筛选脱氮假单胞菌启动子提高维生素B12产量

doi:10.13560/j.cnki.biotech.bull.1985.2017-0010

生物技术通报 ›› 2017, Vol. 33 ›› Issue (8): 159-166.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0010

筛选脱氮假单胞菌启动子提高维生素B₁₂产量

王玲玲¹, 夏苗苗², 董会娜², 朱蓓薇¹, 张大伟^1，2

1. 大连工业大学食品学院，大连 116034；
2. 中国科学院天津工业生物技术研究所，天津 300308

收稿日期:2017-01-16 出版日期:2017-08-01 发布日期:2017-08-01
作者简介:王玲玲，女，硕士研究生，研究方向：微生物代谢；E-mail：wanglingling1122@126.com
基金资助:
天津市自然科学基金（16JCYBJC23500，15JCQNJC09500）

Enhanced Production of Vitamin B₁₂ by Screening the Promoter in Pseudomonas denitrificans

WANG Ling-ling¹, Xia Miao-miao², DONG Hui-na², ZHU Bei-wei¹, ZHANG Da-wei^1，2

1. Dalian Polytechnic University，Dalian 116034；
2. Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308

Received:2017-01-16 Published:2017-08-01 Online:2017-08-01

摘要/Abstract

摘要： 维生素B₁₂（VB₁₂/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B₁₂常用工业菌株之一。通过筛选高表达启动子来过表达VB₁₂合成途径基因，能有效提高菌株的VB₁₂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VB₁₂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B₁₂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B₁₂产量提高21.5 mg/L。筛选高表达启动子用于维生素B₁₂合成途径关键基因的表达，是一种有效的提高维生素B₁₂产量的方法。

关键词: 脱氮假单胞菌, 强启动子, cobA, 维生素B₁₂, Gibson assembly

Abstract: Vitamin B₁₂（VB₁₂/Cobalamin）possesses several physiological functions and is widely used in pharmaceutical and food industries. Pseudomonas denitrificans is commonly employed in the production of VB₁₂. In order to improve the VB₁₂productivity by a strain，the high-expression promoters were screened and then used to express the gene synthetizing VB₁₂. Via online prediction software，analyzing the promoters in promoters-included non-coding sequences that precede the genes encoding heat shock protein and molecular chaperone in P. 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 gene encoding green fluorescent protein. Then the GFP fluorescence signal value was detected by enzyme standard instrument，further for obtaining the promoters with high-expression，the expression levels of promoters in the non-coding sequences preceding the genes encoding heat shock protein and molecular chaperone were evaluated，which then was applied for the overexpression of genes in the VB₁₂ 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 gene cobA in VB₁₂ synthesis pathway，and the fermentation results revealed that the VB₁₂ yield increased by 21.5mg/Lcompared with the control strain. Conclusively，screening high-expression promoter for overexpressing the key gene in VB₁₂ synthesis pathway is an effective approach for improving VB₁₂ production.

Key words: Pseudomonas denitrificans, strong promoter, cobA, vitamin B₁₂, Gibson assembly

王玲玲, 夏苗苗, 董会娜, 朱蓓薇, 张大伟. 筛选脱氮假单胞菌启动子提高维生素B₁₂产量[J]. 生物技术通报, 2017, 33(8): 159-166.

WANG Ling-ling, Xia Miao-miao, DONG Hui-na, ZHU Bei-wei, ZHANG Da-wei. Enhanced Production of Vitamin B₁₂ by Screening the Promoter in Pseudomonas denitrificans[J]. Biotechnology Bulletin, 2017, 33(8): 159-166.

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筛选脱氮假单胞菌启动子提高维生素B₁₂产量

Enhanced Production of Vitamin B₁₂ by Screening the Promoter in Pseudomonas denitrificans

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