RU2139350C1 - Method of effective production of 7-aminodeacetoxycephalosporanic acid through 3-(carboxyethylthio)-propionyl-7-amino- -deacetoxycephalosporanic acid - Google Patents

Abstract

FIELD: biotechnology, antibiotics. SUBSTANCE: invention presents the completed effective process for production and isolation of 7-aminodeacetoxycephalosporanic acid through 3-(carboxyethylthio)-propionyl- -7-aminodeacetoxycephalosporanic acid using the transformant strain of Penicillium chrysogenum expressing expandase in combination with acyltransferase. Method involves the use of a 3,3-thiodipropionic acid as a precursor of by-side chain moiety. 7-Aminodeacetoxycephalosporanic acid is an intermediate compound in production of cephalosporines. EFFECT: increased productivity of strains. 5 cl, 8 dwg, 2 tbl, 5 ex

Description

The scope of the invention and a brief description of the prior art
This invention relates to a biosynthesis process for the preparation and isolation of 7-aminodetacetoxycephalosporanic acid (7-ADCA).

 Betalactam antibiotics form the most important group of antibiotic compounds with a long history of clinical use. Penicillins and cephalosporins are released in this group. These substances are naturally produced by the filamentous fungi Penicillium chrysogenum and Acreonium chrysogenum, respectively.

 As a result of the application of methods for improving the classical strain, the production levels of the antibiotics Penicillinum chrysogenum and Acreonium chrysogenum are markedly increased over the past decades. With an increase in knowledge of the metabolic pathways of biosynthesis leading to the production of penicillins and cephalospirins and the advent of genetic engineering, new means have become available to increase the productivity of strains and to obtain derivatives of compounds in vivo.

 Most of the enzymes involved in the biosynthesis of betalactams have already been identified and their corresponding genes have been cloned, as can be found in Ingolia and Queener, Med. Res. Rev. 9 (1989), 245-264 (biosynthesis pathway and enzymes), and Aharonowitz, Cohen, and Martin, Ann. Rev. Microbiol., 46 (1992), 461-495 (gene cloning).

 The first two steps in the biosynthesis of penicillin in P. chrysogenum are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-α-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) in LLD-ACV tripeptide, followed by cyclization of the tripeptide to form isopenicillin N. This compound contains a typical betalactam structure.

 The third step involves replacing the hydrophilic side chain of 1-5-amino-5-carboxypentanoic acid with a hydrophobic side chain by exposure to the enzyme acetyltransferase (AT). In an industrial process for the production of penicillin G, the side chain of choice is phenylacetic acid (FU). EP-A-0532341 discloses the use as a raw material for the production of adipate (5-carboxypentanoate). The inclusion of this substrate leads to the formation of a penicillin derivative with a 5-carboxypentanoyl side chain, namely 5-carboxypentanoyl-6-aminopenicillanic acid. This inclusion is due to the fact that acetyltransferase has proven broad substrate specificity (Behrens et al., J. Biol. Chem. 175 (1948), 751 - 809; Cole, Process. Biochem. 1 (1966), 334 - 338; Ballio et al. Nature 185 (1960), 97-99). An enzymatic exchange reaction mediated by AT occurs within the cell organelle, the micro-body, as described in EP-A-0448180.

 Cephalosporins are significantly more expensive than penicillins. One of the reasons for this is that some cephalosporins (e.g. cephalexin) are obtained from penicillin through a series of chemical transformations. Another reason is that so far only cephalosporins with the D-5-amino-5-carboxypentanoyl side chain cephalosporin C can be fermented, undoubtedly the most important starting material in this regard, it is very soluble in water at any pH, which implies long and expensive extraction processes using bulky and expensive column technology. The cephalosporin C obtained in this way must be converted into therapeutically applicable cephalosporins using a series of chemical and enzymatic transformations.

 Intermediate 7-ADCA is currently being produced by chemically preparing penicillin G derivatives. The necessary steps in the chemical process for producing 7-ADCA include increasing the 5-membered structure of the penicillin ring to the 6-membered structure of the cephalosporin ring. However, the enzyme expandase from the filamentous bacterium Streptomyces clavuligerus can carry out similar ring enlargements. When introduced into P. chrysogenum, it can convert the penicillin ring structure to a cephalosporin ring structure, as described by Cantwell et al., Proc. R. Sos. Lond. in 248 (1992), 283-289 and in EP-A-0532341 and EP-A-0540210. The expandase enzyme is well characterized (EP-A-0366354) both biochemically and functionally, as well as its corresponding gene. Physical maps of the cefE gene (EP-A-0233715), a DNA sequence, and studies of P. chrysogenum transformation using cefE have been described.

 Another source of a suitable ring expansion enzyme is the filamentous bacterium Nocardia lactamdurans (originally Streptomices lactamdurans). Both the biochemical properties of this enzyme and the DNA sequence of the gene are described (Cortes, et al., J. Gen. Microbiol. 133 (1987), 3165 - 3174; and Coque et al., Mol. Gen. Genet. 236 (1993) , 453 - 458, respectively).

 More specifically, EP-A-0532341 discloses the use of the in vivo expandase enzyme in P. chrysogenum in combination with the 5-carboxypentanoyl side chain as a feed source, which is used as a substrate for the acetyltransferase enzyme in P. chrysogenum. This leads to the formation of 5-carboxypentanoyl-6-APA, which is converted by the expandase enzyme introduced into the P. chrysogenum strain to give 5-carboxypentanoyl-7-ADCA. At the end, removal of the 5-carboxypentanoyl side chain is expected to give 7-ADCA as the final product. Patent application EP-A-0540210 describes a similar process for the preparation of 7-ADCA, including the additional steps of converting the 3-methyl side chain of the ADCC ring to the 3-acetoxymethyl side chain of the ACC. However, the above patent applications do not disclose an effective and cost-effective process, because the problem of timely expression of the expandase enzyme in the cell, concomitant expression of the acetyltransferase enzyme, has not been solved in the first place.

 In contrast, this invention provides an efficient method for the production of 7-ADCA, in which expandase and acetyltransferase are expressed simultaneously.

 In addition, the use of a new side chain precursor, namely, 3,3'-thiodipropionic acid, is also disclosed in this invention. This precursor is very efficiently incorporated by P. chrysogenum into the corresponding penicillins, the ring of which is expanded by the subsequent action of the expandase enzyme.

 In addition to this, an effective method for isolating the 7-ADCA derivative from the fermentation medium before its deacylation has not yet been described. This invention provides an effective solvent extraction procedure for isolating the 7-ADCA derivative.

 Using this invention provides an effective process for producing 7-ADCC, including reaction steps not disclosed and not proposed in the prototypes so far.

 Also, by applying the present invention and similarly to the description given in EP-A-0540210, by the efficiently completed process, in this way, 7-ACC can be obtained.

Brief description of the drawings
Fig. 1: Functional map of plasmid pMcTNE
Fig. 2: Functional map of plasmid pMcTSE
Fig. 3: Functional map of plasmid pMcTNde
Fig. 4: Functional map of the plasmid pGNETA
Fig. 5: Functional map of plasmid pGNETA
Fig. 6: Functional map of the plasmid pANETA
Fig. 7: Functional map of plasmid pASETA
Fig. 8: Nocardia lactamdurans cefE DNA sequence (Coque et al. Above) (bottom lines) compared to PCR product sequence 1 (top lines).

Summary of Sequence Listing
Sequences of NN IDs 1 through 13: oligonucleotides used in the construction of the P. chrysogenum expression cassette for the cefE genes Streptomyces clavuligerus and Nocardia lactamdurans.

 ID 14 sequence: cefE DNA sequence of Nocardia lactamdurans (Coque et al., Supra).

SUMMARY OF THE INVENTION
The present invention thus provides a process for the preparation and isolation of 7-aminodetacetoxycephalosporanic acid (7-ADCA) by:
a) transformation of the Penicillium chrysogenum strain using the expandase gene under transcriptional and translational regulation of filamentous fungus expression signals;
b) fermenting said strain in a culture medium and adding 3,3'-thiodipropionic acid or a salt or ester thereof to said culture medium suitable for the preparation of 3- (carboxyethylthio) propionyl-6-aminopenicillanic acid (3- (carboxyethylthio) propionyl-6 -APA), the ring of which in situ expands to form 3- (carboxyethylthio) propionyl-7-ADCC;
c) isolating 3- (carboxyethylthio) propionyl-7-ADCA from the fermentation broth;
d) deacetylation of said 3- (carboxyethylthio) propionyl-7-ADC; and
e) isolation of crystalline 7-ADCC.

 Preferably, step (e) is a filtration step.

 Preferably, the expression of the expandase gene is under the transcriptional and translational regulation of the corresponding control elements of the AT gene, which ensures simultaneous coordination of the expression of these genes.

 Preferably, 3- (carboxyethylthio) propionyl-7-ADCA is isolated from the liquid fermentation medium by extracting the filtrate of the medium with an organic solvent immiscible with water at a pH below about 4.5 and back extraction of the same substances with water at a pH of from 4 to 10.

 In addition, a recombinant DNA vector containing DNA encoding an expandase functionally linked to transcriptional and translational control elements of the P. chrysogenum AT gene or A. nidulans gpdA gene, and host cells transformed with such DNA are provided.

DETAILED DESCRIPTION OF THE INVENTION
This invention relates to the use of functional gene constructs in P. chrysogenum to expand the in vivo structure of the penicillin ring in combination with the use of a new substrate for biosynthesis enzymes to form a derivative of the key intermediate in the biosynthesis of cephalosporins, 7-aminodetacetoxycephalosporanic acid or 7-ADCA. This derivative has a chemical structure that enables efficient solvent extraction, thereby providing an economically attractive isolation process.

 Transformation of P. chrysogenum can, in principle, be achieved using various DNA delivery vehicles, such as PEC-Ca-mediated uptake of protoplasts, electroporation or particle bombardment methods and selection of transformants. See for example. Van den Hondel en Punt, Gene Transfer and Vector Development for Filamentous Fungi, in: Applied Molecular Genetics of Fungi (Peberly, Laten, Ogden, Bennet, eds.), Cambridge University Press (1991). The use of dominant and non-dominant selection markers has been described (Van den Hondel, supra). Selection markers of both homologous (originating from P. chrysogenum) and heterologous (originating not from P. chrysogenum) origin have been described.

 The use of various selection markers of transformants, homologous or heterologous, in the presence or absence of vector sequences, physically or not physically associated with non-selectable DNA, in the selection of transformants is well known.

 Preferably, a homologous selection marker is used to select P. chrysogenum transformants to limit the amount of heterologous DNA introduced into P. chrysogenum. It is most preferable to use a dominant selection marker that can be selectively removed from the transformed strain, for example, the A. nidulans amdS gene or other filamentous fungi (European Patent Application No. 94201896.1). These preferred characteristics of the P. chrysogenum transformant selection marker are very favorable for the product registration process and procedures, since the antibiotic resistance markers are not involved in the process or they will not be introduced into the environment.

 In the most preferred embodiment, the amdS selection marker, which can be selectively removed from the strain, allows repeated transformation cycles using the same dominant selection again and again. This property of releasing from the marker of selection of new P. chrysogenum strains expressing expansion, is crucial for the rapid development of highly productive strains under the program for improving industrial strains.

 The ability of the ring expansion reaction mediated by the expandase enzyme is introduced and expressed in this way in P. chrysogenum, for example, Wisconsin strain 54-1255. This ring expansion reaction also occurs in their mutants, giving an increased yield of betalactams. It should be understood that in this case, the characteristics of the medium must be slightly adapted to obtain effective growth.

 In addition, the cefE gene is placed under transcriptional and translational control of the corresponding gene-controlling elements of the filamentous fungus, preferably originating from the acetyltransferase (AT) gene of P. chrysogenum, which makes it possible to express them in optimal time frames synchronized with the action of the acetyltransferase enzyme itself. These measures are crucial for the effectiveness of the ring expansion reaction in the penicillin molecule.

 In addition to the synchronized expression of genes encoding expandase and transferase, it could be favorable to develop a cost-effective production process by co-locating a portion of the expandase enzymes with acetyltransferase in microbodies (intracellular localization of acetyltransferase). With these preferred embodiments, the amount of penicillin by-products that are not allowed in the final product of the 7-ADCA by the registration authorities will be greatly reduced.

 In summary, this invention discloses how the action of the expandase enzyme introduced into P. chrysogenum can be aimed at expanding the penicillin ring based on synchronized expression.

 According to this invention, 3- (carboxyethylthio) propionyl-7-ADCA intermediate betalactam compounds are produced by P. chrysogenum by the addition of 3,3'-thiodipropionic acid or a salt or ester thereof. Suitable salts, for example, are sodium or potassium salts. They are effectively isolated from the medium by simple extraction with a solvent, for example, as follows.

 The liquid culture medium is filtered off and an organic solvent immiscible with water is added to the filtrate. The pH is adjusted to extract cephalosporin from the aqueous layer. The pH range should be below 4.5; preferably in the range from 4 to 1, more preferably from 2 to 1. Thus, cephalosporin is separated from many foreign impurities present in the fermentation medium. A small volume of an organic solvent is preferably used, which gives a concentrated solution of cephalosporin, and thus a reduction in volumetric flow rates is achieved. A second possibility is to completely extract the liquid nutrient medium at a pH of 4 or lower. Preferably, the liquid nutrient medium is extracted in the range of 4 to 1 with an organic solvent immiscible with water.

 Any solvent that does not interact with the cephalosporin molecule can be used. Suitable solvents, for example, are butyl acetate, ethyl acetate, methyl isobutyl ketone, alcohols like butanol, etc. 1-butanol or isobutanol are preferably used.

Then, cephalosporin is extracted back with water at a pH of from 4 to 10, preferably from 6 to 9. Again, the final volume is greatly reduced. The selection can be carried out at temperatures from 0 to 50 ^o C, and preferably at ambient temperatures.

 The cephalosporin aqueous solution thus obtained is treated with an appropriate enzyme to remove the 3- (carboxyethylthio) propionyl side chain and obtain the desired 7-ADCA.

 An immobilized enzyme is preferably used so that it is possible to reuse the enzyme. The methodology for producing such particles and immobilizing enzymes has been described in detail in EP-A-0222462. The pH of the aqueous solution should have a value equal, for example, from pH 4 to pH 9, at which the degradation reaction of cephalosporin is minimized, and the desired conversion with the enzyme is optimal. Thus, the enzyme is added to an aqueous solution of cephalosporin while maintaining the pH at an appropriate level by, for example, adding an inorganic base such as a potassium hydroxide solution or using a cation exchange resin. When the reaction is complete, the immobilized enzyme is removed by filtration. Another possibility is to use the immobilized enzyme in a fixed or fluidized bed column, or to use the enzyme in solution and remove products using membrane filtration. Subsequently, the reaction mixture is acidified in the presence of an organic solvent immiscible with water.

