Improvement of microbial strains and fermentation processes.

Improvement of microbial strains for the overproduction of industrial products has been the hallmark of all commercial fermentation processes. Conventionally, strain improvement has been achieved through mutation, selection, or genetic recombination. Overproduction of primary or secondary metabolites is a complex process, and successful development of improved strains requires a knowledge of physiology, pathway regulation and control, and the design of creative screening procedures. In addition, it requires mastery of the fermentation process for each new strain, as well as sound engineering know-how for mediaoptimization and the fine-tuning of process conditions. This review focuses on the various options that may be employed to improve microbial strains and addresses the complex problems of screening, the tools and technology behind the selection of targeted organisms, and the importance of process optimization. Furthermore, this review discusses new and emerging technologies and designing optimized media for tracking mutants with enhanced productivity or other desired attributes.

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway.

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.

Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity.

We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.