title: Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1-dehydrogenase and catalase in Bacillus subtilis.
abstract: OBJECTIVE
  3-ketosteroid-Δ1-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 (H2O2) 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 H2O2to enhance ADD production.
  
  
  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/pMA5-ksddopt-katA to eliminate the toxic effects of H2O2generated 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 H2O2by co-expressing catalase.
  
  
  CONCLUSIONS
  This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry.
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: M
authors: M
authors: Shao
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: Z
authors: Z
authors: Sha
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: X
authors: X
authors: Zhang
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: Z
authors: Z
authors: Rao
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: M
authors: M
authors: Xu
authors: Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: T
authors: T
authors: Yang
authors: Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu Province, China.
authors: Z
authors: Z
authors: Xu
authors: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.
authors: S
authors: S
authors: Yang
mesh_terms: D000730:Androstadienes / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation; D002374:Catalase / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D009170:Nontuberculous Mycobacteria / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011090:Polyenes / Q000378:metabolism
chemical_list: D000730:Androstadienes; D001426:Bacterial Proteins; D011090:Polyenes; C006573:1,4-androstadiene-3,17-dione; D010088:Oxidoreductases; D002374:Catalase; C022656:3-oxosteroid delta(1) dehydrogenase
keywords: 3-ketosteroid-Δ1-dehydrogenase; androst-1,4-diene-3,17-dione; catalase; fed-batch strategy; whole-cell biocatalyst