CN1331751A - Improved i(in vivo) prodn. of cephalosporins - Google Patents

Improved i(in vivo) prodn. of cephalosporins Download PDF

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CN1331751A
CN1331751A CN99814782A CN99814782A CN1331751A CN 1331751 A CN1331751 A CN 1331751A CN 99814782 A CN99814782 A CN 99814782A CN 99814782 A CN99814782 A CN 99814782A CN 1331751 A CN1331751 A CN 1331751A
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aca
nucleotide sequence
acidylate
acid
side chain
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R·A·L·博文博格
R·科克曼
E·科汉
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Koninklijke DSM NV
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

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Abstract

The present invention discloses a process for the production of 7-ACA or a derivative thereof comprising the steps of fermenting a Penicillirsm chrysogenum strain being transformed with an expression construct comprising a nucleotide sequence encoding expandase, hydroxylase and acetyltransferase activity in the presence of a suitable acyl side chain precursor, or a salt or ester thereof, such that an N-acylated 7-ACA compound is produced, N-deacylating the thus produced N-acylated 7-ACA compound and, optionally, acylating the free amino group and/or substituting the 3' acetate group with a side chain suitable to form a cephalosporin antibiotic, characterised in that the nucleotide sequence encoding the acetyltransferase is derived from Acremonium chrysogenum and starts at the second ATG of the open reading frame as present in said nucleotide sequence.

Description

Production method in the body of improved cynnematin
The present invention relates to a kind of method of producing the method for cynnematin and particularly producing the 7-ACA or derivatives thereof; this method may further comprise the steps: at a kind of Penicllium chrysogenum bacterial strain of condition bottom fermentation that has suitable acyl side-chain precursor or its salt or its ester to exist; thereby generation N-acidylate 7-ACA compound; the N-acidylate 7-ACA compound that the N-deacylation so produces and; randomly; acidylate free amine group and/or employing are fit to form the side substitution 3 ' acetoxyl of cephalosporin antibiotics, and wherein the Penicllium chrysogenum bacterial strain is contained code extension enzyme (expandase); the expression construct of the nucleotide sequence of hydroxylase and Transacetylase transforms.
The semi-synthetic approach of preparation cynnematin originates in tunning such as penicillin G mostly, penicillin v, cephalosporin, for example with K.Matsumoto, biological processing technology (Bioprocess.Techn.), 16,67-88 (1993), J.G.Shewale and H.Sivaraman, process biochemistry (ProcessBiochemistry), in August, 1989,146-154, T.A.Sayidge, industrial microbiotic biotechnology (Ed.E.J.Vandamme) Marcel Dekker, New York, 1984, or J.G.Shewale etc., ProcessBiochemistry International, in June nineteen ninety, disclosed method is converted into corresponding beta-lactam parent nucleus with described tunning among the 97-103.The beta-lactam parent nucleus that obtains is subsequently by being converted into needed microbiotic with a suitable side chain coupling, and this is described among JP53005185 and the CH640240 especially at EP0339751.By carrying out the various combination of side chain and beta-lactam parent nucleus, can obtain multiple penicillin and cephalosporin antibiotics.
It is to produce the antibiotic important intermediate that is used for medicine industry that known cephalosporin mother nucleus 7-amino removes acetoxyl Cephalosporanic acid (7-ADCA) and 7-amino-cephalosporanic acid (7-ACA).
Cephalosporin obviously is the most important initiator that cynnematin is used in preparation 7-ACA and other treatment.Yet cephalosporin is all very soluble in water under any pH, therefore means and need remove the cephalosporin that is not transformed in the product by utilizing loaded down with trivial details and expensive column technology to carry out long-time and expensive sepn process.In addition, preparation necessary enzymatic of 7-ACA or chemical cracking are difficult to the alpha-amino group hexanedioyl side chain of cephalosporin is worked.
In order to overcome some above-mentioned defectives of this paper, the fermentation process of a kind of 7-ACA of production is disclosed, this method comprises the cynnematin that some N-of fermentative production replaces, the side chain of this cynnematin can easily be removed by simple enzymatic or chemical cracking reaction.
