摘要
维生素B_(12)(VB_(12)/钴胺素)具有多种生理学功能,广泛应用于制药、食品等行业。脱氮假单胞菌是维生素B_(12)常用工业菌株之一。通过筛选高表达启动子来过表达VB_(12)合成途径基因,能有效提高菌株的VB_(12)生产能力。通过对脱氮假单胞菌中某些编码热激蛋白和分子伴侣基因前含启动子在内的非编码序列用启动子在线预测软件进行分析,选取P_(ibpA)、P_(cbpA)、P_(dnaJ)、P_(htpG)、P_(dnak)、P_(grpE)的非编码区与编码绿色荧光蛋白的GFP报告基因相连,通过酶标仪检测GFP的荧光信号值,对编码热激蛋白和分子伴侣基因前的非编码区所含有的启动子的表达强度进行评估,以获得高表达的启动子对VB_(12)合成途径的基因进行过表达。结果表明,含有启动子P_(dnak)的非编码区,其表达的GFP荧光值最高。进而构建强启动子P_(dnak)与维生素B_(12)合成途径基因cobA的过表达重组菌,发酵数据表明,与对照菌株相比重组菌株维生素B_(12)产量提高21.5 mg/L。筛选高表达启动子用于维生素B_(12)合成途径关键基因的表达,是一种有效的提高维生素B_(12)产量的方法。
Vitamin B_（12）（VB_（12）/Cobalamin）possesses several physiological functions and is widely used in pharmaceutical and foodindustries. Pseudomonas denitrificans is commonly employed in the production of VB_（12）. In order to improve the VB_（12） productivity by a strain,the high-expression promoters were screened and then used to express the gene synthetizing VB_（12）. Via online prediction software,analyzingthe promoters in promoters-included non-coding sequences that precede the genes encoding heat shock protein and molecular chaperone inP. denitrification,the non-encoding sequences of P_（ibpA）,P_（cbpA）,P_（dnaJ）,P_（htpG）,P_（dnak）,and P_（grpE） were selected and ligated to GFP report geneencoding green fluorescent protein. Then the GFP fluorescence signal value was detected by enzyme standard instrument,further for obtainingthe promoters with high-expression,the expression levels of promoters in the non-coding sequences preceding the genes encoding heat shockprotein and molecular chaperone were evaluated,which then was applied for the overexpression of genes in the VB_（12） synthetic pathways.Fluorescence experimental results showed that the expression of GFP fluorescence value for the non-coding sequence with the promoter P_（dnak） was the highest. Subsequently,the strongest P_（dnak） promoter was selected to construct the recombinant P. denitrificans overexpressing genecob A in VB_（12） synthesis pathway,and the fermentation results revealed that the VB_（12） yield increased by 21.5mg/L compared with the controlstrain. Conclusively,screening high-expression promoter for overexpressing the key gene in VB_（12） synthesis pathway is an effective approach forimproving VB_（12） production.
出处
《生物技术通报》
CAS
CSCD
北大核心
2017年第8期159-166,共8页
Biotechnology Bulletin
基金
天津市自然科学基金(16JCYBJC23500
15JCQNJC09500)
