Modification of metabolic pathways of Saccharomyces cerevisiae by the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value

Abstract

Extractive lactic acid fermentation has recently been paid a great deal of attention. The problem with such a process is, however, that only undissociated lactate can be extracted. Therefore, lactic acid fermentation at low pH values is desirable. In the present study, we modified the metabolism of yeast (not lactic acid producing bacteria often cultivated at pH of 6–7) by expressing the lactate dehydrogenase (LDH) gene for the production of lactate at low pH values. For this purpose, the plasmid pADNS which contains the ADH1 promoter was used as a host vector, and a heterologous gene region, cDNA- LDH-A (encoding bovine lactate dehydrogenase) digested from plasmid pLDH12 was digested and ligated into the aforementioned two host vectors. The resultant plasmids were then transformed into Saccharomyces cerevisiae DS37. Using this recombinant S. cerevisiae strain, several batch and fed-batch fermentations at aerobic, microaerobic, and anaerobic conditions were conducted at several pH values (4.5-3.5). Since the recombinant S. cerevisiae produced a considerable amount of ethanol as well as lactate (about 10 g/ l), we disrupted several pyruvate decarboxylase (PDC) genes to suppress the ethanol formation. Among the PDC genes, PDC1, PDC5 and PDC6, PDC1 had the greatest effect on the cell growth and ethanol production. The plasmid which containing the LDH-A structure gene was then transformed into the mutant strain lacking the PDC1 gene. Cultivation of this strain improved the lactate yield from glucose (from 0.155 to 0.20) while suppressing ethanol formation (from 0.35 to 0.20).