Can express enzymic activity by a strain goes acetoxyl cynnematin synthetic enzyme (being also referred to as " extension enzyme "), deacetylate cephalosporin c synthase (" hydroxylase ") and cephalosporin c synthase (" Transacetylase ") reorganization Penicllium chrysogenum bacterial strain (EP00540210) to realize the cynnematin of these N-replacements such as the fermentative production of hexanedioyl 7-ACA.
Utilizing reorganization Penicllium chrysogenum bacterial strain to carry out in the body of hexanedioyl 7-ACA in the production process, observing to exist and compare a large amount of hexanedioyl 7-ACA with hexanedioyl 7-ACA, hexanedioyl-7-amino-cephalosporanic acid (precursor of hexanedioyl-7-ADAC).Obviously, acetyl transferase gene is failed to be enough to that hexanedioyl-7-ADAC is expressed to a large amount of amounts that transform of hexanedioyl-7-ACA.
The invention discloses the Transacetylase expression construct that is designed to obtain the Transacetylase high level expression.Like this, increased the amount that precursor hexanedioyl-7-ADAC is converted into hexanedioyl-7-ACA.
Description is derived from the clone of yellow top spore (Acremonium chrysogenum) acetyl transferase gene (cefG) of product and the document of nucleotide sequence discloses first (EP0437378 that originates in Transacetylase open reading-frame (ORF) (ORF); Gutierrez etc., bacteriology magazine .174:3056-3064 (1992)), or the encoding sequence of second (Mathison etc., Curr Genet.23:33-41 (1992)) or the 3rd ATG (EP0450758).In addition, in the research process that the efficient of different promoters being produced expression Transacetylase in the yellow top spore (A.chrysogenum) is carried out, tested construct is a kind of fusion constructs (Gutierrez etc., applied microbiology biotechnology 48:606-614 (1997)) that contains the Transacetylase encoding sequence that originates in second ATG.
This paper document cited above does not have one piece to disclose the initiator codon of selecting specific cefG 0RF and obtain to carry out the advantage that Transacetylase efficiently expresses in reorganization Penicllium chrysogenum bacterial strain, and this Penicllium chrysogenum bacterial strain is used to produce the fermenting process of 7-ACA or derivatives thereof.
The invention discloses a kind of method of the 7-ACA of production or derivatives thereof; this method may further comprise the steps: under the condition that has suitable acyl side-chain precursor or its salt or its ester to exist; to with containing the code extension enzyme; the Penicllium chrysogenum bacterial strain that the expression construct of the nucleotide sequence of hydroxylase and acetyltransferase activity transforms carries out fermentation culture; produce N-acidylate 7-ACA compound thus; the N-acidylate 7-ACA compound that the N-deacylation so produces; randomly; acidylate free amine group and/or adopt to be fit to form the side substitution 3 ' acetoxyl of cephalosporin antibiotics, the nucleotide sequence of the Transacetylase that it is characterized in that encoding stems from second ATG that produces a yellow top spore and originate in described nucleotide sequence.
Be surprised to find by the present invention, the expression when containing the expression ratio of expression construct when this encoding sequence originates in second ATG of open reading-frame (ORF) (ORF) that stems from the Transacetylase encoding sequence that produces a yellow top spore and originating in first or the 3rd ATG of described ORF is more efficient.One of them effect that Transacetylase more efficiently expresses is that N-acidylate 7-ADAC derivative is converted into N-acidylate 7-ACA derivative more efficiently.
In the method for the invention, the Penicllium chrysogenum bacterial strain of conversion is used to express three kinds of enzymic activitys in the cynnematin biosynthetic pathway that causes the generation of 3 '-acidylate cephalosporin compound.
The suitable source of the gene of the described three kinds of enzymic activitys of encoding is bacterium band spillikin streptomycete or the Nocardia lactamdurans that are used to obtain elongase gene cefE and hydroxylation enzyme gene cefF (relevant cefE referring to EP0341892 and relevant cefF referring to EP0465189), or be used to obtain difunctional extension enzyme/hydroxylation enzyme gene cefEF and acetyl transferase gene cefG (relevant cefEF is referring to EP0281391 and Coque etc., Mol.Gen.Genet.236:453-458 (1993) and about cefG referring to EP0437378 and EP0450758) fungi produce a yellow top spore.
