Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

Abstract

ABSTRACT We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF , pfl , poxB , and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 ( aceEF pfl poxB pps ) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter · h). Ca(OH) 2 was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N 2 for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc ) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD ) reduced succinate formation by 70% to less than 3 g/liter. 13 C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter · h during the anaerobic phase.