Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of Escherichia coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host beta-lacta-mase involved in the degradation of penicillin G and G-7-ADCA. 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 with the starting strain (2.50 mol%).