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Over-expression of Mycobacterium neoaurum 3-ketosteroid-∆ 1-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.
  • Zhang, Xian; Jiangnan University. School of Biotechnology. Key Laboratory of Industrial Biotechnology of Ministry of Education. CN
  • Wu, Dan; Jiangnan University. School of Biotechnology. Key Laboratory of Industrial Biotechnology of Ministry of Education. CN
  • Yang, Taowei; Jiangnan University. School of Biotechnology. Key Laboratory of Industrial Biotechnology of Ministry of Education. CN
  • Xu, Meijuan; Jiangnan University. School of Biotechnology. Key Laboratory of Industrial Biotechnology of Ministry of Education. CN
  • Rao, Zhiming; Jiangnan University. School of Biotechnology. Key Laboratory of Industrial Biotechnology of Ministry of Education. CN
Electron. j. biotechnol ; 19(6): 84-90, Nov. 2016. ilus
Artigo em Inglês | LILACS | ID: biblio-840318
ABSTRACT

Background:

3-Ketosteroid-∆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 asa 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 n) 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 ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.
Assuntos


Texto completo: DisponíveL Índice: LILACS (Américas) Assunto principal: Oxirredutases / Corynebacterium / Androstadienos / Mycobacterium Idioma: Inglês Revista: Electron. j. biotechnol Assunto da revista: Biotecnologia Ano de publicação: 2016 Tipo de documento: Artigo / Documento de projeto País de afiliação: China Instituição/País de afiliação: Jiangnan University/CN

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Texto completo: DisponíveL Índice: LILACS (Américas) Assunto principal: Oxirredutases / Corynebacterium / Androstadienos / Mycobacterium Idioma: Inglês Revista: Electron. j. biotechnol Assunto da revista: Biotecnologia Ano de publicação: 2016 Tipo de documento: Artigo / Documento de projeto País de afiliação: China Instituição/País de afiliação: Jiangnan University/CN