S107
An efficient whole-cell biocatalyst for G-7-ADCA production in Escherichia coli by modifying TCA cycle
Wednesday, July 23, 2014: 10:30 AM
Regency Ballroom A, Second Floor (St. Louis Hyatt Regency at the Arch)
Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). The key step in the synthesis of 7-ADCA in industry has been the chemical ring expansion of penicillin G into phenylacetyl-7-ADCA (G-7-ADCA), which was followed by enzymatic removal of the side chain to obtain 7-ADCA. We developed a new whole-cell biocatalytic process for converting penicillin G to G-7-ADCA that could substitute the expensive and environmentally unfriendly chemical method classically used. Recombinant Escherichia coli expressing deacetoxycephalosporin C synthase (DAOCS) mutant from Streptomyces clavuligerus were constructed and applied as whole-cell biocatalyst to converted penicillin G into phenylacetyl-7-ADCA (G-7-ADCA). DAOCS is 2-oxoglutarate-dependent monocuclear ferrous enzyme. As such, DAOCS catalysis is expected to strongly interact with metabolism as it depends on a tricarboxylic acid (TCA) cycle intermediate as a cosubstrate. By modified TCA cycle, the yield of G-7-ADCA was 2.5-fold than the wild-type strain. Metabolism of acetate was regulated which led to better cell growth and help keeping more stable pH environment in the biocatalytic process. The phenomenon of penicillin G and G-7-ADCA decomposition occurred in E.coli during the bioconversion. The β-lactamase (AmpC) played a major role in the degradation. The ampC gene was knocked out in the recombinant E.coli to reduce the degradation of substrate and product in the bioprocess and the yield of G-7-ADCA was 1.73-fold than non-knockout host. In the whole-cell biocatalytic process, 9.46 g/liter (29 mM) G-7-ADCA was obtained by the engineered Escherichia coli strain. It was the first time that G-7-ADCA was produced in E.coli.