Metabolic Engineering Of Candida Tropicalis To Produce D-lactic Acid

Lactic acid is a kind of?-hydroxy acid,a common carboxylic acid,which is widely applied in biology,pharmaceutical,food and other fields.It can be produced by chemical synthesis and microbial fermentation.Compared with chemical synthesis,microbial fermentation has some advantages such as lower cost of substrates,lower temperature during production,lower energy consumption and higher optical specificity.Yeasts can utilize cheap substrates and has better endurance over pH which could decrease the consumption of neutralizers,so there are more and more researches focused on lactic acid production by yeasts.This research used Candida tropicalis as parental strain and modified its metabolic pathway,finally a genetically engineered strain with considerable D-lactic acid production was obtained.(1)This research isolated and knocked out gene PDC1 and PDC2 encoding pyruvate decarboxylase.Then PDC1 gene knock-out strain AK01?PDC2 gene knock-out strain AK02?PDC1/PDC2 gene knock-out strain AK03 were obtained.Results showed that PDC1 gene deletion can notably decrease the enzyme activity of PDC and production of ethanol.It also decreased the glucose consumption rate and cased growth inhibition to some extent,indicating PDC1 is the key gene of PDC.PDC2 gene deletion does not leave much impact on strain.(2)Genes 2-HAD and D-LDH were integrated into the genome based on PDC gene deletion.Then 2-HAD gene integration with PDC1 gene deletion strain AK04,D-LDH gene integration with PDC1 gene deletion strain AK05,2-HAD gene integration with PDC1/PDC2 gene deletion strain AK06,D-LDH gene integration with PDC1/PDC2 gene deletion strain AK07 have been obtained by homologous recombination.Results showed that D-LDH gene integration strains AK05,AK07 had growth inhibition to some extent compared with 2-HAD gene integration strains AK04,AK06.AK06 produced 0.45 g×L^-1 LA at 48h which is twice as much as that of parental strain.AK05 produced 6.1 g×L^-1 LA at 72h which is 28 times more than that produced by the parental strain.(3)AK05 was chosen to ferment in shake flasks with different concentrations of CaCO₃ as neutralizer.Results showed that pH remained less time in low level during early phase,however it went up quickly in later period.With remarkable reduction of pyruvate,the overall increase of pH contributed to the accumulation of LA.Finally,when the concentration of CaCO₃ was20 g×L^-1,the highest production of LA was raised to 20.25 g×L^-1,96 times as much as that of parental strain.The optical purity of D-LA is 98%.