High-level expression of Cephalosporin C deacetylase from Bacillus subtilis SIL3 in Escherichia coli by a multilevel collaborative strategy

• The highest production of CAH was obtained by the recombinant E. coli DH5a/pB2-CAH-K-SIL3.
• This fermentation process established here is expected to be applicable for CAH production.
• High-level expression of CAH was achieved by a multilevel collaborative strategy.

Cephalosporin C deacetylases (CAH) is employed to hydrolyze 7-aminocephalosporanic acid (7-ACA) to deacetyl-7-aminocephalosporanic acid (D-7-ACA), which can be used to produce the 3-vinyl substituted cephalosporin antibiotics. In this study, a multilevel collaborative strategy was conducted to achieve high-level production of CAH by recombinant Escherichia coli (E. coli). Inducible and constitutive expression systems harboring four Bacillus subtilis (B. subtilis) CAH genes were constructed and expressed in E. coli. Effects of expression system, spacing sequence and terminator on the CAH expression were investigated. Obvious increase in CAH activity demonstrated that the recombinant E. coli strain of DH5α/pB2-CAH-K-SIL3 was the optimal CAH producing strain, and the CAH activity produced increased approximately 3 times after systematic optimization. Using glycerol feeding with pH control, an effective fermentation process for recombinant CAH production was established in 7.0 L fermenter. The cell density (OD600), CAH activity and productivity reached 60, 1340 U/mL and 55 U/mL/h, which are the highest values reported in E. coli. The fermentation process established in this work is expected to be applicable for industrial CAH production.