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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

dc.creatorZhang,Xian
dc.creatorWu,Dan
dc.creatorYang,Taowei
dc.creatorXu,Meijuan
dc.creatorRao,Zhiming
dc.date2016-11-01
dc.date.accessioned2019-05-03T12:45:28Z
dc.date.available2019-05-03T12:45:28Z
dc.identifierhttps://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000600013
dc.identifier.urihttp://revistaschilenas.uchile.cl/handle/2250/85587
dc.descriptionBackground: 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.
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dc.languageen
dc.publisherPontificia Universidad Católica de Valparaíso
dc.relation10.1016/j.ejbt.2016.10.004
dc.rightsinfo:eu-repo/semantics/openAccess
dc.sourceElectronic Journal of Biotechnology v.19 n.6 2016
dc.subjectAndrost-1,4-diene-3,17-dione
dc.subjectBioconversion
dc.subjectCodon optimization
dc.subjectFlavoprotein enzyme
dc.subjectHeterologous expression
dc.subjectMycobacterium neoaurum
dc.subjectOverexpression
dc.subjectRecombinant Corynebacterium
dc.subjectSterol catabolism
dc.subjectWhole-cell catalyst
dc.titleOver-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


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