 Suitable enzymes, for example, are obtained from a Pseudomonas SY77 microorganism having a mutation in one or more positions: 62, 177, 178 and 179. Enzymes from other Pseudomonas microorganisms, preferably Pseudomonas SE83, having, optionally, a mutation of one or more positions, can also be used. corresponding to 62, 177, 178 and 179 in Pseudomonas SY77. After adjusting the pH to about 0.1 to 1.5, the layers are separated and the pH of the aqueous layer is adjusted to about 2-5. The crystalline 7-ADCA is then filtered off.

Deacylation can also be carried out chemically, in a known manner, for example, through the formation of an iminochloride side chain by adding phosphorus pentachloride at a temperature below 10 ^° C. and then isobutanol at ambient temperature or below.

 The following examples are offered by way of illustration, but not limitation.

Example 1
Expression of the cefE Streptomyces and Nocardia gene in Penicillium chrysogenum
a. General gene cloning and gene transformation procedures
This application uses the usual techniques used in gene cloning procedures. These techniques include polymerase chain reactions (PCR), synthetic oligonucleotide synthesis, DNA nucleotide sequence analysis, enzymatic ligation and DNA restriction, vector subcloning in E. coli, transformation and selection of transformants, DNA isolation and purification, DNA characterization using blot analysis by blot analysis Southern and ³² P-labeled probes, labeling ³² P DNA by random priming. All these techniques are very well known in this field of technology and are accordingly described in many sources. See, e.g., Sambrook et al. Molecular Cloning :, a Laboratory Manual, Cold Spring Harbor, USA (1989), Innes et al., PCR protocols, a Guide to Methods and Applications, Academic Press (1990), and McPherson et al., PCR, a Practical Approach, IRL Press (1991).

 General procedures used in the transformation of filamentous fungi and selection of transformants include obtaining protoplasts of fungi, DNA transfer and protoplast regeneration mode, purification and determination of the properties of transformants. All of these procedures are well known in this field of knowledge and are very well documented in: Finkelstein and Ball (eds), Bio-technology of Filamentous Fungi, technology and products, Butterworth-Heinemann (1992); Bennet and Lasure (eds). More Gene Manipulations in Fungi, Academic Press (1991); Turner, in Puhler (ed.), Biotechnology, second completely revised edition, VCH (1992).

 More specific applications of gene cloning technology and gene transformation for Penicillinum chrysogenum are very well documented by Bennet and Lasure (above) and Finkelstein and Ball (above).

 - Synthetic oligonucleotide DNAs were synthesized using a commercial DNA synthesizer (Applied Biosistems, CA, U.S.A.) in accordance with the manufacturer's instructions.

 - PCR is performed using a commercial automatic PCR apparatus (Perkin Elmer, U.S.A.) and DNA polymerase (Perkin Elmer) according to the manufacturer's instructions.

 - The hGC PCR procedure (Dutton et al., Nucleic Acids Res. 21, (no 12) (1993) 2953-2954) was used to allow amplification of the cefE coding regions of the chromosomal DNA of N. lactamdurans and S. clavuligerus.

 - Restriction enzymes and other enzymes for DNA modification obtained from BRL (MD, U.S.A.) and used in accordance with the manufacturer's instructions.

- Vector pBluescript ^® E. coli obtained from Stratagene (CA, USA).

 - The other chemicals used are all of analytical purity and obtained from different suppliers.

 - DNA nucleotide sequence analysis was performed using an apparatus for automatic DNA sequence analysis (Applied Biosistems), based on the detection of a sequence-specific fluorescent label in accordance with the manufacturer's instructions.

b. Microorganism cultivation
Streptomyces clavuligerus ATCC 27064 is grown in trypsinized soy broth (Difco). The chromosomal DNA of this strain was used to isolate the cefE gene (Kovacevic et al., J. Bacteriol. (1989), 754-760).

 Nocardia lactamdurans ATCC 27382 is also grown in trypsinized soy broth (Difco). The chromosomal DNA of this strain was used to isolate the cefE gene (Coque et. Al., Supra).

 Penicillium chrysogenum Wisconsin 54-1255 (ATCC 28089) is grown in complete YPD medium (YPD; 1% yeast extract, 2% peptone, 2% glucose). The chromosomal DNA of this strain was used to isolate the 5 'and 3' regulatory regions of the penDE gene necessary for expression of the cefE gene. Penecillium chrúsogenun ATCC 28089 is also used as a host for experiments with transformation of the cefE gene. Other strains of Penicillium chrisogenum are also suitable, including mutants of the Wisconsin strain 54-1255 giving an increased yield of betalactams. Depending on the transformant selection marker used, P. chrysogenum strains containing mutations in the pyrG, niaD or facA gene can be used. These mutant strains can be obtained using methods well known in the art (Cantoral, Bio / Technol. 5 (1987), 494–497; Gouka et al., J. Biotechn. 20 (1991), 189–200; and Gouka et al, Appl. Microbiol. Biotechnol. (1993), 514-519).

