Whole-Cell Biotransformation of Penicillin G by a Three-Enzyme Co-expression System with Engineered Deacetoxycephalosporin C Synthase

Graphical Deacetoxycephalosporin C synthase (DAOCS) requires 2-oxoglutarate (2OG) as a co-substrate to catalyze the conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporin acid (G-7-ADCA). Catalase and L-GOX were introduced into the catalytic process of ring expansion to construct a 2OG production module to form a three-enzyme co-expression system, so as to realize the conversion of penicillin G to G-7-ADCA in vivo . Deacetoxycephalosporin C synthase (DAOCS) catalyzes the transformation of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) for which it depends on 2-oxoglutarate (2OG) as co-substrate. However, the low activity of DAOCS and the expense of 2OG restricts its practical applications in the production of G-7-ADCA. Herein, a rational design campaign was performed on a DAOCS from Streptomyces clavuligerus (scDAOCS) in the quest to construct novel expandases. The resulting mutants showed 25∼58 % increase in activity compared to the template. The dominant DAOCS variants were then embedded into a three-enzyme co-expression system, consisting of a catalase and an L-glutamic oxidase for the generation of 2OG, to convert penicillin G to G-7-ADCA in E. coli . The engineered whole-cell enzyme cascade was applied to an up-scaled reaction, exhibiting a yield of G-7-ADCA up to 39.21 mM (14.6 g ⋅ L −1 ) with a conversion of 78.42 mol %. This work highlights the potential of the integrated whole-cell system that may inspire further research on green and efficient production of 7-ADCA.