基于回补途径的TCA循环改造对克雷伯氏菌生长和甘油代谢的影响

TCA cycle modification based on the anaplerotic reaction and its effects on growth and glycerol metabolism in Klebsiella pneumonia

回补途径和TCA循环在克雷伯氏菌(Klebsiella pneumoniae)的中心代谢中扮演着十分重要的角色.通过过表达回补途径的关键酶磷酸烯醇式丙酮酸羧化酶(PPC)和柠檬酸(TCA)循环的关键酶柠檬酸合成酶(GLTA)将磷酸烯醇式丙酮酸(PEP)和乙酰辅酶A节点的碳流引入TCA循环,以考察基于回补途径的TCA循环强化对K. pneumoniae生长和甘油代谢的影响.结果显示:单独过表达ppc或gltA基因,TCA循环还原和氧化分支的中间代谢产物琥珀酸和α-酮戊二酸分别增加了8.3倍和1.2倍,甘油利用能力明显增强,除2,3-丁二醇外,其他副产物积累量均有所下降,但1,3-PDO产量分别降低了23.3%、5.9%.共表达ppc和gltA基因后,与对照菌相比,生物量降低了35.7%,甘油利用能力进一步增强,1,3-丙二醇产量提高了10.2%,所有副产物积累量均有所减少;与单独过表达ppc或gltA基因相比,乙醇、乳酸、乙酸积累量未有明显变化,但生物量略有提高,2,3-丁二醇积累量显著降低.上述结果表明通过共表达磷酸烯醇式丙酮酸羧化酶和柠檬酸合成酶强化TCA循环能够提高菌株的甘油利用能力,弱化副产物合成,强化1,3-丙二醇的合成.

The anaplerotic reaction and the tricarboxylic acid (TCA) cycle play important roles in the central metabolism of Klebsiella pneumoniae. Phosphoenolpyruvate carboxylase (PPC) and citrate synthase (GLTA) are key enzymes of the anaplerotic reaction and TCA cycle, respectively. This study aimed to investigate the effects of strengthening the TCA cycle on cell growth and glycerol metabolism of recombinant K. pneumoniae based on the anaplerotic reaction. To fulfill these objectives, PPC encoded by ppc and GLTA encoded by gltA were expressed individually or co-expressed in K. pneumoniae. Overexpressing ppc and gltA genes led to a 8.3-fold and 1.2-fold increase in the production of succinate and α-ketoglutarate, respectively. Succinate and α-ketoglutarate are the intermediate metabolites of the reductive and oxidative branches of the TCA cycle. The glycerol utilization ability of the recombinants was significantly enhanced. The accumulation of by-products-except 2,3-butanediol-decreased, but the yield of 1,3-propanediol (1,3-PDO) was decreased by 23.3% and 5.9%, respectively. Compared to the control strain, co-expression of ppc and gltA resulted in a 35.7% decrease in biomass, but the glycerol utilization ability was further enhanced and 1,3-PDO titer was increased by 10.2%. In addition, the accumulation of all by-products decreased. Production of ethanol, lactate, and acetate did not significantly differ between the strains that overexpressed ppc or gltA;however, the biomass increased slightly and the accumulation of 2,3-butanediol decreased significantly. Enhancing the TCA cycle by co-overexpressing ppc and gltA could improve the glycerol utilization ability of recombinant K. pneumoniae, weaken the synthesis of by-products, and enhance the production of 1,3-PDO.