HK1041610A1 - Improved in vivo production of cephalosporins - Google Patents

Abstract

The present invention discloses a process for the production of 7-amino cephalosporanic acid (7-ACA) or a derivative thereof comprising the steps of fermenting a P. 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

Improved in vivo production of cephalosporins

The present invention relates to a process for the production of cephalosporins and in particular 7-ACA or derivatives thereof, comprising the steps of: fermenting a Penicillium chrysogenum strain transformed with an expression construct comprising nucleotide sequences encoding an elongase (expandase), a hydroxylase and an acetyltransferase in the presence of a suitable acyl side chain precursor or a salt or ester thereof, thereby producing an N-acylated 7-ACA compound, N-deacylating the N-acylated 7-ACA compound so produced and, optionally, acylating the free amino group and/or substituting the 3' acetate group with a side chain suitable for forming a cephalosporin antibiotic.

The semisynthetic route to cephalosporin is mostly initiated by fermentation products such as penicillin G, penicillin V, cephalosporin C, e.g. by the methods disclosed in k.matsumoto, bioprocessing technology (bioprocess. techn.), 16, 67-88(1993), j.g. shewale and h.sivaraman, processing biochemistry (process biochemistry), 8.1989, 146. 154, t.a.sayidge, industrial antibiotic biotechnology (ed.e.j.vandame) Marcel Dekker, new york, 1984, or j.g. shewale et al, process biochemistry International, 1990, 6.months, 97-103, to convert the fermentation products to the corresponding β -lactam parent nucleus. The resulting β -lactam nucleus is subsequently converted into the desired antibiotic by coupling with a suitable side chain, as described in particular in EP0339751, JP53005185 and CH 640240. By making different combinations of side chains and beta-lactam parent nucleus, a variety of penicillin and cephalosporin antibiotics can be obtained.

Cephalosporin parent nucleus 7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-aminocephalosporanic acid (7-ACA) are known to be important intermediates for the production of antibiotics for use in the pharmaceutical industry.

Cephalosporin C is apparently the most important starting material for the preparation of 7-ACA and other therapeutic cephalosporins. However, cephalosporin C is very soluble in water at any pH, thus implying that unconverted cephalosporin C in the product needs to be removed by long and costly separation processes using cumbersome and expensive column technology. In addition, the enzymatic or chemical cleavage necessary for the preparation of 7-ACA hardly works on the α -aminoadipoyl side chain of cephalosporin C.

To overcome some of the disadvantages described herein above, a fermentative process for the production of 7-ACA has been disclosed which comprises the fermentative production of certain N-substituted cephalosporins whose side chains can be easily removed by simple enzymatic or chemical cleavage reactions.

Fermentative production of these N-substituted cephalosporins, such as adipoyl 7-ACA, is achieved by a recombinant P.chrysogenum strain capable of expressing the enzymatic activities desacetoxycephalosporin synthase (also known as "elongase"), deacetylcephalosporin C synthase ("hydroxylase") and cephalosporin C synthase ("acetyltransferase") (EP 00540210).

In the in vivo production of adipoyl 7-ACA using the recombinant P.chrysogenum strain, the presence of a large amount of precursors of adipoyl 7-ACA, adipoyl-7-aminocephalosporanic acid (adipoyl-7-ADAC) was observed compared to adipoyl 7-ACA. Apparently, the acetyltransferase gene was not expressed in an amount sufficient for the mass transformation of adipoyl-7-ADAC into adipoyl-7-ACA.

The present invention discloses an acetyltransferase expression construct designed to obtain high levels of expression of an acetyltransferase. Thus, the amount of conversion of the precursor adipoyl-7-ADAC to adipoyl-7-ACA was increased.

A literature describing the cloning and nucleotide sequence of the acetyltransferase gene from Acremonium chrysogenum (cefG) discloses a coding sequence starting from the first of the acetyltransferase Open Reading Frames (ORF) (EP 0437378; Gutierrez et al, J. Bacteriol. 174: 3056-3064(1992)), or the second (Mathison et al, Curr Genet. 23: 33-41(1992)) or the third ATG (EP 0450758). In addition, during the study of the efficiency of the expression of acetyltransferase from different promoters in A.chrysogenum, the construct tested was a fusion construct containing the acetyltransferase coding sequence starting from a second ATG (Gutierrez et al, applied microbiology Biotechnology 48: 606-614 (1997)).

