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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
Zhang, Xian; Wu, Dan; Yang, Taowei; Xu, Meijuan & Rao, Zhiming
Abstract
Background: 3-Ketosteroid-Δ1-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 (ksddII) 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 Km and Vmax 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.
Keywords
Androst-1,4-diene-3,17-dione; Bioconversion; Codon optimization; Flavoprotein enzyme; Heterologous expression; Mycobacterium neoaurum; Overexpression; Recombinant Corynebacterium; Sterol catabolism; Whole-cell catalyst
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