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Efficient androst‐1,4‐diene‐3,17‐dione production by co‐expressing 3‐ketosteroid‐Δ 1 ‐dehydrogenase and catalase in Bacillus subtilis
Author(s) -
Shao M.,
Sha Z.,
Zhang X.,
Rao Z.,
Xu M.,
Yang T.,
Xu Z.,
Yang S.
Publication year - 2017
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13336
Subject(s) - bacillus subtilis , biochemistry , bioconversion , dehydrogenase , catalase , biotransformation , enzyme , chemistry , biology , fermentation , bacteria , genetics
Aims 3‐ketosteroid‐Δ 1 ‐dehydrogenase ( KSDD ), a flavin adenine dinucleotide ( FAD )‐dependent enzyme involved in sterol metabolism, specifically catalyses the conversion of androst‐4‐ene‐3,17‐dione ( AD ) to androst‐1,4‐diene‐3,17‐dione ( ADD ). However, the low KSDD activity and the toxic effects of hydrogen peroxide (H 2 O 2 ) generated during the biotransformation of AD to ADD with FAD regeneration hinder its application on AD conversion. The aim of this work was to improve KSDD activity and eliminate the toxic effects of the generated H 2 O 2 to enhance ADD production. Methods and Results The ksdd gene obtained from Mycobacterium neoaurum JC ‐12 was codon‐optimized to increase its expression level in Bacillus subtilis , and the KSDD activity reached 12·3 U mg −1 , which was sevenfold of that of codon‐unoptimized gene. To improve AD conversion, catalase was co‐expressed with KSDD in B. subtilis 168/ pMA 5‐ ksdd opt ‐ katA to eliminate the toxic effects of H 2 O 2 generated during AD conversion. Finally, under optimized bioconversion conditions, fed‐batch strategy was carried out and the ADD yield improved to 8·76 g l −1 . Conclusions This work demonstrates the potential to improve enzyme activity by codon‐optimization and eliminate the toxic effects of H 2 O 2 by co‐expressing catalase. Significance and Impact of the Study This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry.