Europaisches Patentamt J) European Patent Office Publication number: 0 280 051 Office europden des brevets A1 EUROPEAN PATENT APPLICATION © Application number: 88101105.0 © int. cia C07K 5/06 , C12P 37/00 , C12P 35/00, C12N 9/00, @ Date of filing: 26.01.88 //C12R1/75 ® Priority: 17.02.87 US 15062 © Applicant: QUEEN'S UNIVERSITY AT KINGSTON © Date of publication of application: 31.08.88 Bulletin 88/35 Kingston Ontario K7L 3N6(CA) © Designated Contracting States: Applicant: MASSACHUSETTS INSTITUTE OF AT CH DE FR GB IT LI SE TECHNOLOGY 77 Massachusetts Avenue Cambridge, MA 02139(US) © Inventor: Banko, Gerald. Chemie Linz A.G. CEC-BI StPeter Strasse 25 A-4026 Llnz(AT) Inventor: Wolfe, Saul R.R.Number 1 Kingston Ontario K/L 4V1(CA) Inventor: Demain, Arnold 65 Grove Street, Apt 251 Wellesley, Massachusetts(US) © Representative: Popp, Eugen, Dr. et al MEISSNER, BOLTE & PARTNER Widenmayerstrasse 48 Postfach 86 06 24 D-8000 MUnchen 86(DE) ACV synthetase. r"® A process for producing peptides and /S-lactam antibiotics from their amino acid precursors, and analogs ^thereof, is described. A mixture of ^ -a-aminoadipic acid (A), J» -cysteine (C) and L, -valine (V) is en- ^zymatically converted into L L D -ACV by a glycerol stabilized cell free extracted of Cephalosporium macremonium (Acremonium chrysogenum ATCC48272). The cell free extract is a single enzyme defined as ACV- O synthetase which requires all three ^ -amino acids for maximum activity. O 00 a. UJ Xerox Copy Centre 0 280 051 ACV SYNTHETASE Background of the Invention 1 . Field of the Invention 5 This invention relates to a cell free process for producing peptides and jS-lactam antibiotics from the amino acid precursors and analogs thereof. More particularly this invention relates to enzymatic synthesis of 5-( L-a-aminoadipyl)- j^-cysteinyl- £) -valine (ACV) and analogs thereof for subsequent conversion to a penicillin, cephalosporin or cephamycin. m 2. The Prior Art. The cell free synthesis of penicillin and certain related cephalosporins from ACV tripeptide precursors is thereof using an extract from the eukaryotic organism Cephalosporium acremonium is known, and attention is directed to Demain et al U.S. patents 4,178,210 issued 11 December 1979, 4,248,966 issued 3 February 1981, 4,307,192 issued 22 December 1981. The lability of the epimerase agent described in these references is believed to preclude use of cell free extracts of C; acremonium for high yield commercial production of cephalosporins. This belief led Wolfe et al to investigate the production of stable cell free 20 extracts from prokaryotic organisms such as Streptomyces clavuligerus, Streptomyces lipmanii and Strep- tomyces cattleya, and attention is directed to Wolfe et al U,S. patents 4,510,246 issued 9 April 1985, 4,536,476 issued 20 August 1985, and 4,579,818 issued 1 April 1986, and use of these extracts to produce the desired penicillins and related cephalosporins from the tripeptide precursors. Each of these processes, however, requires the peptide precursor and generally the chemical synthesis thereof is relatively costly. 25 It is, therefore, an object of the present invention to provide a biosynthetic process for producing ACV and analogs thereof. Summary of the Invention 30 It has now been discovered that appropriately treated extracts of C.acremonium convert a mixture of the three amino acids i. -a-aminoadipic acid (A), j^ -cysteine(C) and ^ -valine (V) into ACV. Thus, by one aspect of this invention there is provided a process for providing peptides from the amino acid precursors thereof, comprising: treating a mixture of said amino acids with a disrupted cell preparation 35 of an organism producing at least one of penicillins, cephalosphorins, and cephamycins. By another aspect there is provided A-C-V synthetase. By yet another aspect a process for producing peptides from the amino acid precursors thereof, comprising: treating a mixture of said amino acids with a disrupted cell preparation of an organism producing at least one of penicillins cephalosporins and cephamycins. 40 Description of the Preferred Embodiments The biosynthesis of penicillins and cephalosporins is a linear process in both eukaryotic and prokaryotic 45 organisms. The process begins, at the amino acid oxidation level, with the coupling of j- -a-aminoadipic and to form the -valine acid, L. -cysteine =^-valine tripeptide 5-( JL-a-aminoadipyl)- 4-cysteinyl- Q kLB- ACV). This peptide 50 0 280 051 (1) SH 1 •' 1 n n 1 ; ° C02H //~ H^Y^ w CC^H is then converted sequentially into isopenicillin N, (2) 15 H H 20 penicillin N, (3) 25 H H 30 desacetoxycephalosDorin C, (4a) X = H, H hi 35 \5^\x-^N 4 \^ (4) HoNi2r mu— 40 desacetylcephalosporin C (4b): X = OH, and cephalosporin C (4c): X = OAc or carbamoylox- ycephalosporin C (4d): X = OCONHS, the nature and oxidation level of the ultimate product depending 