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Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus : Kinetics of adipoyl‐7‐aminodeacetoxycephalosporanic acid and byproduct formations
Author(s) -
Robin J.,
Bruheim P.,
Nielsen M. L.,
Noorman H.,
Nielsen J.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10678
Subject(s) - streptomyces clavuligerus , penicillium chrysogenum , chemostat , biochemistry , chemistry , chromatography , high performance liquid chromatography , bioreactor , enzyme kinetics , amidase , biology , stereochemistry , enzyme , bacteria , organic chemistry , clavulanic acid , genetics , amoxicillin , active site , antibiotics
The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl‐7‐aminodeacetoxycephalosporanic acid (ad‐7‐ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high‐performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC‐MS) showed that this byproduct has a six‐membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as‐yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various β‐lactams produced showed higher instability for compounds with a five‐membered thiazolidine ring than those with a six‐membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h −1 . The specific productivity of total β‐lactams was compared with that of the penicillin‐G‐producing host strain, and it was found to be lower at dilution rates of <0.06 h −1 . Quantification of the fluxes through the pathway leading to ad‐7‐ADCA showed a decrease in flux toward ad‐7‐ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad‐7‐ADCA pathway decreased as the dilution rate increased. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 353–360, 2003.