None of the documents cited herein above discloses the advantage of selecting the start codon of a particular cefG 0RF to obtain efficient expression of acetyltransferases in recombinant P.chrysogenum strains for fermentation processes for the production of 7-ACA or derivatives thereof.

The invention discloses a method for producing 7-ACA or derivatives thereof, which comprises the following steps: a Penicillium chrysogenum strain transformed with an expression construct comprising nucleotide sequences encoding an elongase, a hydroxylase and an acetyltransferase activity, is subjected to fermentative culture in the presence of a suitable acyl side chain precursor or a salt or ester thereof, thereby producing an N-acylated 7-ACA compound, N-deacylating the N-acylated 7-ACA compound thus produced, and optionally acylating the free amino group and/or substituting the 3' acetate group with a side chain suitable for forming a cephalosporin antibiotic, characterized in that the nucleotide sequence encoding the acetyltransferase is derived from Acremonium chrysogenum and starts at the second ATG of said nucleotide sequence.

It has surprisingly been found by the present invention that expression constructs comprising an acetyltransferase coding sequence derived from A. chrysogenum express more efficiently when the coding sequence starts at the second ATG of an Open Reading Frame (ORF) than when it starts at the first or third ATG of said ORF. One of the effects of more efficient expression of acetyltransferase is that N-acylated 7-ADAC derivatives are converted into N-acylated 7-ACA derivatives more efficiently.

In the process of the invention, the transformed P.chrysogenum strain is used to express three enzyme activities in the cephalosporin biosynthesis pathway leading to the production of the 3' -acylated cephalosporin compound.

Suitable sources of the genes encoding the three enzymatic activities are the bacterial Streptomyces clavuligerus or Nocardia lactamdurans for obtaining the elongase and hydroxylase genes cefE and cefF (see EP0341892 for cefE and EP0465189 for cefF) or the fungal Acremonium chrysogenum for obtaining the bifunctional elongase/hydroxylase genes cefEF and acetyltransferase genes cefG (see EP0281391 for cefEF and Coque et al, mol.Gen.Genet.236: 453) 458(1993) and EP0437378 and EP0450758 for cefG).

According to the invention, the acetyltransferase activity is provided by the cefG gene obtained from A. chrysogenum. In particular, the present invention shows that it is advantageous to use the second ATG of the cefG ORF as the start codon. The use of the second ATG of the cefG ORF as initiation codon means that the acetyltransferase used in the method of the invention has an N-terminal amino acid sequence which starts with methionine-leucine-arginine-aspartic acid-serine.

In the method of the present invention, the genes encoding the three enzymatically active elongases, hydroxylating enzymes and acetyltransferases may carry the 5 'and 3' regulatory sequences of the gene itself under consideration or may carry regulatory sequences heterologous to the said genes.

Examples of suitable 5 'and 3' regulatory sequences, promoters and terminators provided for recombinant Gene expression in filamentous fungal host cells, are mentioned in Van den Hondel et al (in: More Gene management in Fungi, eds. Bennett and Lasure, 396-427(1991)) or in applied molecular genetics in filamentous Fungi (Kinghorn, Turner (eds.), Blackie, Glasgow, UK, 1992). Preferred promoters are the A.niger glucoamylase promoter or the P.chrysogenum promoter from the genes encoding ACV synthase, isopenicillin N synthase, acetyltransferase, phosphoglycerate kinase or gene Y. The transcription terminator can also be obtained from the same gene.

In one embodiment of the invention, a method for the fermentative production of 7-ACA or derivatives thereof is provided comprising the use of a strain of penicillium chrysogenum transformed with an expression construct in which the coding sequence for the gene encoding elongase, hydroxylase and/or acetyltransferase activity is fused to a promoter sequence heterologous to said coding sequence. The heterologous promoter sequence is, for example, the IPNS (pcbC) promoter from Penicillium chrysogenum.

In another embodiment of the invention, precise fusion between the selected promoter sequence and the start codon of the coding sequence encoding the activity of the elongase, hydroxylase and/or acetyltransferase can be conveniently achieved by employing PCR techniques.

Transformation of a P.chrysogenum host cell can generally be accomplished by different means of DNA delivery, like PEG-Ca mediated protoplast uptake, electroporation or particle gun techniques, and subsequent selection of transformants. See, for example, Van den Hondel en Punt, the development of genes and transfer and vectors for filamentous fungi, in: application of the fungus molecular genetics (Peberdy, Laten, Ogden, Bennett, eds.), Cambridge university Press (1991). The use of dominant and non-dominant selection markers has been described (Van den Hondel, supra). Selection markers both homologous (derived from P.chrysogenum) and heterologous (not derived from P.chrysogenum) have also been described (Gouka et al, J.Biotechnol. (J.Biotechnol.). 20189-200 (1991)).

