ABSTRACT
When Escherichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively). In order to decrease the byproduct levels, we inactivated succinate and acetate synthesis in B0013-030. Two recombinant plasmids containing mutation cassettes of frdA::difGm and tdcDE::difGm respectively were constructed first. The mutation cassettes were used to delete the target genes on the chromosomal by Red recombination. Subsequently, the antibiotic resistance gene was excised from the chromosomal by Xer recombination. Thereby, mutants B0013-040B (B0013-030, frdA) and B0013-050B (B0013-040B, tdcDE) were produced. D-lactate producing abilities of the engineered strains were tested both in shake flasks and in bioreactors using two-phase fermentation (aerobic growth and anaerobic fermentation) with glucose as the sole carbon source. When fermentation was carried out in shake flasks, inactivation of frdA in B0013-030 to produce B0013-040B reduced succinate accumulation by 80.8%. When tested in a 7-liter bioreactor, B0013-040B accumulated 114.5 g/L D-lactate of over 99.9% optical purity. However, 1.0 g/L succinate and 5.4 g/L acetate still remained in the broth. Further inactivation of tdcD and tdcE genes in B0013-040B to produce B0013-050B decreased acetate and succinate accumulation to 0.4 g/L and 0.4 g/L respectively, and lactate titer was as much as 111.9 g/L (tested in the 7-liter bioreactor). In lightof the lower byproduct levels and high lactate production, strain B00 13-050B may prove useful for D-lactate production.