Efficient Succinic Acid Production from Glucose through Overexpression of Pyruvate Carboxylase in an Escherichia coli Alcohol Dehydrogenase and Lactate Dehydrogenase Mutant

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• 作者：Ailen M. Sá ; nchez ; George N. Bennett ; and Ka-Yiu San
• 刊名：Biotechnology Progress
• 出版年：2005
• 出版时间：April 2005
• 年：2005
• 卷：21
• 期：2
• 页码：358 - 365
• 全文大小：176K
• 年卷期：v.21,no.2(April 2005)
• ISSN：1520-6033

文摘

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has beenconstructed to produce succinic acid in the presence of heterologous pyruvatecarboxylase (PYC). The strategic design aims at diverting maximum quantities ofNADH for succinate synthesis by inactivation of NADH competing pathways toincrease succinate yield and productivity. Additionally an operational PFL enzymeallows formation of acetyl-CoA for biosynthesis and formate as a potential source ofreducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favorsuccinate generation. SBS110MG harboring plasmid pHL413, which encodes theheterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM)of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphereof CO₂ yielding 1.3 mol of succinate per mole of glucose. This molar yield exceededthe maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work furtherexplores the importance of the presence of formate as a source of reducing equivalentsin SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway(FDH) in this strain significantly reduced succinate yield, suggesting that reducingpower was lost in the form of formate. Additionally we investigated the effect of ptsGinactivation in SBS110MG(pHL413) to evaluate the possibility of a further increasein succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4mol/mol at the expense of a reduction in glucose consumption of 33%. In the presenceof PYC and an efficient conversion of glucose to products, the ptsG mutation is notindispensable since PEP converted to pyruvate as a result of glucose phosphorylationby the glucose specific PTS permease EIICB^glu can be rediverted toward OAA favoringsuccinate production.