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
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 usua...
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Pontificia Universidad Católica de Valparaíso
2016
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oai:scielo:S0717-345820160006000132017-01-25Over-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-dioneZhang,XianWu,DanYang,TaoweiXu,MeijuanRao,Zhiming Androst-1,4-diene-3,17-dione Bioconversion Codon optimization Flavoprotein enzyme Heterologous expression Mycobacterium neoaurum Overexpression Recombinant Corynebacterium Sterol catabolism Whole-cell catalyst 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.6 20162016-11-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000600013en10.1016/j.ejbt.2016.10.004 |
| institution |
Scielo Chile |
| collection |
Scielo Chile |
| language |
English |
| topic |
Androst-1,4-diene-3,17-dione Bioconversion Codon optimization Flavoprotein enzyme Heterologous expression Mycobacterium neoaurum Overexpression Recombinant Corynebacterium Sterol catabolism Whole-cell catalyst |
| spellingShingle |
Androst-1,4-diene-3,17-dione Bioconversion Codon optimization Flavoprotein enzyme Heterologous expression Mycobacterium neoaurum Overexpression Recombinant Corynebacterium Sterol catabolism Whole-cell catalyst Zhang,Xian Wu,Dan Yang,Taowei Xu,Meijuan Rao,Zhiming 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 |
| description |
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. |
| author |
Zhang,Xian Wu,Dan Yang,Taowei Xu,Meijuan Rao,Zhiming |
| author_facet |
Zhang,Xian Wu,Dan Yang,Taowei Xu,Meijuan Rao,Zhiming |
| author_sort |
Zhang,Xian |
| title |
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 |
| title_short |
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 |
| title_full |
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 |
| title_fullStr |
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 |
| title_full_unstemmed |
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 |
| title_sort |
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 |
| publisher |
Pontificia Universidad Católica de Valparaíso |
| publishDate |
2016 |
| url |
http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000600013 |
| work_keys_str_mv |
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| _version_ |
1718441939551911936 |