产酸克雷伯氏菌乳酸脱氢酶基因敲除提高2,3-丁二醇产量  

Knockout of lactate dehydrogenase gene in Klebsiella oxytoca to enhance 2,3-butanediol production

作　　者：叶广彬 银联飞 王长丽 孙珊珊 杨睿睿 葛菁萍[2] YE Guangbin;YIN Lianfei;WANG Changli;SUN Shanshan;YANG Ruirui;GE Jingping(Science Experiment Center,Youjiang Medical University for Nationalities,Baise 533000,China;Key Laboratory of Microbiology of Heilongjiang University,College of Life Science,Heilongjiang University,Harbin 150080,China)

机构地区：[1]右江民族医学院科学实验中心,广西百色533000 [2]黑龙江大学生命科学学院微生物黑龙江省高校重点实验室,黑龙江哈尔滨150080

出　　处：《中国酿造》2020年第11期158-162,共5页China Brewing

基　　金：国家自然科学基金项目(31570492);黑龙江省教育厅重点项目(HDJCCX-2016Z05);广西自然科学基金(2017GXNSFAA198101);广西高校中青年教师基础能力提升项目(2019KY0580,2020KY13007)。

摘　　要：乳酸脱氢酶(LDH)是乳酸生成途径中的关键酶,敲除乳酸脱氢酶基因,理论上可以减少甚至消除乳酸的产生,提高2,3-丁二醇的产量和得率。该研究采用体外拼接方式构建乳酸脱氢酶基因的同源线性片段ldhL-Cmr-ldhR,将其电转化至Klebisella oxytoca HD79中,通过Red同源重组技术筛选敲除成功的重组菌株,经聚合酶链式反应(PCR)、荧光定量-聚合酶链式反应(FQ-PCR)及2,3-丁二醇产量检测验证。结果表明,重组菌株2,3-丁二醇产量为40.20 g/L、转化率为0.38 g/g和生产强度为0.48 g/(L·h),相比供试菌株分别提高了26.8%、11.8%和45.5%;而乳酸产量则由4.83 g/L下降至2.45 g/L,相比供试菌株降低了49.3%。该实验为后续进一步提高2,3-丁二醇的产量和扩大菌株选择范围奠定基础。Lactate dehydrogenase(LDH)is a key enzyme in lactic acid production pathway,knockout of LDH gene can theoretically reduce or even eliminate the production of lactic acid,thereby increasing the production and yield of 2,3-butanediol.In this study,the homologous recombination linear sequence ldhL-Cmr-ldhR of LDH gene was constructed by the method of digestion and connection in vitro.After that,the ldhL-Cmr-ldhR was electrotransformed into Klebisella oxytoca HD79.The recombination strain was successfully screened by Red homologous recombination technology,and verified by polymerase chain reaction(PCR),fluorogenic quantitative-polymerase chain reaction(FQ-PCR)and 2,3-butanediol production detection.The results indicated that the 2,3-butanediol production,convert ratio and production intensity of the recombinant strain were 40.20 g/L,0.38 g/g and 0.48 g/(L·h),respectively,which were 26.8%,11.8%,45.5%higher than that of the tested strain,respectively.However,the lactic acid production of recombinant strain decreased from 4.83 g/L to 2.45 g/L,which was 49.3%lower than that of the tested strain.It laid a foundation for further increasing the production of 2,3-butanediol and expanding the range of strain selection.

关 键 词：产酸克雷伯氏菌 乳酸脱氢酶 基因敲除 2 3-丁二醇 产量 

分 类 号：TQ923[轻工技术与工程—发酵工程]