The use of homologous or heterologous, with or without vector sequences, different transformant selection markers physically linked or not to the non-selective DNA during selection of transformants is known in the art.

The fermentative culture of the transformed P.chrysogenum strain can be carried out in any suitable fermentation medium known in the art, provided that the fermentation is carried out in the presence of a suitable side chain precursor. In this respect, a suitable side chain precursor is defined as an N-acyl side chain precursor capable of producing the N-acyl side chain of a fermentatively-produced cefepime compound, said N-acyl side chain being capable of simple chemical or enzymatic removal. In particular, suitable side chain precursors are dicarboxylic acids, more particularly dicarboxylic acids of formula (1)

HOOC-X-(CH₂)_n-COOH (1)

Wherein

n is an even number of at least 2, and

x is (CH)₂)_p-A-(CH₂)_qWherein

p and q are each 0, 1, 2, 3 or 4, respectively, and

a is CH-CH, C.ident.C, CHB, C.ident.O, O, S, NH, nitrogen being optionally substituted or sulfur being optionally also oxidized, and B is hydrogen, halogen, C_1-3Alkoxy, hydroxy or optionally substituted methyl, with the proviso that p + q should be 0 or 1 when a is CH-CH, p + q should be 2 or 3 when a is CH or C or p + q should be 3 or 4 when a is CHB, C ═ O, S or NH, or a salt or ester thereof.

Examples of suitable side chain precursors according to formula (1) are adipic acid, 3 '-carboxymethylthiopropionic acid (WO95/04148), 3' -thiodipropionic acid (WO95/04149) or side chain precursors as provided in WO98/48034 or WO 98/48035. Preferred side chain precursors are adipic acid or trans-beta-hydromuconic acid.

N-acylated 7-ACA compounds, such as adipoyl-7-ACA, obtained by fermentation in the presence of a suitable acyl side chain precursor, such as adipic acid, can be efficiently recovered from the fermentation medium by employing conventional recovery techniques, such as the following simple solvent extraction methods:

the culture solution was filtered and an organic solvent immiscible with water was added to the filtrate. The pH was adjusted to extract the cephalosporin from the aqueous layer. The pH range must be below 4.5; preferably between 4 and 1, more preferably between 2 and 1. This separates the cephalosporin from many other impurities present in the fermentation broth. Preferably, a small amount of organic solvent is used, resulting in a more concentrated cephalosporin solution and thus a reduced volumetric flow rate. A second possibility is to perform the extraction of the whole broth at a pH of 4 or lower. It is preferable that the culture broth is extracted with a water-immiscible organic solvent at a pH in the range of 4 to 1.

Any solvent that does not affect the cephalosporin molecule may be used. Suitable solvents are, for example, butyl acetate, ethyl acetate, methyl isobutyl ketone, alcohols such as butanol, etc.

Thereafter the cephalosporin is back-extracted with water between pH4 and pH10, preferably between pH6 and pH 9. The final volume can be reduced again. The recovery may be carried out at a temperature of from 0 ℃ to 50 ℃, preferably from 0 ℃ to 10 ℃.

Since the 7-amino group is suitably protected by the presence of a suitable acyl side chain, the N-acylated cephalosporin derivatives produced by the process of the invention can be conveniently used as intermediates in the chemical synthesis of semi-synthetic cephalosporins.

Alternatively, to remove the N-acyl group, e.g. adipoyl, the side chain and to obtain the desired 7-ACA, the aqueous solution of the N-acylated cephalosporin thus obtained is treated with a suitable enzyme.

Preferably, an immobilized enzyme is used so that the enzyme can be reused. Methods for the preparation of such particles and the immobilization of enzymes have been extensively described in EP 0222462. The pH of the aqueous solution is, for example, from pH4 to pH9, in which range the degradation reaction of the cephalosporin is minimized and the required enzymatic conversion is optimized. The enzyme is added to an aqueous solution of cephalosporin while the pH is maintained at an appropriate level, for example by addition of an inorganic base such as potassium hydroxide solution or by use of a cation exchange resin. When the reaction was complete, the immobilized enzyme was removed by filtration. Another possibility is to apply the immobilized enzyme in the form of an immobilized or fluidized bed column, or to use the enzyme in the form of a solution and to remove the product by membrane filtration. The pH of the aqueous solution is then adjusted to a value between 2 and 5, preferably between 3 and 4. Then the crystalline 7-ACA is filtered off.

