (New England Nuclear Company). Southern In the Brod method,cefAs a probe of E gene sequence,³²PCR labeled with P2 Hybridize with product. Of the purified PCR2 product³²P-labels are commercially available Α using the labeling kit (Boehringer Mannheim)³²In the presence of P dCTP, Random primer labeling was used.   ThecefThe transformant containing the E coding sequence iscefTest expression of E gene product Tested, and here refers to extended enzyme activity.   The selected transformant is cultured in a penicillin production medium (see Example 2).   For time course experiments, mycelium samples are taken after fermenting for 48, 72 and 96 hours. Fungus A filamentous extract was prepared and the paper of Rollins et al. (Can. J. Microbiol.34: 1196-1202 (1 988)) and measured the expanded enzyme activity substantially in the crude extract. I do. Transformants with expanded enzymatic activity are described in Alvarez et al. (Antimicrob. Ag. ent Chem.31: 1675-1682 (1987)). Measure the enzyme activity.   From these analyses, different levels of acyltransferase and Transformants with enzymatic activity are selected for fermentative production of 7-ADCA derivatives. You.                                 Example 2 2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propio Fermentative production of nil-7-ADCA and its isolation   P. chrysogenum strain Wisconsin 54-1255 (ATCC 28089) is described in Example 1. Transformed with one of the DNA constructs and seeded in 2 * 10⁶ Planted with conidia / ml Wearing. At this time, the seeding medium consisted of (g / l): glucose, 30; (NH_Four)₂SO_Four , 10 ； KH₂PO_Four, 10; Trace element solution I (MgSO_Four・ 7H₂O, 25; FeSO_Four・ 7H₂O, 10; CuS O_Four・ 5H₂O, 0.5; ZnSO_Four・ 7H₂O, 2; Na₂SO_Four, 50; MnSO_Four・ H₂O, 2; CaCl₂・ 2H₂O , 5), 10 (ml / l) (pH before sterilization is 6.5).   The seeding medium was incubated for 48-72 hours at 25-30 ° C, followed by And inoculate 10-20 volumes of production medium. At this time, the medium should contain (g / l) Tose, 80; Maltose, 20; CaSO_Four, 4; urea, 3; MgSO_Four・ 7H₂O, 2; KH₂PO_Four , 7; NaCl, 0.5; (NH_Four)₂SO_Four, 6; FeSO_Four・ 7H₂O, 0.1; 3'-carboxymethylthio Propionic acid, 5; Trace element solution II (CuSO_Four・ 5H₂O, 0.5; ZnSO_Four・ 7H₂O, 2; MnS O_Four・ H₂O, 2; Na₂SO_Four, 50), 10 (ml / l) (pH before sterilization is 5.5-6.5). Then, Incubate for 96-120 hours.   At the end of the production fermentation, the mycelium is removed by centrifugation or filtration and 2- ( Ciethylthio) -acetyl- and 3- (carboxymethylthio) propionyl-7- Analysis of ADCA by high performance liquid chromatography (HPLC) on a reverse phase column I do. The HPLC equipment used is a Beckman System Gold company Gram capable solvent system (model 126), autosampler (model 507), dio Door Array Detector (Model 168) and System Gold Data System (5.10 ) Consists of. Chromspher C18 cartridge column (10 Use 0x3mm, 2 Chrompacks. Mobile phase is 0.07M phosphoric acid 100% buffer solution (pH 4.8), 25% acetonitrile and phosphate buffer solution (pH 4.8) Consisting of a linear gradient of up to 75%, flow rate 0.5 ml / min, 15 min. 2- ( Ruboxyethylthio) acetyl- and 3- (carboxymethylthio) propionyl- The amount of 7-ADCA produced was determined using the synthetic 2- (carboxyethylthio) acetate as a reference substance. With cetyl- and 3- (carboxymethylthio) propionyl-7-ADCA , 260 nm.   The peaks are identified by online comparison of UV and NMR spectra. .   After filtering the broth, about 0.1 volume of 1-butanol is added to the filtrate. The pH value Was adjusted to 2 with dilute hydrochloric acid, and the mixture was stirred at room temperature for 5 minutes. Yes after separating The engineered layer is evaporated and then used for chemical deacylation (Example 3), or Back-extract with 0.33 volume of water (pH 8) and use for further enzymatic deacylation ( Perform either of Examples 4 and 5).                                 