[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0 g/L增加到15 g/L,而乙醇浓度则从27.3 g/L降为16.2 g/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15 g/L增加到20 g/L,乙醇浓度从16.2 g/L降为8.2 g/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+.
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