 Cultivation of P. chrysogenum to form the protoplasts used in the transformation was also performed in YPD medium.

 It is well known that protoplast formation and regeneration procedures may vary slightly depending on the specific use of the Penicillium chrysogenum strain and the transformant selection procedure used.

 E. coli WK6 (Zell and Fritz, EMBO J. 6 (1987), 1809 - 1815), XLI-Blue (Stratagene) and HB101 (Boyer and Roulland-Dussoix, J. Mol. Biol., 41 (1969), 459 ; Bolivar and Backman, Messages Ensymol. 68 (1979), 2040) were preserved and cultured using standard E. coli culture medium (Sambrook, supra).

with. Cultivation of cefE expression cassettes
CefE expression cassettes are listed in Table 1, which also explains the nomenclature that was used for these plasmids.

 Published nucleotide sequences of the S. clavuligerus cefE gene (Kovacevic, supra); cefE gene N. lactamdurans (Coque, supra); A. nidulans gpdA gene (Punt et al, Gene 69 (1988)); and the penDE gene of P. chrysogenum (Barredo et al., Gene 83 (1989), 291-300; Dies et al., Mol. Gen. Genet. 218 (1989), were used to create the synthetic oligonucleotides listed in Table II.

c1 .. Construction of E. coli cefE expression plasmids pMCTSE and pMCTNE
PCR, 1: cefE N. lactamdurans
In the first PCR using chromosomal DNA N.lactamdurans and oligonucleotides 1 and 2, an open reading frame of cfeE N.lactamdurans was obtained as a 0.9 kb PCR product containing a unique Ndel restriction site at the 5'-end and a unique Xbal site at 3 '-end.

PCR 2: cefE S. clavuligerus
In the second PCR using S. clavuligerus chromosomal DNA and oligonucleotides 3 and 4, an open reading frame of S. clavuligerus cefE was obtained as a 0.9 kb PCR product also containing a unique NdeI restriction site at the 5'-end and a unique Xbal restriction site at the 3'-end.

 In order to obtain expression of cefE and E. coli genes and characterize PCR products using DNA sequence analysis, PCR products 1 and 2 were cloned into pMCTNde vector derived from pMC-5 (Stanssens et al., Nucleic Acids Res, 17 (1989) , 4441), Plasmid pMCTNde was obtained from pMC5-8 (European Patent Application No. 0351029) by inserting a fragment encoding the tac promoter followed by the RBS site and the Ndel cloning site (Fig. 3).

PCR products 1 and 2 were digested with NdeI and XbaI and ligated into the pooled NdeI-XbaI vector pMCTNde. The ligation mixture was used to transform E. coli WK6. Transformants were selected for chloramphenicol resistance. These transformants were used to isolate plasmid DNA. The insertion of cefE expression cassettes was first analyzed by digestion with a restriction enzyme to predict the generation of restriction fragments. Plasmids containing the predicted restriction enzyme sites are finally analyzed by automatic DNA sequence analysis,
The DNA sequence of the open reading frame of cefE S. clavuligerus in plasmid pMCTSE (Fig. 2) was 100% identical to the published sequence (Kovacevic, supra).

 The DNA sequence (Fig. 8) of all the clones that were analyzed containing the open reading frame of cefE N. lactamdurans differed from the nucleated sequence (Coque, supra).

 The amino acid sequence derived from the published cefE gene N. lactamdurans has a proline at amino acid position 41 (see Last ID N 14). This proline is omitted in clones obtained by PCR 1. This plasmid is named pMCTNE (Fig. 1).

c2. Construction of plasmids for expression of cefE P. chrysogenum
PCR 3: gpdA promoter
In this third PCR using pAN7-1 plasmid DNA (Punt et al., Gene 56 (1987) 117 - 124) containing the E. coli hph gene under the control of the A. nidulans gpdA promoter and oligonucleotides 5 and 6, the gpdA promoter was learned as a 0.9 kb PCR product containing a single EcoRI restriction site at the 5'-end and a single NdeI site at the 3'-end.

PCR 4: AT promoter
In the fourth PCR, P. chrysogenum chromosomal DNA and oligonucleotides 7 and 8 were used to obtain an AT promoter fragment of 1.5 kb, which also contains a single EcoRI restriction site at the 5'-end and a single NdeI site at the 3'-end.

PCR 5: AT terminator and 3'-end of the cefE gene
N.lactamdurans.

 In the fifth PCR, 0.5 kb of the penDE terminator region (AT) was obtained using P. chrysogenum chromosomal DNA and oligonucleotides 9 and 10, and 11 and 10, respectively. These PCR products thus contain the 3'-terminal sequence of the cefE gene with or without a targeting signal to the microbody consisting of the C-terminal amino acid sequence of ARL (Muller et al., Biochimica et Biophysica Acta 1116 (1992), 210 - 213) .

 Oligonucleotides are designed so that a single BspEl site is inserted at the 5'-end of the PCR product and a single SpeI site is introduced at the 3'-end of the PCR product.

