化工学报 ›› 2016, Vol. 67 ›› Issue (7): 2956-2962.DOI: 10.11949/j.issn.0438-1157.20151935

• 生物化学工程与技术 • 上一篇    下一篇

黄色短杆菌ilvN基因定点突变和ilvBNilvC串联表达对L-缬氨酸产量的影响

曾邦定1, 黄钦耿1, 梁玲2, 郭小雷2, 王明兹1, 施巧琴1, 吴松刚1   

  1. 1. 福建师范大学生命科学学院, 工业微生物教育部工程研究中心, 福建 福州 350117;
    2. 福建省麦丹生物集团有限公司 福州研究中心, 福建 福州 350008
  • 收稿日期:2015-12-21 修回日期:2016-04-17 出版日期:2016-07-05 发布日期:2016-07-05
  • 通讯作者: 王明兹

Effects of Brevibacterium flavum with directed mutagenesis of ilvN and co-expression of ilvBNC cluster on L-valine production

ZENG Bangding1, HUANG Qingeng1, LIANG Ling2, GUO Xiaolei2, WANG Mingzi1, SHI Qiaoqin1, WU Songgang1   

  1. 1. College of Life Sciences, Engineering Research Center of Industrial Microbiology of Ministry of Education, Fujian Normal University, Fuzhou 350117, Fujian, China;
    2. Fuzhou Research Center of Fujian Maidan Biology Group Co., Ltd., Fuzhou 350008, Fujian, China
  • Received:2015-12-21 Revised:2016-04-17 Online:2016-07-05 Published:2016-07-05

摘要:

ilvBNilvC基因编码的乙酰羟酸合成酶(AHAS)和乙酰羟酸异构还原酶(AHAIR)是L-缬氨酸合成途径的两个关键酶。本实验以黄色短杆菌Brevibacterium flavum MD515为出发菌株,通过PCR技术扩增其ilvBNilvC基因,对调节亚基ilvN进行定点突变,获得抗反馈抑制突变型编码基因ilvBNrC;然后将其插入穿梭表达载体pZ8-1中,构建串联表达质粒pZ8-1-ilvBNrC并转化出发菌株,筛选获得工程菌株B.flavum MD515/pZ8-1-ilvBNrC。摇瓶发酵该工程菌株L-缬氨酸产量达29.5 g·L-1,较出发菌株提高27.7%,同时生长速度和生物量也比出发菌株有所提高,丙氨酸含量降低,L-亮氨酸及L-异亮氨酸含量提高。在30 L发酵罐连续补料发酵60 h后L-缬氨酸产量达61.7 g·L-1,糖酸转化率为39.2%。菌株MD515/pZ8-1-ilvBNrC发酵液透光率较出发菌株高且蛋白含量低,这些特性有利于发酵液后期的分离提取。

关键词: L-缬氨酸, 定点突变, 乙酰羟酸合成酶, 乙酰羟酸异构还原酶, 共表达

Abstract:

Acetohydroxy acid synthase (AHAS) and acetohydroxy acid isomeroreductase (AHAIR) encoded by ilvBN and ilvC are two key enzymes which play important roles in the biosynthetic pathway of L-valine. Brevibacterium flavum MD515 was used as the origin strain and site-specific mutagenesis was performed in its ilvN gene which coded for the regulatory subunit of AHAS, resulting in the obtainment of an anti-feedback inhibition gene, named ilvBN'C. Then, the ilvBNrC gene was ligased to plasmid pZ8-1 for construction of the recombinant plasmid pZ8-1-ilvBNrC, which was subsequently transfored into B. flavum MD515. With this method, the targeted transformant B. flavum MD515/pZ8-1-ilvBNrC showed better L-valine producing capacity of 29.5 g·L-1, 27.7% increase than that of original strain when it was cultured in 250 ml shake flasks. Meanwhile, the yield of leucine and isoleucine also increased while the alanine decreased. The biomass and growth rate were also increased. Moreover, fermentation experiments was performed in a 30 L fermentor and the results indicated that the L-valine productivity of B. flavum MD515/pZ8-1-ilvBNrC reached to 61.7 g·L-1 while the conversion rate of glucose/valine was up to 39.2%. The work finally made some simple investigations about the purification of L-valine in B. flavum MD515/ pZ8-1-ilvBNrC and B. flavum MD515. The results showed that the light transmittance of strain B. flavum MD515/pZ8-1-ilvBNrC was higher while the protein content was lower than that of B. flavum MD515, which was beneficial for subsequent separation process.

Key words: L-valine, site-specific mutagenesis, acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, co-expression

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