烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002

摘要:

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+，引起胞内辅酶NAD(H)的不平衡，最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因，通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+，从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而，BA014在厌氧发酵过程中有大量丙酮酸积累，为进一步提高菌株的丁二酸生产能力，减少副产物丙酮酸的生成，共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc，构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明，BA016发酵112 h后，共消耗了35.00 g/L的葡萄糖。发酵结束时，菌体OD600为4.64，产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因，使BA016的丙酮酸积累进一步降低，丁二酸产量进一步提高。

Abstract:

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.