PCR 6: AT terminator and 3'-end of the cefE gene of S. clavuligerus
In this sixth PCR, a 0.5 kb penDE terminator region (AT) was obtained using P. chrysogenum chromosomal DNA and oligonucleotides 12 and 10, and 13 and 10, respectively. These PCR products thus contain the 3'-terminal sequence of the cefE gene of S. clavuligerus with or without a targeting signal to the microbody consisting of the SKL C-terminal amino acid sequence (De Hoop et al., Biochem. J. 286 (1992), 657-669).

 Oligonucleotides are designed so that a single BqlI restriction site is introduced at the 5'-end of the PCR product and a single SpeI site is obtained at the 3'-end of this PCR product.

 In order to obtain expression of cefE genes in P. chrysogenum, the gpdA promoter and the AT promoter fragment were ligated to cefE fragments from plasmids pMCTNE and pMCTSE. These ligated fragments were cloned into the pBluescript II KS vector.

 The PCR 3 product was digested with EcoRI and Ndel. pMCTNE and pMCTSE were digested with NdeI and XbaI. Restriction fragments were separated by agarose gel electrophoresis. The coding fragments of 0.9 kb cefE were purified from agarose gel. The EcoRI-Ndel promoter fragment was ligated together with NdeI-XbaI cefE fragments into EcoRI-XbaI digested pBluescript II KS vector. Thus, the following plasmids were obtained: pGSE and pGNE.

 To obtain optimal expression of cefE genes in P. chrysogenum, we selected the cloning of the AT termination signal sequence after cefE genes in the Penicillium expression plasmids mentioned above.

pGNETA-pGNEWA
PCR 5 products were digested with BspEI and SpeI and ligated into the digested BspEI and SpeI pGNE vector. Ligation mixtures were used to transform E. coli HB101. Transformants were selected for ampicillin resistance. Plasmids isolated from these transformants were characterized by analysis of restriction fragments and then by DNA sequence analysis. Thus, the following plasmids were obtained: pGNEWA and pGNETA (Fig. 4).

pGSETA-pGSEWA
PCR 6 products were digested with BqlI and SpeI and ligated into the digested BqlI and SpeI pGSE vector. Ligation mixtures were used to transform E. coli HB101. Transformants were selected for ampicillin resistance. Plasmids isolated from these transformants were also characterized by analysis of restriction fragments and then DNA sequence analysis. Thus, the following plasmids were obtained: pGSWA and pGSETA (Fig. 5).

pANETA, pANEWA and pASETA and pASEWA
Plasmids pGNETA, pGNEWA, pGSETA and pGSEWA were digested with EcoRI and NdeI. Restriction fragments were separated by agarose gel electrophoresis and 4.5 kb fragments were purified from the gel.

 The PCR 4 product was digested with EcoRI and NdeI and ligated to the purified fragments mentioned above. After transformation of the ligation mixtures into E. coli HB101, transformants were selected for ampicillin resistance.

 Transformants were grown and their plasmids were isolated and characterized by analysis of restriction fragments and, finally, by DNA sequence analysis. Thus, the desired designs were obtained, namely pANETA (Fig. 6), pANEWA, pASETA (Fig. 7) and pASEWA.

d. Transformation of P. chrysogenum
The protoplast transformation procedure mediated by Ca-PEG was used.

 According to the methods described by Cantoral (see above), Gouka et al. (J. Biotechn., See above) and Gouka et al. (Appl. Mirobiol. Biotechnol., See above) to transform the P. chrysogenum strain, a whole plasmid or purified cefE expression polygenic cluster (lacking E. coli vector sequences) with pyrG, niaD, facA or amdS genes (Beri et al., Curr. Genet. 11 (1987) 639 - 641), respectively, as selection markers.

 By using homologous selection markers of pyrG, niaD or facA, in pure form, devoid of E. coli vector sequences, transformed P. chrysogenum strains that do not contain bacterial resistance genes were obtained.

 European Patent Application No. 94201896.1 describes a method for producing recombinant strains lacking selection marker genes. This method has been successfully used on P. chrysogenum transformants containing the A. nidulans amdS gene as a dominant selection marker.

 In this case, the only elements of heterologous origin are the 0.9 kb cefE coding region and, optionally, the 0.9 kb gpdA promoter region.

e. Analysis of transformants
Transformants of P. chrysogenum are purified by re-cultivation on selective medium. Single resistant colonies were used to prepare agar stocks for obtaining spores and for testing for transformants containing the cefE expression cassette. The boiling of a fragment of fresh mycelium from transformants on an agar plate was used to obtain a sufficient amount of template DNA to efficiently test hundreds of transformants for the presence of the cefE gene using the PCR technique. (Seth, Fungal Genetis Conference, Asilomar (1991), Fungal Genetics Newsletter 38, 55). Acting in this way established the effectiveness of the transformation.

 The selection of transformants was also performed using biological research. Transformants were grown on an agar medium that contained selectable side chain precursors. E. coli ESS2231 was used as an indicator bacterium in the topcoat of agar, which also contained Bactopenase, so that it was possible to differentiate the production of penicillin and cephalosporin according to methods well known in this field of science and described, for example, by Guttierez et al., Mol. Gen. Genet. 225 (1991), 56 - 64.

