Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae

• Published in: Applied microbiology and biotechnology Vol. 100; no. 6; pp. 2737 - 2748
• Main Authors: Baek, Seung-Ho, Kwon, Eunice Y, Kim, Yong Hwan, Hahn, Ji-Sook
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.03.2016; Springer; Springer Nature B.V
• Subjects: Acids; Adaptation, Biological; alcohol dehydrogenase; Amino acids; Applied Genetics and Molecular Biotechnology; Bacteria; batch fermentation; biodegradability; Biodegradable materials; Biodegradation; Biomedical and Life Sciences; Biopolymers; Biotechnology; Dehydrogenase; Dehydrogenases; Engineering; Ethanol; ethanol production; Evolution & development; evolutionary adaptation; Fermentation; Gene Deletion; Gene Expression; Genes; Genetic engineering; Glucose; Glycerol; glycerol-3-phosphate dehydrogenase; isomers; Lactic acid; Lactic Acid - metabolism; Leuconostoc - enzymology; Leuconostoc - genetics; Leuconostoc mesenteroides; Leuconostoc mesenteroides subsp. mesenteroides; Life Sciences; metabolic engineering; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Metabolism; Microbial Genetics and Genomics; Microbiology; Neutralization; Observations; Physiological aspects; Plasmids; polylactic acid; Properties; pyruvate decarboxylase; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; stress response; Studies; transactivators; Yeast; South Korea; Adaptive evolution; lactic acid; Metabolic engineering; Acid tolerance; D-lactic acid; Saccharomyces cerevisiae
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-7174-0

There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h).