双层面调控Saccharomyces cerevisiae碳流促进L-乳酸积累  

Modification of carbon flux in Sacchromyces cerevisiae to improve L-lactic acid production

作　　者：赵亮亮[1] 汪军[1] 周景文[2] 刘立明[1,2] 堵国成[2] 陈坚[1,2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,无锡214122 [2]江南大学工业生物技术教育部重点实验室,无锡214122

出　　处：《微生物学报》2011年第1期50-58,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金重点项目(20836003);国家自然科学基金(20706025);霍英东教育基金项目(11075);全国优秀博士学位论文作者专项资金(200962)~~

摘　　要：【目的】调控Sacchromyces cerevisiae丙酮酸节点碳流分布促进L-乳酸积累。【方法】利用同源重组方法,将来源于Bovine的乳酸脱氢酶基因LDH整合到S.cerevisiae CEN.PK2-1C基因组中,同时敲除丙酮酸脱羧酶基因PDC1,将碳流导向L-乳酸的积累,构建了基因工程菌S.cerevisiae CEN.PK2-1C[LDH]。在此基础上,通过分析丙酮酸节点处关键酶对NADH的Km值不同,而将来源于Streptococcus pneumoniae的NADH氧化酶(nox)过量表达于CEN.PK2-1C[LDH]中,构建基因工程菌S.cerevisiae CEN.PK2-1C[LDH]-nox。【结果】与出发菌株S.cerevisiae CEN.PK2-1C比较,S.cerevisiae CEN.PK2-1C[LDH]发酵液中L-乳酸积累量从0g/L增加到15g/L,而乙醇浓度则从27.3g/L降为16.2g/L;过量表达了nox的S.cerevisiae CEN.PK2-1C[LDH]-nox的发酵液中L-乳酸浓度则进一步从15g/L增加到20g/L,乙醇浓度从16.2g/L降为8.2g/L。【结论】本研究通过构建S.cerevisiae L-乳酸合成途径和降低NADH/NAD+比率,在引入外源途径和调控辅因子浓度两个层面上成功实现了S.cerevisiae丙酮酸代谢节点碳流的重新分布,促进了L-乳酸的积累。[Objective]We developed an engineered Sacchromyces cerevisiae strain to produce L-lactic acid efficiently by using glucose as carbon source.[Methods]For construction of the strain CEN.PK2-1C[LDH],we integrated an LDH gene coding L-lactic acid dehydrogenase from bovine into the genome of S.cerevisiae via homologous recombination and meanwhile knocked out a PDC1 gene coding pyruvate decarboxylase.The carbon fluxes were led into L-Lactic acid.We analyzed the Km value of these key enzymes to NADH and over-expressed an NADH oxidase （nox） from Streptococcus pneumoniae into the cytoplasm for the construction of S.cerevisiae CEN.PK2-1C[LDH]-nox.[Results]Compared to the initial strain,the yield of L-lactic acid in CEN.PK2-1C[LDH]fermentation broth increased from 0 g /L to 15 g /L and the concentration of ethanol decreased from 27.3 g /L to 16.2 g /L.Compared to CEN.PK2-1C[LDH],the yield of L-lactic acid in CEN.PK2-1C[LDH]-nox fermentation broth increased from 15 g /L to 20 g /L and the concentration of ethanol decreased from 16.2 g /L to 8.2 g /L.[Conclusions ] The carbon metabolic flux was redistributed to efficient accumulation of L-lactic acid through two-sided control that heterologous expression of the gene LDH and decreasing the ratio of NADH /NAD ＋.

关 键 词：S.cerevisiae L-乳酸 碳代谢流 NADH 

分 类 号：Q93[生物学—微生物学]