title: 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.
authors.affiliation: Center for Process Biotechnology, BioCentrum-DTU, Building 223, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Robin
authors.forename: P
authors.initials: P
authors.lastname: Bruheim
authors.forename: M L
authors.initials: ML
authors.lastname: Nielsen
authors.forename: H
authors.initials: H
authors.lastname: Noorman
authors.forename: J
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D018929:Cell Culture Techniques / Q000379:methods*; D002511:Cephalosporins / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000096:biosynthesis*; D007700:Kinetics; D007769:Lactams / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010408:Penicillium chrysogenum / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013302:Streptomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics
chemical_list: D002511:Cephalosporins; D007769:Lactams; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; C424182:adipoyl-7-amino-3-deacetoxycephalosporanic acid; C014320:7-aminodesacetoxycephalosporanic acid; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase