Metabolic Engineering Of Saccharomyces Cerevisiae For D-Lactic Acid Production

D-lactic acid(D-LA),an isomer of L-lactic acid,is one of the most valuable bio-based chemicals,which can be used as an intermediate for producing chiral drugs,pesticides,and other chemicals.Particularly,D-LA is gaining increased attention as it can improve the thermostability of poly lactic acid(PLA).Microbial-production of D-LA is environment friendly and can avoid environmental pollution caused by chemical process.Acid-resistant Saccharomyces cerevisiae,because it is generally recognized as safe and easily to be edited,is suitable for industrial-scale production of D-LA.However,S.cerevisiae produces D-LA are limited by the heterologous expression level of the stereospecific D-lactate dehydrogenase(D-LDH),growth retardation of engineered strain,and acidic toxicity.In this work,we optimized the expression level of D-LDH,enhanced the acid-tolerance,and improved the productivity of D-LA by metabolic engineering.Firstly,a synthetic biology approach was used to construct high-producing D-LA strains by optimization of D-LDH expression through combinations of four promoters,two terminators and five D-LDHs.The pyruvate decarboxylase-deficient mutant strain TAMH was used as host strain for optimizing the 40 D-LDH expression cassettes.The TCSt strain harboring P:TEF1-E.coli D-LDH-Tsynth25 produced 5.8 g/L D-LA with an optical purity of 99.9%.The D-LA production was further improved by integrating this cassette into the Tyl transposable element of the YIP-01 strain with the deletion of Pdcland Pdc6 genes.The integrated strains were screened by a double enzyme-coupled system.Genome sequencing of the resulting strain YIP-pTCSt-301 showed that there were three copies of the D-LDH expression cassette.In fed-batch fermentation,the strain produced 35 g/L D-LA,with a yield of 0.45 g/g and a productivity of 0.9 g/L/h under acid conditions.D-LA production was further improved by deleting the Jenl(a monocarboxylate transporter),D-Lactate dehydrogenasel(Dldl),L-lactate cytochrome-c oxidoreductase(Cyb2),and alcohol dehydrogenase 1(Adhl)genes of the YIP-pTCSt-301 strain,and the resulting strain YIP-JCDA1 produced 80.0 g/L D-LA with a yield of 0.6 g/g and a productivity of 1.1 g/L/h in fed-batch fermentation.Then,a heterologous glycosylphosphatidylinositol-anchored protein,IoGasl,required for resistant to low pH and salt stress,was overexpressed in the YIP-JCDA1 strain.D-LA production of the resulting strain YIP-IJCDA1 was improved to 85.3 g/L with a yield of 0.71 g/g and a productivity of 1.20 g/L/h.Next,the D-LA production was further increased by attenuating the ethanol pathway and disrupting glycerol-3-phosphate dehydrogenases(Gpd).The resulting strain YIP-A15G12 with the deletion of Adhl,Adh5,Gpdland Gpd2 genes,produced 92.0 g/L D-LA with a yield of 0.70 g/g and a productivity of 1.21 g/L/h in fed-batch fermentation under acid conditions.The optical purity of D-LA reached 99.7%.Subsequently,the acetic acid-resistant genes Acs2 and Haal were overexpressed in the YIP-IJCDA1 strain,respectively.D-LA production of the resulting strains YIP-JlA and YIP-CH was not improved.Further overexpressing the cytosolic aldehyde dehydrogenase 6(Ald6)gene in the YIP-J1A strain showed that the D-LA production of the resulting strain was not improved.Overexpression of another acetic acid-resistant gene,Whi2,can significantly improve the glucose fermentation of the resulting strain under acetic acid stress,which suggested that the Whi2 protein combined with IoGas1 protein can significantly improve the glucose fermentation under acetic acid stress.Finally,to increase acetyl-CoA synthesis in S.cerevisiae,the bacterial acetylating acetaldehyde dehydrogenase(A-ALD)genes mhpF and eutE were expressed in the yeast strain which was attenuated acetaldehyde dehydrogenase(Ald)/acetyl-CoA synthetase(Acs).Growth rate of the resulting strains,YIP-m-ald6 and YIP-e-ald6,were significantly increased.Under fed-batch fermentation condition,YIP-m-ald6 produced 92.6 g/L D-LA with a yield of 0.70 g/g and a productivity of 1.68 g/L/h.The productivity of this strain was 1.4 times of YIP-A15G12.The optical purity of D-LA reached 99.8%.