Expression of bovine lactate dehydrogenase gene in yeast Pichia pastoris and lactic acid production

Lactic acid has the possibility to use in a wide range, for example medicine, food, cosmetics, chemical industry, etc. Lactic acid has two optical isomers: L (+)-lactic acid and D (-)-lactic acid. Pure production of these isomers is possible only with fermentation. D (-)-lactic acid can cause acidosis and osteoclasis in human metabolism. However, L (+)-lactic acid is important for its bioavailability and for pharmaceutical use. Pichia pastoris is an expression system required for recombinant protein production in the biotechnology and pharmaceutical industry, and is frequently used by researchers. P. pastoris, like bacteria, can grow rapidly in cheap media containing methanol, glucose, glycerol or ethanol as a carbon source. In this thesis study, production of L (+)-lactic acid in P. pastoris was aimed. This is why this yeast can exhibit activity at much lower pH than the microorganisms used to produce lactic acid up to now. Furthermore, the use of L (+) - lactic acid compared to D (-) - lactic acid is more common. The L-lactate dehydrogenase gene from bovine genome was used as a gene source for lactic acid production. In the first step of the study, P. pastoris ΔPDC (pyruvate decarboxylase) gene was inactivated. The L-lactate dehydrogenase gene, which oxidizes pyruvate to L-lactic acid, was expressed under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) and pyruvate decarboxylase (PDC) promoters. Preliminary production studies were done in shake flask scale in a shaker incubator. L-lactic acid production was determined to be approximately 9,5 g/L (0,19 g/g) by X33 pGAP-LDH and FA115 pPDC-LDH single copy P. pastoris strains in batch fermentation conditions. In fed-batch fermentation studies, the highest L-lactic acid production was 38,0 g/L (0,27 g/g) by FA115 pPDC-LDH two-copy strain. However, further studies are expected to yield higher yields with modifications such as optimizing the fermentation conditions, redesigning the promoters, using new promoters and increasing the gene copy number of the LDH. The results obtained from this study will shed light on similar work in the future.