Systematically redesigning and optimizing the expression of _D-lactate dehydrogenase efficiently produces high-optical-purity _D-lactic acid in Saccharomyces cerevisiae

_D-lactic acid ( _D-LA) is gaining increased attention as it can improve the thermostability of poly lactic acid. Acid-tolerant Saccharomyces cerevisiae is a good host for _D-LA fermentation. High catalytic efficiency of _D-lactate dehydrogenase ( _D-LDH, EC 1.1.1.28) is crucial for the production of _D-LA in yeast. Here, a synthetic biology approach was used to construct high-producing _D-LA strains by redesigning and optimization of _D-LDH expression by a combination of different promoters, terminators and _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 the pTCSt plasmid with the TEF1 promoter, E. coli _D-LDH and Synth25 synthetic short terminator produced 5.8 g/L _D-LA with an optical purity of 99.9%. The production of _D-LA was further improved by integrating this high expression cassette into the Ty1 transposable element of the YIP-01 strain with deleted Pdc1and Pdc6. The resulting strain YIP-pTCSt-301(CGMCC2.5726) was screened by a double enzyme-coupled system. Genomes sequencing of the strain revealed three copies of the _D-LDH expression cassette. This strain was further improved by deleting the Jen1, Cyb2, Dld1, and Adh1 genes and the resulting strain YIP-J-C-D-A1 (CGMCC2.5783) produced 80.0 g/L _D-LA with a yield of 0.6 g/g glucose and a volumetric productivity of 1.1 g/L/h in fed-batch fermentation under non-neutralization conditions.