Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Abstract

Background: 3-Ketosteroid-Δ 1 -dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd ( ksdd II ) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The K m and V max values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production. Normal 0 21 false false false ES X-NONE X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif;}