Suitable enzymes are, for example, derived from a pseudomonas SY77 microorganism having mutations in one or more of positions 62, 177, 178 and 179. Enzymes derived from other Pseudomonas microorganisms, preferably Pseudomonas SE83, may also be used, optionally with mutations at one or more of the positions corresponding to positions 62, 177, 178 and 179 of Pseudomonas SY 77.

Deacylation can also be carried out by chemical methods known in the art, for example, by addition of phosphorus pentachloride at a temperature below 10 ℃ followed by addition of an alcohol such as isobutanol at or below room temperature to form the side chain of the imminichloride.

In one embodiment of the invention, an aqueous solution containing the N-acylated 7-ACA derivative or the 7-ACA obtained after deacylation may be treated with a suitable acylating agent in order to convert any (acyl) -7-ADAC which may be present in said aqueous solution into the corresponding (acyl) -7-ACA derivative. Said acylation can for example be carried out by using acetic anhydride, for example by the method disclosed in US5221739, or by using a suitable lipase or esterase, for example the method disclosed in EP 667396.

In a further step, the 7-ACA compounds obtained by the process of the invention are used as starting, end and intermediate products for the preparation of various cephalosporin antibiotics. The free amino group of 7-ACA can be acylated, for example, by any suitable side chain, by employing well known chemical or enzymatic coupling procedures, resulting in the production of N-acylated 7-ACA derivatives. In addition, substitution reactions may occur at the 3' position. Examples of cephalosporin compounds thus prepared are cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cefprozil, ceftazidime and cefchlor.

Example 1

pICG for expressing acetyltransferase in P.chrysogenum₁WA、pICG₂WA and pICG₃WA constructs

Deacetylcephalosporin C acetyltransferase (cefG) expression cassette pICG containing the wild-type A. chrysogenum cefG gene comprising the P.chrysogenum pcbC promoter and the penDE terminator₁WA WAs constructed as follows. The N-terminal part of the cefG gene, i.e.the beginning of the first ATG of the ORF, was obtained from chromosomal DNA of A. chrysogenum by PCR using primers #1 and #2 (SEQ ID NO1 and 2, respectively). The C-terminal part of the cefG gene was obtained from the same template by PCR reaction using primers #3 and #4 (SEQ ID NO3 and 4, respectively). After fusion PCR using primers #1 and #4 and the above fragments as templates, an intact cefG gene (further denoted cefG herein) was generated₁Gene) in which the cefG gene is deleted. SfiI and HimdIII sites to create a new MsiI site.

In the next step, the first part of the pcbC promoter was PCR-amplified with primers #5 and #6 (SEQ ID NO5 and 6, respectively) and introduced directly into cefG after fusion PCR with primers #5 and #4₁In front of the gene. After digestion with PstI/NsiI, a 1592bp fragment was ligated to the PstI/NsiI vector fragment of a 4.3kb pISEWA-N (vector previously described in WO 98/46772) to yield the Penicillium transformation vector pICG₁WA。

Construction of pICG₂cefG required for WA₂The N-terminal part of the gene, i.e.the start of the second ATG of the ORF, was derived from pICG in a PCR reaction using primers #5 and #7(SEQ ID NO7)₁WA。CefG₂The C-terminal part of the gene was derived from PCR reaction using primers #8 and #9 (SEQ ID NO8 and 9, respectively)The same template. Fusion PCR using primers #5 and #9 and the above fragment as templates yielded intact cefG₂A gene.

To construct pICG₃WA, in which the cefG gene starts at the third ATG, using the same pICG₂Construction of WA the same procedure. To accomplish this, primers #5/#10 (SEQ ID NO5/10, respectively) and #11/#9 (SEQ ID NO 11/9, respectively) were used.

After digestion of the internal PstI/NcoI site, cefG₂/cefG₃The fusion fragment was ligated to pICG₁On the PstI/NcoI vector fragment of WA, the Penicillium transformation vector pICG WAs generated₂WA and pICG₃WA。

Example 2

Effect of different ATG initiation codon choices on the expression of acetyltransferases

After NotI digestion of the different pICWGA constructs, the isolated cefG fragment was introduced into P.chrysogenum by Ca-PEG mediated protoplast transformation as described in EP 635574.

