Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Patent
1997-06-19
1999-08-24
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
435 43, 435 47, 435 48, 435 711, 435 712, 435 713, 435183, 4352523, 43525231, 43525233, 43525235, 43525411, 4352543, 4352545, 4353201, 536 232, C12P 3506, C12P 3700, C12N 120, C07H 2104
Patent
active
059424117
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to biosynthesis of .beta.-lactam antibiotics. More specifically, the invention relates to in vivo and in vitro processes of producing .beta.-lactam antibiotics.
Also contemplated is a novel enzyme which advantageously can be used in the .beta.-lactam antibiotic biosynthesis. Further the invention relates to a DNA construct encoding said novel enzyme, a recombinant vector or transformation vehicle comprising said DNA construct, and finally a cell comprising said DNA construct or recombinant vector.
BACKGROUND OF THE INVENTION
The first step in the biosynthesis of penicillin involves the formation of the tripeptide .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine (ACV) from L-.alpha.-aminoadipic acid, L-cysteine and L-valine (Fawcett et al., Biochem. J., 157, p. 651-660, 1976). The reaction is catalyzed by a multifunctional enzyme .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase) with ATP and Mg.sup.2+ as co-factors (Banko et al., J. Am. Chem. Soc., 109, p. 2858-2860, 1987).
ACV synthetase (ACVS) has been purified from Aspergillus nidulans (Van Liempt et al., J. Biol. Chem., 264, p. 3680-3684, 1989), Cephalosporium acremonium (Baldwin et al., J. Antibiot., 43, p. 1055-1057, 1990) and Streptomyces clavuligerus (Jensen et al., J. Bacteriol., 172, p. 7269-7271, 1990, and Zhang et al., Biotechnol. Lett., 12, p. 649-654, 1990). The purification of ACV synthetase from Penicillium chrysogenum has not been published. However, ACV synthetase from P. chrysogenum has been cloned by Diez et al. (J. Biol. Chem., 265, p. 16358-16365, 1990).
The linear tripeptide, ACV, is converted to isopenicillin N (IPN) in the presence of isopenicillin N synthase (also referred to as cyclase or isopenicillin N synthetase (IPNS)), ferrous ions, oxygen and an electron donor (e.g. ascorbate). Isopenicillin N synthase was first isolated from P. chrysogenum by Ramos et al. (Antimicrobial Agents and Chemotherapy, 27, p. 380-387, 1985) and the isopenicillin N synthase structural gene from P. chrysogenum cloned by Carr et al. (Gene, 48, p. 257-266, 1986).
These first two steps in the biosynthesis of penicillins are common in penicillin and cephalosporin producing fungi and bacteria.
In some fungi, for example in P. chrysogenum and in A. nidulans, the .alpha.-aminoadipyl side chain of isopenicillin N can be replaced by other side chains of intracellular origin or exogenously supplied. The exchange is catalyzed by an acyltransferase (referred to as acyl-coenzyme A: isopenicillin N acyltransferase or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase). It is still unclear whether this conversion proceeds in vivo by a two-step reaction in which first the L-.alpha.-aminoadipyl side chain is removed to yield 6-aminopenicillanic acid (6-APA) followed by the acylation step, or the conversion is a direct exchange of the side chains. Purified acyltransferase from P. chrysogenum has both an isopenicillin N-amidohydrolase activity and an acyl-coenzyme A:6-aminopenicillanic acid acyltransferase activity (Alvarez et al. Antimicrobial Agents and Chemotherapy, 31, p. 1675-1682, 1987).
The genes coding for ACV synthetase (pcbAB), isopenicillin N synthase (pcbC) and acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (penDE) are found in the same cluster in P. chrysogenum and A. nidulans (Diez et al., J. Biol. Chem., 265, p. 16358-16365, 1990, and Smith et al., Bio/Technology, 8, p. 39-41, 1990).
Amplification of the pcbC-penDE gene cluster of P. chrysogenum Wis 54-1255, coding for isopenicillin N synthase (IPNS) and acyltransferase (AT), respectively, led to as much as a 40% improvement in production yields (Veenstra et al., J. Biotechnol., 17, p. 81-90, 1991). Increased antibiotic yields were also reported in A. nidulans transformants containing multiple copies of pcbAB (coding for ACV synthetase (ACVS)) and pcbC genes (coding for isopenicillin N synthetase (INPS)) (McCabe et al., J. Biotechnol., 17, p. 91-97, 1991).
EP 200425 (Eli Lilly) discloses vectors en
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Kaasgaard Svend
Kristiansen Klaus N.
M.o slashed.lgaard Henrik
Gist-Brocades
Stole Einar
Wax Robert A.
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