Spores were used for plating P. chrysogenum into the culture medium as described in section d. After 72 hours of cultivation (at 25 ^° C.), chromosomal DNA is isolated from the mycelium. DNA is digested with a 6 bp restriction enzyme, similar to EcoRI or PstI.

DNA fragments were separated by agarose gel electrophoresis and transferred to Gene screen (New England Nuclear) nylon membranes. Southern blotting was performed by hybridization with a ³² P-labeled PCR 2 product as a sample for cefE gene sequences. Labeling ³² P of purified PCR 2 product is accomplished by random priming labeling in the presence of α ³² P dTCP using a commercial labeling kit (Boeringer Mannheim).

 Transformants containing the cefE coding sequence were tested for expression of the cefE gene product, referred to herein as expandase activity.

 Selected transformants are cultured in penicillin production medium (see example 2).

 In an experiment to evaluate the progress of the process over time, mycelium samples were taken after 48, 72, and 96 hours of fermentation. Mycelium extracts are prepared, and expandase activity is determined in the crude extracts as described by Rollins et al., Can. J. Microbiol. 34 (1988), 1196-1202. Transformants with expandase activity were tested for acyltransferase activity also by the methods described by Alvares et al., Antimicrob. Agent Chem. 31 (1987), 1675 - 1682).

 Transformants with different levels of acitransferase and expandase enzymatic activity for the enzymatic production of 7-ADCA derivatives were selected from these analyzes.

Example 2
Enzymatic preparation of 3- (carboxyethylthio) propionyl-7-ADCA and its isolation
P. chrysogenum strain Wisconsin 54-1255 (ATCC 28089) is transformed using one of the DNA constructs, as described in example 1, and inoculated at a concentration of 2 • 10 ⁶ conidia / ml inoculated medium consisting of (g / l): glucose, thirty; (NH ₄ SO ₄ , 10; KH ₂ PO ₄ , 10; trace element solution I (MgSO ₄ • 7H ₂ O, 25; FeSO ₄ • 7H ₂ O, 10; CuSO ₄ •• 5H ₂ O, 0.5; ZnSO ₄ • 7H ₂ O, 2; Na ₂ SO ₄ , 50; MnSO ₄ • H ₂ O, 2; CaCl ₂ • 2H ₂ O, 5), 10 (ml / l) (pH 6.5 before sterilization).

The seed culture was incubated for 48 - 72 hours at 25-30 ^o C and then used for sowing 10-20 volumes of production medium containing (g / l) lactose, 80; maltose, 20; CaSO ₄ , 4; urea, 3; MgSO ₄ • 7H ₂ O, 2; KH ₂ PO ₄ , 7; NaCl, 0.5; (NH ₄ ) ₂ SO ₄ , 6; FeSO ₄ • 7H ₂ 0, 0.1; 3,3'-thiodipropionic acid, 5; trace element solution II (CuSO ₄ • 5H ₂ O, 0.5; ZnSO ₄ • 7H ₂ 0, 2; MnSO ₄ • H ₂ O, 2; Na ₂ SO ₄ , 50), 10 (ml / l) (pH to sterilization 5.5-6.0). Then the incubation continues for another 96-120 hours.

 At the end of production fermentation, the mycelium is separated by centrifugation or filtration, and 3- (carboxyethylthio) propionyl-7-ADCA is analyzed by high performance liquid chromatography (HPLC) on a reverse phase column. The Beckman System Gold HPLC apparatus was used, consisting of a programmable solvent system 168, an automatic sampler Model 507, a diode mesh detector Model 168, and a System Gold scorecard (5.10). As the stationary phase, two (2) Chromspher C18 cartridge columns (100 x 3 mm, Chrompack) in a row were used. The mobile phase consists of a linear gradient from 100% 0.07M phosphate buffer pH 4.8 to 25% acetonitrile and 75% phosphate buffer pH 4.8 after 15 minutes at a flow rate of 0.5 ml / min. The production of 3- (carboxyethylthio) propionyl-7-ADCA is quantified at 260 nm using 3- (carboxyethylthio) propionyl-7-ADCA as a reference substance.

 The identity of the peak is confirmed by comparing the curves of the ultraviolet and NMR spectra.

 After filtering the liquid culture medium, approximately 0.1 volumes of 1-butanol are added to the filtrate. The pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was stirred for 5 minutes at room temperature. After separation, the organic layer is either evaporated or further used for chemical deacylation (example 3) or is back extracted with a volume of water equal to 0.33 with a pH of 8 and then used for enzymatic deacylation (example 4).