The Penicillium chrysogenum strains used have previously been transformed with an expression construct derived from A. chrysogenum, containing the bifunctional elongase/hydroxylase coding sequence (cefEF), under the control of the P.chrysogenum pcbC promoter and the penDE terminator.

Said fragment was co-transformed with amdS (EP635574), which enables the growth of Penicillium chrysogenum transformants on selective media containing acetamide as sole nitrogen source. The transformants were purified by repeated culturing on a selective medium. Single stable colonies were used for further screening for the presence of cefG gene obtained by PCR cefG-7-ACA production by determining the transformants and cefG-positive colonies were used for further screening for cefG expression.

For this purpose, the transformants were inoculated into liquid medium as described in WO95/04149, supplemented with 0.5-3mg/ml of sodium adipate as side chain precursor for the preparation experiments. The well-grown culture filtrate was analyzed for adipoyl-7-ACA production by HPLC and NMR. The results shown in Table 1 clearly show an increase in adipoyl-7-ACA production by transformants containing cefG starting from the second ATG of the ORF (indicated as "ATG 2") compared to transformants containing cefG starting from the first ATG (indicated as "ATG 1") or the third ATG (indicated as "ATG 3").

TABLE 1

adipoyl-7-ACA yields from P.chrysogenum transformants incorporating the cefG coding sequence starting from the first, second or third ATG.

CefG initiation codon	Yield (%)
		ATG1	49
ATG2	100
		ATG3	77

In vivo production of improved cephalosporins of sequence Listing <110> DSM N.V. <120> method <130> acetyltransferase <140> <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> description of the artificial sequence: primer # 1;

description of 5 '5' cefG1, delta SfiI <400>1tgctgccgtc cgcccaagtg gcccgtctaa ag 32<210>2<211>33<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: primer # 2;

3 '5' cefG1, delta HindIII <400>2aggcgacata tgggtgtcta gaaaaataat ggt 33<210>3<211>27<212> DNA <213> Artificial sequence <220> <223> description of the artificial sequence: primer # 3;

5 '3' cefG1, delta HindIII <400>3gacacccata tgtcgcctca gatcgcc 27<210>4<211>33<212> DNA <213> Artificial sequence <220> <223> description of the artificial sequence: primer # 4;

3 '3' cefG1, a description of NsiI <400>4cttttggaca cggatagctt agcctggatt gtc 33<210>5<211>32<212> DNA <213> artificial sequence <220> <223> artificial sequence was introduced: primer # 5; 5'

Description of the Universal Pipns primer <400>5ccaggctaag ctatccgtgt ccaaaagtat tc 32< 32 >6<211>53<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: primer # 6;

description of 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: primer # 7;

description of the 3 '5' cefG2 primer <400>7cacacaggaa gagagctcag 20<210>8<211>36<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: primer # 8;

description of 5 '3' cefG2 primer <400>8ggacggcagc atatgggtgt ctagaaaaat aatggt 36<210>9<211>34<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: primer # 9; 3'

Description of the Universal cefG primer <400>9ccgcagcata tgggtgtcta gaaaaataat ggtg 34< 34 >10<211>29<212> DNA <213> Artificial sequence <220<223 Artificial sequence: primer # 10;

description of 3 '5' cefG3 primer <400>10gacacccata tgctgcggga tagcctcac 29<210>11<211>20<212> DNA <213> Artificial sequence <220> <223> Artificial sequence: primer # 11;

5 '3' cefG3 primer <400>11aggcccattc cagagtgtgc 20

Claims (3)

1. A method for producing 7-ACA or a derivative thereof, comprising the steps of: fermentation of a strain of penicillium chrysogenum transformed with an expression construct comprising a nucleotide sequence encoding an elongase, a hydroxylase and an acetyltransferase in the presence of a suitable acyl side chain precursor or a salt or ester thereof, thereby producing an N-acylated 7-ACA compound, N-deacylating the N-acylated 7-ACA compound thus produced, and optionally acylating the free amino group and/or substituting the 3' acetate group with a side chain suitable for forming a cephalosporin antibiotic, characterized in that the nucleotide sequence encoding the acetyltransferase is derived from acremonium chrysogenum and starts at the second ATC of the open reading frame present in said nucleotide sequence.

2. The method of claim 1 wherein the side chain precursor is selected from the group consisting of adipic acid, 3 '-carboxymethylthiopropionic acid, 3' -thiodipropionic acid, and trans- β -hydromuconic acid.

3. The method of claim 1, wherein the side chain precursor is adipic acid.