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

Zhong, Wei, Yang, Maohua, Mu, Tingzhen, Wu, Fan, Hao, Xuemi, Chen, Ruonan, Sharshar, Moustafa Mohamed, Thygesen, Anders, Wang, Qinhong, Xing, Jianmin
Biochemical engineering journal 2019 v.144 pp. 217-226
Saccharomyces cerevisiae, acid tolerance, batch fermentation, catalytic activity, genes, glucose, lactate dehydrogenase, lactic acid, mutants, plasmids, pyruvic acid, synthetic biology, thermal stability, transposons, yeasts
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 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. coliD-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.