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

Abstract

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the transformation of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) for which it depends on 2oxoglutarate (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 À 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.

Combining Cell-Free Expression and Multifactor Optimization for Enhanced Biosynthesis of Cinnamyl Alcohol

“…Biotransformation is the enzymatic conversion process utilizing either in vivo engineered microbial whole cells or in vitro purified enzymes. − Due to complete cellular structures and the detrimental effect of toxic intermediates/products on microbial cell growth, the final yield of whole-cell catalysis is often low and unsatisfactory. , In spite of the high efficiency of purified enzyme catalysis, the enzyme purification process is laborious, time-consuming, and expensive, preventing mass production . Recently, cell-free protein synthesis (CFPS) systems have emerged as an alternative that can avoid the disadvantages of the aforementioned two approaches.…”