According to the present invention, acetyltransferase activity is to provide by the cefG gene that obtains from produce a yellow top spore.Especially, the present invention shows that adopting second ATG of cefG ORF is favourable as initiator codon.Second ATG of cefG ORF means that as the use of initiator codon the Transacetylase that is used for the inventive method has the n terminal amino acid sequence with methionine(Met)-leucine-arginine-aspartic acid-Serine beginning.
In the method for the invention, three kinds of enzymic activitys of coding gene of extending enzyme, hydroxylase and Transacetylases can have and is in 5 ' and the 3 ' regulating and controlling sequence that this gene among the meter opinion itself has and maybe can has and the allogenic regulating and controlling sequence of described gene.
Example that 5 ' and 3 ' suitable regulating and controlling sequence is promotor that the recombinant gene expression in filamentous fungal host cell provides and terminator at Van den Hondel etc. (in More Gene Manipulationsin Fungi, Eds.Bennett and Lasure, 396-427 (1991)) in or at the application molecular genetics (Kinghorn of filamentous fungus, Turner (eds.), Blackie, Glasgow, UK, 1992) in mention.Preferred promotor is aspergillus niger glucoamylase promotor or Penicllium chrysogenum promotor or the gene Y that stems from the gene of coding ACV synthetic enzyme, isopenicillin N synthase, Transacetylase, phosphoglyceric kinase.Also can from identical gene, obtain transcription terminator.
In one embodiment of the invention, the method of a kind of fermentative production 7-ACA or derivatives thereof that is provided comprises the Penicllium chrysogenum bacterial strain that employing transforms through expression construct, the encoding sequence of the gene of code extension enzyme, hydroxylase and/or acetyltransferase activity in this expression construct be fused to the allogenic promoter sequence of described encoding sequence on.Described allogeneic promoter sequence for example is IPNS (pcbC) promotor that stems from Penicllium chrysogenum.
In another embodiment of the invention, the accurate fusion between the initiator codon of the encoding sequence by the promoter sequence that adopts round pcr to realize expediently to select and code extension enzyme, hydroxylase and/or acetyltransferase activity.
The conversion of Penicllium chrysogenum host cell can resemble protoplastis picked-up, electroporation or the particle gun technology of PEG-Ca mediation and the screening of subsequent transformation body and finish by the means of different of DNA transmission usually.For example see Van den Hondel en Punt, gene and transfer and the development that is used for the carrier of filamentous fungus, in: the application molecular genetics of fungi (Peberdy, Laten, Ogden, Bennett, eds.), Cambridge University Press (1991).The existing description of the application of dominance and non-dominant selectable marker (Van den Hondel sees above).The selective marker of homology (deriving from Penicllium chrysogenum) and allos (the non-Penicllium chrysogenum that derives from) also all has description (Gouka etc., biotechnology magazine (J.Biotechnol.) 20 189-200 (1991)).
In the chosen process of transformant, homology or allogenic has or the carrier free sequence, is known in the art with being applied in of different transformant selective markers non-selective DNA physical connection or non-physical connection.
As long as fermentation is to carry out under the condition that has suitable side chain precursor to exist, the fermentation culture of the Penicllium chrysogenum bacterial strain that has transformed can be carried out in any suitable fermention medium known in the art.In this respect, suitable side chain precursor is defined as a kind of energy and produces the N-acyl side-chain precursor that sieves the N-acyl side-chain of compound through the cephalo of fermentative production, and described N-acyl side-chain can carry out simple chemistry or enzymatic is removed.Particularly, suitable side chain precursor is a dicarboxylic acid, more particularly dicarboxylic acid as the formula (1)
HOOC-X-(CH 2) n-COOH?????????????????????????????(1)
Wherein
N be at least 2 even number and
X is (CH 2) p-A-(CH 2) q, wherein
P and q each be respectively 0,1,2,3 or 4 and
A is CH=CH-CH=CH, CH=CH, and C ≡ C, CHB, C=O, O, S, NH, nitrogen are selectively replaced or sulphur is hydrogen, halogen, C by selectivity also oxidation and B 1-3The methyl that alkoxyl group, hydroxyl or selectivity replace, condition is that p+q should be 0 or 1 when A is CH=CH-CH=CH, p+q should be 2 or 3 or to work as A be CHB when A is CH=CH or C=C, C=O, O, p+q should be 3 or 4 when S or NH, or its salt or its ester.
