Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

Abstract

AbstractBACKGROUNDThe 3‐ketosteroid‐Δ1‐dehydrogenase (KSDD) expressed in Escherichia coli was mainly in the form of inclusion bodies and the KSDD enzyme activity was at a low level. Therefore, the regulation of process conditions and supplementation of natural osmolytes were carried out to improve the soluble expression of KSDD in E. coli.RESULTSEffects of temperature, inducer concentration, and osmolytes (K‐glutamate, proline, and betaine) supplementation on cell growth and soluble KSDD production in recombinant Escherichia coli were investigated. With the decrease of cultivation temperature, lower isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) concentration, and betaine addition, high intracellular soluble KSDD production was realized in the recombinant E. coli BL21/pET28a‐ksdd and the KSDD enzyme activity reached 11.7 U mg−1, which is the highest KSDD activity ever reported. In addition, the whole‐cell biocatalyst of this recombinant strain was used for bioconversion of androst‐4‐ene‐3,17‐dione (AD) to androst‐1,4‐diene‐3,17‐dione (ADD). By optimization of the transformation conditions and applying a fed‐batch strategy, a final ADD yield of 5.7 g L−1 was achieved with a productivity of 0.158 g (L h)−1, which is the highest reported productivity using a microbial process. More importantly, no by‐products were detected during the whole bioconversion process.CONCLUSIONThese results provide the basis for industrial scale production of ADD. © 2016 Society of Chemical Industry