Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional chemical process to produce 7-ADCA by ring expansion of penicillin G is expensive and polluted; thus, a bioconversion process is highly desirable.

Dr. LIN Baixue and her colleagues led by Prof. TAO Yong in collaboration with Prof. Yang Keqian from the Institute of Microbiology, CAS, Beijing, have recently developed an efficient whole-cell biocatalytic process for converting penicillin G to G-7-ADCA that could substitute the expensive and environmentally unfriendly chemical method classically used.

In this work, Escherichia coli expressing deacetoxycephalosporin C synthase (DAOCS) was developed as a whole-cell biocatalyst to convert penicillin G to G-7-ADCA. The major strategy used was to reconstitute the TCA cycle of E. coli with DAOCS catalyzed reaction; thus the metabolic flux of central metabolism was forced to go through DAOCS catalyzed reaction to produce G-7-ADCA. This strategy was combined with engineering efforts to reduce the accumulation of acetate and the degradation of penicillin G and G-7-ADCA to improve the conversion rate of penicillin G by DAOCS significantly. By combining these manipulations in an engineered strain, The yield of G-7-ADCA was increased from 2.50±0.79 mM (0.89±0.28 g/L, 0.07±0.02 g/gDCW) to 29.01±1.27 mM (10.31±0.46 g/L, 0.77±0.03 g/gDCW) with a conversion rate  of 29.01 mol%, representing an 11-fold increase compared to the starting strain (2.50 mol%).

This work describes an elegant strategy to modify the native TCA cycle in E. coli such that it cannot efficiently convert 2DOG to succinate. They then engineer a “succinate diversion” via a coupled biochemical reaction that represents a highly valuable biotransformation. This work demonstrates the feasibility to redirect the TCA cycle to drive a desired enzyme reaction, and this strategy could be applied to other enzymes that catalyze TCA cycle-coupleable reactions.

This work entitled “Reconstitution of TCA cycle with DAOCS to engineer E. coli into an efficient whole cell catalyst of penicillin G” have recently been published online by Proceedings of the National Academy of Sciences of the United States of America on July 27, 2015.  (doi:10.1073/pnas.1502866112).