Example suc as formula the suitable side chains precursor of (1) is hexanodioic acid, 3 '-carboxymethyl propane thioic acid (WO95/04148), 3,3 '-thio-2 acid (WO95/04149) or the side chain precursor as providing among WO98/48034 or the WO98/48035.Preferred side chain precursor is hexanodioic acid or trans-β-hydrogenation muconic acid.
By the N-acidylate 7-ACA compound that ferments and obtained under the suitable acyl side-chain precursor condition that for example hexanodioic acid exists is being arranged; for example hexanedioyl-7-ACA can be by adopting conventional recovery technology, and for example following simple solvent extraction method reclaims from fermention medium efficiently:
Filtering nutrient solution also will be added in the filtrate with the organic solvent of water immiscibility.Regulate pH so that from water layer, extract cynnematin.The pH scope must be lower than 4.5; Preferably between 4 to 1, more preferably between 2 to 1.Separate in many other impurity in making cynnematin like this and being present in fermentation culture.The a small amount of organic solvent of preferred use, the result obtains more spissated cynnematin solution, thereby volumetric flow rate is reduced.Second possibility be pH be 4 or lower pH under carry out the extraction of whole nutrient solutions.Preferably nutrient solution is to use with the immiscible organic solvent of water under 4 to 1 the condition to extract in the pH scope.
Any solvent that does not influence the cynnematin molecule can use.Suitable solvent is, for example, butylacetate, ethyl acetate, mibk, alcohol are as butanols etc.
After this cynnematin is between pH4 to pH10, and preferably water is stripped between pH6 to pH9.Final volume can be reduced once more.Can under 0 ℃ to 50 ℃ temperature, preferably under 0 ℃ to 10 ℃ temperature, reclaim.
Because 7-amino has been carried out suitable protection by the existence of suitable acyl side-chain, so can be used as the intermediate of the semisynthetic cynnematin of chemosynthesis easily by the N-acidylate cephalosporins derivatives of the inventive method production.
Perhaps, in order to remove the N-acyl group, hexanedioyl for example, side chain and obtain required 7-ACA is handled the aqueous solution of thus obtained N-acidylate cynnematin with suitable enzyme.
Preferably, use immobilized enzyme so that can reuse enzyme.Be used for the preparation of this particle and the immobilized method of enzyme and carried out a large amount of descriptions at EP0222462.PH value of aqueous solution is, pH4 to pH9 for example, and it is optimised that the DeR of cynnematin is reduced to minimum and required enzymatic conversion in this scope.Enzyme is added in the aqueous solution of cynnematin simultaneously by for example adding mineral alkali such as potassium hydroxide solution or adopting Zeo-karb that pH is maintained proper level.When reaction is finished, remove by filter immobilized enzyme.Another kind may be with the applying immobilized enzyme of form of a kind of immobilization or fluidisation column, perhaps to use enzyme and remove product by membrane filtration with the form of solution.Then, the pH of the aqueous solution is transferred to value between 2 and 5, preferably between 3 and 4.Crystal 7-ACA is filtered then.
Suitable enzyme is for example to stem from the pseudomonas SY77 microorganism that has sudden change on one or more positions of position 62,177,178 and 179.Also can use to derive from other pseudomonas microbial, the enzyme of pseudomonas SE83 preferably optionally has sudden change on the one or more positions corresponding to 62,177,178 and 179 positions of pseudomonas SY77.
Deacylation also can carry out with chemical process known in the art, for example, and by adding phosphorus pentachloride under 10 ℃ the temperature and then under room temperature or subambient temperature, add alcohol forms the imino-choride side chain as isopropylcarbinol method being lower than.
In one embodiment of the invention, the aqueous solution that contains the 7-ACA that obtains behind N-acidylate 7-ACA derivative or the deacylation can adopt suitable acylating agent to handle so that any (acyl group)-7-ADAC that may be present in the described aqueous solution changes into (acyl group)-7-ACA derivative accordingly.Described acylation reaction for example can for example by disclosed method among the US5221739, or adopt suitable lipase or esterase by adopting diacetyl oxide, and for example disclosed method is finished among the EP667396.