Example 3 2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propio Deacylation of nil-7-ADCA   2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propyi Onyl-7-ADCA, 3 g (8 mmol), N, N-dimethylaniline 3.5 ml (36 mmol ), 13 ml of methylene chloride, and 2.6 ml (21 mmol) of trimethylchlorosilane at room temperature. Add to form a mixed solution. After stirring for 30 minutes, the reaction mixture was cooled to about -50 ° C, Immediately, 1.8 g (8.5 mmol) of phosphorus pentachloride are added. Maintain the temperature at -40 ℃ for 2 hours And subsequently the reaction mixture is cooled to -65 ° C. Next, do not raise the temperature above -40 ° C. Treat the mixture with 12 ml (137 mmol) isobutanol at such a rate. 2 more After stirring for 15 hours, 15 ml of water was poured into the solution, and immediately thereafter, 5 ml of 4.5N ammonia was added. Add. The pH was adjusted by slowly adding solid ammonium bicarbonate. Adjust to 4. After cooling to 5 ° C, the mixture was filtered to remove crystalline 7-ADCA. Isolate by washing with water and 5 ml of acetone (1: 1).                                 Example 4 2- (Carboxyethylthio) using a mutant of Pseudomonas SY77 acylase Acetyl- and 3- (carboxymethylthio) propionyl-7-ADCA enzyme Deacylation   2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propyi The conversion of onyl-7-ADCA uses a specific acylase to produce a single enzymatic step. Conduct on the floor. At this time, the acylase passes through the mutagenizing region. It is derived from Pseudomonas SY77 acylase. 2- (Carboxyethylthio) acetyl Improved activity towards ru- and 3- (carboxymethylthio) propionyl side chains For the construction and identification of the mutated Pseudomonas SY77 acylase It is disclosed in Kai 0453048. In the mutant, Pseudomonas SY77 Asila The tyrosine at position 178 of the α-subunit of the enzyme is replaced with histidine. You. The mutant acylase isE. coliProduced in. Cells are harvested by centrifugation Collect and resuspend in 10 mM phosphate buffer (pH 7.4) containing 140 mM NaCl. continue, The cells are disrupted by sonication. Acylase activity after removing cell debris Collect the supernatant containing. Furthermore, the purification of the acylase was performed by the following series of chromatographic The process is as follows: (1) Ion exchange with Q-sepharose high-speed flow at pH 8.8 Chromatography; (2) Hydrophobic bond chromatography with phenyl-Sepharose Ruffy; and (3) Gel-permeation chromatography on Sephacryl S200HR column. It's Ruffy.   Immobilize purified acylase on particles composed of a mixture of gelatin and chitosan Become The particles are treated with glutaraldehyde just before adding the enzyme.   2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propyi Onyl-7-ADCA conversion is carried out in a stirred tank reactor. First, Cephalos An aqueous porin solution is added to the reactor. Subsequently, the temperature of the solution is brought to 30 ℃ and adjust the pH to 8 with potassium hydroxide. Next, add the immobilized enzyme, Start conversion. The pH in the reactor is continuously recorded and maintained at 8 during conversion . The 3'-carboxymethylthiopropionic acid liberated during the reaction is titrated with KOH. You. Show the total amount of KOH added and record with a flatbed recorder. Inversion is the Reac A sample from the monitor and monitored at this time Tylthio) acetyl- and 3- (carboxymethylthio) propionyl-7-ADC A And 7-ADCA are analyzed by HPLC as described in Example 2.   When the reaction was completed, the immobilized enzyme was filtered off and the filtrate was butyl acetate. The pH of the filtrate is adjusted to 1 while containing. Separate the layers to separate the aqueous layer containing 7-ADCA Adjust pH to 3. The crystalline 7-ADCA is then filtered off.                                 Example 5 2- (Carboxyethylthio) acetyl using Pseudomonas SE83 acylase -And 3- (Carboxymethylthio) propionyl-7-ADCA enzymatic deacylation Conversion   2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) propyi Conversion of onyl-7-ADCA is carried out as in Example 4, but with the acylase The same results were obtained using Pseudomonas SE83.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI C12R 1:82) (C12P 35/00 C12R 1:82) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), BR, CA, CN, CZ, HU, JP, KR, NO, PL, RO, RU , SK, US (72) Inventor Kukuman Berthus Peter Holland Enuel-2636 Behässitz Pryden Prunesstraat 8 (72) Inventor Fukema Andreas Netherlands Enuel-2341 Carix Woofsjoest Warmonderwech 66 (72) Inventor van der Laan Jan Metzke Netherlands Enuel 4839 BP Blader Reul Severn 364 (72) Inventor Verway Jan The Netherlands Enuel-2313 AES Leiden Paje Brockstraat 13 (72) Inventor De Frome Eric Netherlands Enuel-2313 JEM Leiden De Mayfantstreff Kerkstraat 65

Claims (1)

[Claims] 1. 7-aminodesacetoxy cephalosporanic acid (having the following steps: 7-ADCA) production and recovery method:     a) Using the expansion enzyme gene under the transcriptional and translational control of filamentous fungal expression signals Transforming a Penicillium chrysogenum strain;   b) fermenting the strain in a culture medium and expanding in situ, 2- (carboxyethylen (Luthio) acetyl- and 3- (carboxymethylthio) propionyl-7-ADCA 2- (carboxyethylthio) acetyl- and 3- (carboxy Methylthio) propionyl-6-aminopenicillanic acid (2- (carboxyethylthio O) acetyl- and 3- (carboxymethylthio) propionyl-6-APA) 3'-carboxymethylthiopropionic acid or a salt thereof, which is suitable for Adding an ester to the culture medium;   c) 2- (carboxyethylthio) acetyl- and 3- (carboxyl) from fermentation broth Recovering (cimethylthio) propionyl-7-ADCA;   d) the 2- (carboxyethylthio) acetyl- and 3- (carboxymethylthio) E) deacylating propionyl-7-ADCA; and   e) a step of recovering crystalline 7-ADCA. 2. The Penicillium chrysogenum strain is characterized by transcription of AT gene expression signals and The transformant according to claim 1, which is transformed with an expanding enzyme gene under translational control. Method. 3. The method according to claim 1 or 2, wherein step (e) is a filtration step. 4. The step (c) is a filtration step, the pH is about 4.5 or less, and the organic solvent is immiscible with water. By extracting the broth filtrate with a medium and back-extracting it with water having a pH of 4-10 The method according to any one of claims 1 to 3. 5. The expansion enzyme gene is Streptomyces crabligeras or Nocardia lac The method according to any one of claims 1 to 4, which is derived from Tamdurans. 6. Expanded fermentation functionally linked to transcriptional and translational regulation of filamentous fungal expression signals A recombinant DNA vector containing DNA encoding the element. 7. The DNA encoding the expansion enzyme is responsible for the transcription and translation of AT gene expression signals. 7. A recombinant DNA vector according to claim 6, which is operatively associated with a node. 8. The DNA encoding the expansion enzyme is Streptomyces crabligeras or The recombinant DN according to claim 6 or 7, which is derived from Cardia lactamdurans. A vector. 9. Transformed with the vector according to any one of claims 6, 7 or 8 Host cells.