Metabolic engineering of sugar pathways in Escherichia coli

Abstract

A consortium of two strains of Escherichia coli was used to convert a mixture of xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, succinate production from xylose or glucose alone was compared using E. coli expressing either heterologous pyruvate carboxylase or heterologous ATP-forming PEP carboxykinase. Expression of PEP carboxykinase resulted in higher succinate yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase resulted in higher succinate productivity (76 mg/gh) for glucose conversion although the succinate yield (0.91-0.99 g/g) was about the same whether pyruvate carboxylase or PEP carboxykinase was expressed. Then, processes using the xylose-selective strain TXXP pTrc99A-pck and the glucose-selective strain TXG0 pTrc99A-pyc as a consortium were designed for two different feed ratios of glucose:xylose. For either feed ratio, the consortium generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of the consortium approach for the conversion of sugar mixtures: the ability to optimize the pathway for each sugar-to-product conversion independently, and the ability to adjust the consumption rate for each sugar independently, for example, by altering the biomass concentration of each consortium member strain. Escherichia coli unable to metabolize D-glucose (knockouts in ptsG, manZ, glk) accumulates a small amount of D-glucose (yield of about 0.01 g/g) during growth on the pentoses D-xylose or L-arabinose as a sole carbon source. Additional knockouts in zwf and pfkA genes encoding respectively D-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143) increased accumulation to greater than 1 g/L D-glucose and about 100 mg/L D-mannose from 5 g/L D-xylose or L-arabinose. Knockouts of other genes associated with interconversions of D-glucose-phosphates demonstrate that D-glucose is formed primarily by the dephosphorylation of D-glucose-6P. Under controlled batch conditions with 20 g/L D-xylose, MEC143 generated 4.4 g/L D-glucose and 0.6 g/L D-mannose. The results establish a direct link between pentoses and hexoses, and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.