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 beta-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 beta-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.

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h(-1). After three to five residence times, adipate degradation and ad-7-ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h(-1). Both variables decreased when the dilution rate was increased above 0.065 h(-1). A correlation between mean total hyphal length and productivity of ad-7-ADCA was found.

No abstract

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and byproducts of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.