45 upon the organism. As described in the references above, each of the steps has been observed under cell-free conditions, using homogeneous enzymes, functionally purified enzymes, or mixtures of enzymes, and the sequence 1— 2— 3— 4(a)-*4(b) has also been accomplished in quantitative yield on a single immobilized enzyme reactor. A variety of analogs of ACV, in which the L -a-aminoadipyl and/or £ -valinyl moieties have been 50 altered by chemical synthesis, are accepted by one or more of the antibiotic-forming enzymes, and converted into nuclear and/or side chain modified penicillins/cephalosporins. Several of these nuclear modified cephalosporins have also been prepared by multi-step chemical syntheses from penicillin precursors. It has thus been demonstrated that novel ring systems, in sufficient quantities for biological evaluation, are more readily accessible from peptide precursors and the appropriate combinations of 55 enzymes and cofactors, than by the more traditional methods of organic chemistry. It must be emphasized, however, that heretofore a peptide precursor has always been required. Nonetheless, the strategy just described still requires a peptide precursor. Although the requisite chemical syntheses have been solved, and ^ -carboxymethylcysteine found to be an effective alternative 0 280 051 to L.-a-aminoadipic acid, a replacement of the valinyl moiety of the peptide still requires the synthesis, resolution and incorporation of a £ -amino acid. In addition, an average of 10-13 protection, coupling and deprotection steps are necessary, from the amino acids, for each new peptide. An enzymatic synthesis of tripeptide analogs of k.k,B -ACV would, therefore, be of value. 5 The formation of LLD -ACV from its amino acid precursors has received only sporadic biosynthetic attention. In an early study, a tripeptide was reported to be formed by a cell-free preparation from Penicillium chrysogenum, but the configurations of the amino acids in this peptide were not determined. Experiments with particulate fractions of Cephalosporium sp. suggested that i -a-aminoadipyl- j^-cysteine (LL-AC) is an intermediate in the formation of lld -ACV and that k. -to Q -epimerization of valine 10 occurs during its attachment to LL-AC. More recently, AC-synthetase activity in cell-free extracts of P. chrysogenum has been confirmed, and soluble extracts of a non-antibiotic producing mutant of Cephalosporium acremonium were found to give 0.1-0.4% incorporation of labelled amino acids into LLD -ACV, and 0.03-0.15% incorporation into LL-AC. It was thought that LLD -ACV biosynthesis parallels glutathione biosynthesis, and involves the action of two separate enzymes. ?5 It has now been found, however, that the ACV-synthetase of C; acremonium C-10 is a single multifunctional enzyme, with broad substrate specificity, whose behavior is more properly compared to that of the multifunctional enzymes associated with the biosyntheses of the peptide antibiotics gramicidin S, tyrocidine, bacitracin, polymyxin and enniatin. It was first thought that the ATP-and Mn2+-or Mg2+ -dependent activity that is stabilized by addition of 20 glycerol during the preparation of cell-free extracts, was caused by the presence of a barely detectable AC- synthetase. If two enzymes were required for k^k-D -ACV synthesis, and the first exhibited inhibition by its product, lld -ACV formation via the reaction LL-AC + k -valine would be faster than LL-AC formation from j^ -a-aminoadipate + k -cysteine. That this is the case is seen in Table I, which shows data for the two reactions. 25 TABLE I. Peptide Formation by ACV Synthetasea Conversion ( g/ml) 30 Reactants Product 15 rain 30 min 60 rain A + C LL-AC 0.2 35 LL-AC+V LLD-ACV 0.9 1.1 A+C+V LL£-ACV 5.2 10.2 40 — — @ a Typical reaction mixtures contained desalted extract (about 0.5 mg protein), 20 ag cycloheximide, 10 mM ATP, 10 mM MgCij, 5 mM dithiothreitol, 5 mM k. -a-aminoadipic acid, I mM k. -cysteine and 5 mM k. -valine, all components dissolved in 100 ul of 100 mM MOPS/KOH at pH 7.5. Reactions were performed 45 at 25° C in a water bath shaker, and terminated by addition of 25 til of 20% trichloroacetic acid. Precipitated protein was removed by centrifugation, and the supernatant stored at -20 °C prior to analysis. (MOPS = 3- [N-Morpholino] propanesulfonic acid, - a biological buffer available from Sigma Chemical Co. However, Table I also shows the unexpected finding that the conversion of L, -a-aminoadipic acid + k -cysteine + JL, -valine to LLD -ACV is far more rapid. These observations are not compatible with 50 the hypothesis that two enzymes are required are required for the synthesis of LLD -ACV from its components. Rather, a single enzyme, hereinafter ACV-synthetase, must carry out the synthesis of lld -ACV, and this enzyme requires binding of all three amino acids for maximum activity.