In further step, the 7-ACA compound that obtains by the inventive method is used as the initiator for preparing multiple cephalosporin antibiotics, end product and intermediate.By adopting known chemistry or enzymatic coupling method, the free amine group of 7-ACA for example can be by any suitable side chain institute acidylate, and the result produces the derivative of N-acidylate 7-ACA.In addition, on 3 ' position substitution reaction may take place.So the example of the cephalosporin compound of preparation is cefotaxime, cephalo azoles beautiful jade, ceftriaxone, cefuroxime, cefprozil, ceftazidime and cephalo chlorine.
Embodiment 1
Be used for expressing the pICG of Transacetylase at Penicllium chrysogenum 1WA, pICG 2WA and pICG 3The WA construct
Contain the wild-type that comprises Penicllium chrysogenum pcbC promotor and penDE terminator and produce deacetyl cephalosporin C Transacetylase (cefG) the expression cassette pICG of a yellow top spore cefG gene 1WA makes up in accordance with the following methods.The N-terminal portions of cefG gene, promptly the beginning of first ATG of ORF is to obtain from the chromosomal DNA that produces a yellow top spore by the PCR reaction of adopting primer #1 and #2 (being respectively SEQ ID NO1 and 2).The C-terminal portions of cefG gene is to obtain from identical template by the PCR reaction of adopting primer #3 and #4 (being respectively SEQ ID NO3 and 4).After adopting primer #1 and #4 and above-mentioned fragment to merge PCR, produced a complete cefG gene and (further be expressed as cefG here as template 1Gene), lacked in this cefG gene in.SfiI and HimdIII site and set up a new MsiI site.
In next procedure, the first part of pcbC promotor adopts primer #5 and #6 (being respectively SEQ IDNO5 and 6) PCR-amplification, and after adopting primer #5 and #4 to merge PCR, is introduced directly into cefG 1The front of gene.With after the PstI/NsiI digestion, 1592bp fragment be connected to a 4.3kb pISEWA-N (carrier of in WO98/46772, describing in the past) thus the PstI/NsiI carrier segments on generation Penicillium conversion carrier pICG 1WA.
Make up pICG 2The needed cefG of WA 2The N-terminal portions of gene i.e. the beginning of second of ORF ATG, derives from the pICG in the PCR reaction of adopting primer #5 and #7 (SEQ ID NO7) 1WA.CefG 2The C-terminal portions of gene derives from the same template in the PCR reaction of adopting primer #8 and #9 (being respectively SEQ ID NO8 and 9).After adopting primer #5 and #9 and above-mentioned fragment to merge PCR, just produced complete cefG as template 2Gene.
In order to make up pICG 3WA, wherein the cefG gene originates in the 3rd ATG, has adopted and described pICG 2The step that the structure of WA is identical.In order to finish this structure, primer #5/#10 (being respectively SEQ ID NO5/10) and #11/#9 (being respectively SEQ ID NO 11/9) have been adopted.
After having carried out the digestion in interior PstI/NcoI site, cefG 2/ cefG 3Merge fragment and be connected to pICG 1On the PstI/NcoI carrier segments of WA, produced Penicillium conversion carrier pICG 2WA and pICG 3WA.
Embodiment 2
Different ATG initiator codons are selected the influence to the expression of Transacetylase
After different pICGWA constructs have carried out NotI digestion,, isolating cefG fragment is introduced in the Penicllium chrysogenum by the protoplast transformation that the Ca-PEG that describes among the EP635574 mediates.
Employed Penicllium chrysogenum bacterial strain transforms with stemming from the expression construct that contains difunctional extension enzyme/hydroxylase encoding sequence (cefEF) of producing a yellow top spore in advance, and this conversion is to carry out under the regulation and control of Penicllium chrysogenum pcbC promotor and penDE terminator.
Described fragment is with amdS cotransformation (EP635574), and it can make the Penicllium chrysogenum transformant grow on the selection substratum that contains as the ethanamide of only nitrogen source.Transformant obtains purifying by cultivating repeatedly on the selection substratum.Single stable bacterium colony is used to the existence of the cefG gene that further examination obtains by PCR by measuring the ability that transformant produces hexanedioyl-7-ACA, and the positive bacterium colony of cefG is used to the expression of further examination cefG.
