Ailen Sanchez

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia colistrain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl‐CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous 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 atmosphere of CO2 yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the 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 further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsGinactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICBglu can be rediverted toward OAA favoring succinate production.

Effect of Overexpression of a Soluble Pyridine Nucleotide Transhydrogenase (UdhA) on the Production of Poly(3-hydroxybutyrate) in Escherichia coli

A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phboperon from Alcaligenes eutrophusH16 and the native udhAfrom E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.

Manipulating respiratory levels in Escherichia coli for aerobic formation of reduced chemical products

Optimizing the productivity of bioengineered strains requires balancing ATP generation and carbon atom conservation through fine-tuning cell respiration and metabolism. Traditional approaches manipulate cell respiration by altering air feeding, which are technically difficult especially in large bioreactors. An approach based on genetic regulation may better serve this purpose. With excess oxygen supply to the culture, we efficiently manipulated Escherichia coli cell respiration by adding different amount of coenzyme Q1 to strains lacking the ubiCA genes, which encode two critical enzymes for ubiquinone synthesis. As a proof-of-concept, the metabolic effect of the ubiCA gene knockout and coenzyme Q1 supplementation were characterized, and the metabolic profiles of the experimental strains showed clear correlations with coenzyme Q1 concentrations. Further proof-of-principle experiments were performed to illustrate that the approach can be used to optimize cell respiration for the production of chemicals of interest such as ethanol. This study showed that controlled respiration through genetic manipulation can be exploited to allow much larger operating windows for reduced product formation even under fully aerobic conditions.