Example 3
Deacylation of 3- (carboxyethylthio) propionyl-7-ADC
To a mixture of 3 g (8 mmol) of 3- (carboxyethylthio) propionyl-7-ADCA, 3.5 ml (36 mmol) of N, N-dimethylaniline, 13 ml of methylene chloride, and 2.6 ml (12 mmol) of trimethylchlorosilane is added at room temperature . After stirring for 30 minutes, the reaction mixture was cooled to about -50 ^° C and 1.8 g (8.5 mmol) of phosphorus pentachloride were added, all at once. The temperature is maintained at -40 ^o C for two hours and then the reaction mixture is cooled to -65 ^o C. Then it is treated with 12 ml (137 mmol) of isobutanol at such a rate that the temperature does not rise above -40 ^o C. After further stirring for two hours, the solution is poured into 15 ml of water and then 5 ml of 4.5 N ammonia are immediately added. The pH is adjusted to 4 by slowly adding solid ammonium bicarbonate. After cooling to 5 ^° C., the mixture was filtered and the crystalline 7-ADCA was washed with 5 ml of aqueous acetone (1: 1) and separated.

Example 4
Enzymatic deacylation of 3- (carboxyethylthio) propionyl-7-ADCA using the mutant acylase Pseudomonas SY77
The conversion of 3- (carboxyethylthio) propionyl-7-ADCA is carried out in a single enzymatic step using specific acylase, which is produced from Pseudomonas SY77 acylase by targeting a specific area of mutagenesis. The construction and identification of the Pseudomonas SY77 mutant acylase with improved activity on the 3- (carboxyethylthio) propionyl side chain has been described in EP-A-0453048. In the mutant, tyrosine at position 178 in the alpha subunit of the acylase of Pseudomonas SY77 is replaced by histidine. Mutant acylase is produced by E. coli. Cells are harvested by centrifugation and resuspended in 10 mM phosphate buffer pH 7.4 containing 140 mM NaCl. Then the cells are destroyed by ultrasound. After removal of cell debris, supernatants with acylase activity were collected. Further purification of the acylase was performed using a number of chromatographic steps:
(1) ion exchange chromatography on a Q-sephazor with fast flow at pH 8.8;
(2) chromatography of hydrophobic interaction on phenylsepharose; and
(3) Sephacryl gel column chromatography.

 Purified acylase was immobilized on particles consisting of a mixture of gelatin and chitosan. Particles were treated with glutaraldehyde just before the enzyme was added.

The conversion of 3- (carboxyethylthio) propionyl-7-ADCA was performed in a stirred reactor. First, an aqueous solution of cephalosporin is added to the reactor. Then the temperature of the solution is brought to 30 ^o C with constant stirring and the pH is fixed at 8 with potassium hydroxide. Then the immobilized enzyme is added and the conversion begins. During the conversion, the pH in the reactor is continuously monitored and maintained at 8. 3.3'-thiodipropionic acid, which is released during the reaction, is titrated with KOH. The amount of KOH that is added, added, and recorded on a flat-panel recorder. The conversion is controlled by sampling from the reactor, which are analyzed for 3- (carboxyethylthio) propionyl-7-ADCA and 7-ADCC using HPLC, as described in example 2.

 When the reaction is complete, the immobilized enzyme is removed by filtration and the pH of the filtrate is adjusted to 1, while the filtrate contains butyl acetate. The layers are separated, and the pH of the aqueous phase, which contains 7-ADCA, is adjusted to 3. The crystalline 7-ADCA is then filtered off.

Example 5
Enzymatic deacylation of 3- (carboxyethylthio) propionyl-7-ADCA using Pseudomonas a83 acylase
The conversion of 3- (carboxyethylthio) propionyl-7-ADCA is carried out as in Example 4, however, when using the Pseudomonas SE83 acylase as the acylase to obtain the same result.

Claims (5)

 1. The method of obtaining and isolation of 7-aminodetacetoxycephalosporanic acid (7-ADCA) by a / transformation of the Penicillium chrysogenum strain of the expandase gene under the control of transcriptional and translational signal sequences of expression of filamentous fungi, b / growing the specified strain in culture medium and adding N- to the indicated medium acyl of the side chain precursor or a salt or ester thereof suitable for the formation of N-acyl-6-aminopenicillanic acid (N-acyl-6-APA), the ring of which in situ expands to form N-acyl-7-ADCA, iv I N-acyl-7-ADCA from the fermentation medium, g / deacylation of the indicated N-acyl-7-ADCA and d / isolation of crystalline 7-ADCA, characterized in that the N-acyl side chain precursor is 3,3'-thiodipropionic an acid suitable for the formation of 3- (carboxyethylthio) propionyl-6-APA, the ring of which in situ expands to form 3- (carboxyethylthio) propionyl-7-ADCA.  2. The method according to p. 1, characterized in that said Penicillium chrysogenum strain is transformed by the expandase gene under the control of transcriptional and translational signal sequences of the AT gene expression.  3. The method according to claim 1 or 2, characterized in that stage (d) is a filtration stage.  4. The method according to any one of the preceding paragraphs, characterized in that stage (c) is a filtration step, and with extracting the filtrate of the liquid nutrient medium with an organic solvent not miscible with water at a pH below about 4.5, and back extraction of the same substances with water at a pH of 4 to 10.  5. The method according to any one of the preceding paragraphs, characterized in that the expandase gene is obtained from Streptomyces clavuligerus or Nocardia lactamdurans.

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