GENETIC MODIFICATION OF Escherichia coli FOR L-LACTIC ACID PRODUCTION

Abstract

Lactic acid is a chemical compound that has been used in many industrial applications. Therefore, effective process of lactic acid production is required to meet the higher demand of PLA plastic. The lactic acid can be produced by microbial fermentation including lactic acid bacteria (LAB) and fungi. Currently, Rhizopus oryzae has been interested due to its pure L(+)-lactic acid production. However, without additional applications, its morphology is difficult to control during fermentation process because its mycelium can cause a problem in stirred-tank bioreactor. To overcome such problem, genetic engineering technique for generating genetically modified Escherichia coli will be used. In this research, the gene replacement method and applied different plasmid system were used. According to a limitation of gene replacement technique, the DNA fragment for L(+)-lactic acid production was designed by containing ORF of R. oryzae ldhA gene flanked by upstream and downstream region of E. coli ldhA gene and planned to replace E. coli chromosomal ldh gene by linear transformation technique with plasmid pKD46. However, the construction of DNA fragment for gene replacement was unsuccessful. For applied different plasmid system, the pUC19 and pBluescript II KS(+) were used to harbor R. oryzae ldhA gene. The recombinant E. coli strains were fermented at 37 oC for 48 hours under anaerobic condition. The recombinant E. coli strain name TW2 which applied pUC19 vector harboring R. oryzae ldhA gene was successfully produced L(+)-lactic acid with higher yield and LDH activity, but lower intracellular pyruvate concentration than RB24 (pBluescript II KS(+) harboring R. oryzae ldhA gene). The better performances than pBluescript II KS(+) vector on ldhA expression may resulted from lower promoter strength. However, L(+) lactic acid production from TW2 strain was lower than wild type strain. This may come from the function of the other lactate dehydrogenase (LLDH) in wild type strain, and the shift to other fermentative pathways in TW2 strain.