For this reason, transformant is inoculated into as in the described liquid nutrient medium of WO95/04149, has replenished the side chain precursor of the hexanodioic acid sodium of 0.5-3mg/ml as the preparation test.Well-grown culture filtrate is carried out the analysis of hexanedioyl-7-ACA output by HPLC and NMR.Table 1 result displayed clearly illustrates that, compare with the transformant that contains the cefG that originates in first ATG (being expressed as " ATG1 ") or the 3rd ATG (being expressed as " ATG3 "), the hexanedioyl-7-ACA output of transformant that contains the cefG of second ATG (being expressed as " ATG2 ") that originates in ORF increases to some extent.
Table 1
Combine originate in first, the hexanedioyl-7-ACA productive rate of the Penicllium chrysogenum transformant of the cefG encoding sequence of second or the 3rd ATG.
The CefG initiator codon Productive rate (%)
????ATG1 ????49
????ATG2 ????100
????ATG3 ????77
Sequence table＜110〉DSM N.V.＜120〉production method＜130〉Transacetylase＜140 in the body of improved cynnematin〉＜141＜150〉EP98204469.5＜151〉1998-12-22＜160〉11＜170〉PatentIn Ver.2.1＜210〉1＜211〉32＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #1;
5 ' 5 ' cefG1, delta SfiI＜400〉1tgctgccgtc cgcccaagtg gcccgtctaa ag 32＜210〉2＜211〉33＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #2;
3 ＇, 5 ' cefG1, delta HindIII＜400〉2aggcgacata tgggtgtcta gaaaaataat ggt 33＜210〉3＜211〉27＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #3;
5 ' 3 ＇ cefG1, delta HindIII＜400〉3gacacccata tgtcgcctca gatcgcc 27＜210〉4＜211〉33＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #4;
3 ' 3 ' cefG1 introduces NsiI＜400〉4cttttggaca cggatagctt agcctggatt gtc 33＜210〉5＜211〉32＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #5; 5 '
General Pipns primer＜400〉5ccaggctaag ctatccgtgt ccaaaagtat tc 32＜210〉6＜211〉53＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #6;
3 ' 5 ' cefG1 primer＜400〉6tgcattggct cgtcatgaag agcctatcac attaatgact gatcgaggaa tcc 53＜210〉7＜211〉20＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #7;
3 ' 5 ' cefG2 primer＜400〉7cacacaggaa gagagctcag 20＜210〉8＜211〉36＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #8;
5 ' 3 ' cefG2 primer＜400〉8ggacggcagc atatgggtgt ctagaaaaat aatggt 36＜210〉9＜211〉34＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #9; 3 '
The description of general cefG primer＜400〉9ccgcagcata tgggtgtcta gaaaaataat ggtg, 34＜210〉10＜211〉29＜212〉DNA＜213〉artificial sequence＜220＜223 artificial sequences: primer #10;
3 ' 5 ' cefG3 primer＜400〉10gacacccata tgctgcggga tagcctcac 29＜210〉11＜211〉20＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer #11;
5 ＇, 3 ' cefG3 primer＜400〉11aggcccattc cagagtgtgc 20

Claims (3)

1. method of producing the 7-ACA or derivatives thereof; comprise step: under the condition that has suitable acyl side-chain precursor or its salt or its ester to exist; to with containing the code extension enzyme; the Penicllium chrysogenum bacterial strain that the expression construct of the nucleotide sequence of hydroxylase and Transacetylase transforms ferments; produce N-acidylate 7-ACA compound thus; the N-acidylate 7-ACA compound that the N-deacylation so produces; randomly; acidylate free amine group and/or adopt to be fit to form the side substitution 3 ' acetoxyl of cephalosporin antibiotics, the nucleotide sequence of the Transacetylase that it is characterized in that encoding stems from second ATC that produces a yellow top spore and originate in the open reading-frame (ORF) that appears in the described nucleotide sequence.
2. the method for claim 1, wherein said side chain precursor is selected from by hexanodioic acid, 3 '-carboxymethyl propane thioic acid, 3, one group of material that 3 '-thio-2 acid and trans-β-hydrogenation muconic acid is formed.
3. the method for claim 1, wherein said side chain precursor is a hexanodioic acid.
CN99814782A 1998-12-22 1999-12-21 Improved i(in vivo) prodn. of cephalosporins Pending CN1331751A (en)

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EP98204469.5 1998-12-22

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JP (1) JP2002533092A (en)
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AU (1) AU3042600A (en)
HK (1) HK1041610A1 (en)
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