Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl‐7‐aminodeacetoxycephalosporanic acid and byproduct formations

Abstract

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.

Conceptual Process Design of Integrated Fermentation, Deacylation, and Crystallization in the Production of β-Lactam Antibiotics

“…The fermentation product of the ADCA process, adADCA, has a degradation rate that follows first-order kinetics with a value of the rate constant of 5.3 × 10 -4 h -1 at 25 °C and pH 6.5. 19 This value is in agreement with those from other literature 13 and will be used for the model calculations.…”

“…The production rate equations for Pen G and adADCA were obtained from the experimental data published. 26,19 The equation for q PenG is valid for specific growth rate (µ) values between 0 and 0.075 h -1 and q adADCA for values between 0.03 and 0.05 h -1 . The current industrial production rates for Pen G and adADCA production are probably much higher, but they have not been reported.…”

“…Therefore, 60% of the published yield of Pen G on substrate was used for the adADCA fermentation model. This assumption was based on the fact that the published maximum productivity of the adADCA fermentation process is 60% of the maximum productivity of the Pen G fermentation . Also the maintenance coefficient and the K s for glucose are not known for the transformed strain and have been assumed the same as for the nontransformed, Pen G producing strain.…”

The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics

“…intermediates of cephem antibiotics. In contrast to penicillins, like penicillin G or penicillin V, which have PAA and POA as a sidechain precursor respectively, microbial semi-synthetic cephalosporin (SSC) precursor biosynthesis uses adipic acid as side-chain precursor resulting in formation of adipoyl-7-ACA or adipoyl-7-ADCA (Crawford et al, 1995;Robin et al, 2003b). In a subsequent enzymatic step, the adipoyl side-chain can be easily removed by an acylase leading to formation of cephalosporin precursors (7-ACA, 7-ADCA).…”

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

“…The secondary structures of residues 81-90 and 167-177 were highly disordered and were not determined [84]. The cylinders, arrows, and black lines represent -helices, -sheets, and coil structures, respectively expression in E. coli [28,34,49], S. lividans [22,33], Penicillium chrysogenum [13,24,68,[70][71][72][73], Pseudomonas putida [47], and Pichia pastoris [3]. High level expression of recombinant scDAOCS has often been carried out in E. coli cells together with the use of traditional extraction to purify scDAOCS from E. coli cell lysate for biochemical studies [57,58,90].…”