LACTIC ACID PRODUCTION IN GENETICALLY MODIFIED Escherichia coli

Abstract

Lactic acid is an organic acid, which can be classified into 2 isomers: L (+)-lactic acid and D (-)-lactic acid. It is widely used in various industrial applications, including as a potential precursor to synthesize polylactides (PLA) for biodegradable plastic which the production of optically pure monomers is essential. Although Rhizopus oryzae is a potential candidate for L-lactic acid production due to the production of an optically pure L-isomer of lactic acid, it has some limitations such as its unsuitable morphology for fermentation in bioreactors. The aim of this research is to study lactic acid production from genetically engineered Escherichia coli with inactivated genes involved in fermentative pathways and such mutants harboring plasmid with R. oryzae ldhA gene. The strains of deactivated pta ldhA with additional knocked out pflB or adhE could not grow anaerobically. Moreover, the exogenous ldhA from R. oryzae on the plasmid could not complement the growth under this condition. The deactivated pta ldhA frdC was able to grow, but poorly, under anaerobic condition, while R. oryzae ldhA gene could restore the fermentative growth. The E. coli strain of knocked out pta ldhA frdC with plasmid containing ldhA gene from R. oryzae (AP20) verified the expression of R. oryzae ldhA gene by qRT-PCR. Nevertheless, LDH activity and growth rate were lower than those of the wild type strain. After fermented in media with 2% initial glucose, a small amount of L-lactic acid was detected, but other fermentation byproducts seemed to be reduced, except for ethanol. This could be resulted from inadequate NADH recycle, inhibition of phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for glucose consumption, or expression of eukaryotic protein in prokaryotic host.