双层面调控S. cerevisiae碳流促进L-乳酸积累

Manipulation of Carbon flux in S. cerevisiae to improve L-lactic acid production

摘要:

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(n

Abstract:

Abstract: [Objective] The target of this study was to develop an engineered Sacchromyces cerevisiae strain to produce L-lactic acid efficiently using glucose as carbon source. [Methods] For construction of the strain CEN.PK2-1C[LDH], we integrated a 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. As this, the carbon fluxes were led into L-Lactic acid. On the basis, we analyzed the Km value of these key enzymes to NADH and successfully over-expressed a NADH oxidase (nox) from Streptococcus pneumoniae into the cytoplasm for the construction of S. cerevisiae CEN.PK2-1C[LDH]-nox. [Results] Compared to initial strain, the yield of L-lactic acid in CEN.PK2-1C[LDH] fermentation liquor 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. Furthermore, the concentration of L-lactic acid increased further to 20 g/L and the concentration of ethanol decreased to 8.2 g/L. [Conclusions] The results indicated that 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+.