Oxido reductase enzyme system obtained from P. chrysogenum

Details Oxido reductase enzyme system obtained from P. chrysogenum Oxido reductase enzyme system obtained from P. chrysogenum

1992-03-24

1994-07-12

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

435189, 435935, C12N 906, C12N 902, C12N 114

Patent

active

053288394

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to an enzyme system involved in the production of .beta.-lactam compounds and to the use of enzyme systems for increasing antibiotic production.
Penicillins and cephalosporins are the most widely used antibacterial agents. Penicillins and cephalosporins are secondary metabolites which are industrially produced by filamentous fungi like Penicillium chrysogenum and Acremonium chrysogenum, respectively, in several enzymatic steps (J. R. Miller and T. D. Ingolia, Mol. Microbiol. (1989) 3:689-695).
The main steps in the biosynthetic pathways leading to cephalosporins and penicillins have been elucidated in the past 30 years. The pathways share two enzymatic steps. In the first step a tripeptide is formed from .alpha.-aminoadipic acid, cysteine and valine. The enzyme which is responsible for this step is .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase. In the second step the ACV is cyclised by the action of isopenicillin N synthetase (hereinafter referred to as IPNS) or cyclase. The reaction product is isopenicillin N, a compound which contains the typical .beta.-lactam ring structure and which possesses antibacterial activity. The biosynthesis of penicillin involves a unique third and last step in which the .alpha.-aminoadipic acid side-chain of isopenicillin N is exchanged for a hydrophobic side-chain. The hydrophobic side-chains commonly used in industrial production are phenylacetic acid (PAA) and phenoxyacetic acid (POA) yielding penicillin G and penicillin V, respectively. The side-chain exchange has been proposed to be a reaction catalyzed by a single enzyme referred to as acyltransferase (AT). Cephalosporins are formed from isopenicillin N in a number of steps including epimerization of isopenicillin N to penicillin N, ring expansion and hydroxylation.
The biosynthetic pathways to penicillins and cephalosporins have almost completely been elucidated and most of the enzymes involved in the biosynthesis towards penicillins and cephalosporins have been purified and characterized (Ingolia et al., Med. Res. Rev., (1989) 9:245-256). The genes encoding these enzymes have been cloned and it is commonly accepted that the introduction of expression vectors containing DNA encoding these enzymes into production strains may improve the yield of .beta.-lactam compounds. Recently, an example of the successful application of the expression of extra copies of genes has been described (Skatrud et al., Biotechnology, (1989) 7:477-485).
It has been recognized that maintaining reducing conditions is important for producing .beta.-lactam compounds. For instance, under non-reducing conditions the tripeptide ACV dimerizes into ACV disulphide (bis-ACV) or possibly into mixed disulphides with other thiol-containing compounds. These disulphides are not used as substrates by IPNS. Furthermore, the enzyme IPNS is less active in non-reducing conditions (Perry et al, Biochem. J. (1988) 255:345-351). Reducing compounds such as dithiothreitol (DTT) are used to maintain reducing conditions in vitro. In this way a process for producing cephalosporins from tripeptide precursors as well as unnatural penicillin and cephalosporin derivatives using isolated enzymes has been developed (U.S. Pat. Nos. 4,510,246 and 4,536,476). Moreover, a process for producing tripeptides using the enzyme ACVS has been described in EP-A-280051. At this moment it is not known how enzymes and disulphide compounds, for instance ACV, involved in the production of .beta.-lactam compounds are maintained in a reduced state in P. chrysogenum or other .beta.-lactam producing microorganisms. Surprisingly a yet unreported enzyme system with reducing properties which is able to reduce bis-ACV amongst other disulphide compounds, has been isolated. The enzyme system comprises two polypeptide components, a high and a low molecular weight polypeptide, and an electron carrier, such as nicotinamide adenine dinucleotide phosphate (NADPH), in its reduced form. The polypeptide components share similarity with thioredoxin reductase

REFERENCES:
patent: 4250258 (1981-02-01), Szarka et al.
patent: 4373026 (1983-02-01), Yamada et al.
Russel et al, J. Biol. Chem. vol. 263, No. 18, Jun. 25, 1988, pp. 9015-9019.
Holmgren, A. Ann. Rev. Biochem "Thioredoxin" 54:237-271 (1985).

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