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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

Authors
  • Zhong, Wei
  • Yang, Maohua
  • Mu, Tingzhen
  • Wu, Fan
  • Hao, Xuemi
  • Chen, Ruonan
  • Sharshar, Moustafa Mohamed
  • Thygesen, Anders
  • Wang, Qinhong
  • Xing, Jianmin
Publication Date
Apr 15, 2019
Source
Institutional Repository of Institute of Process Engineering, CAS (IPE-IR)
Keywords
License
Unknown
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Abstract

(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 Pdc1 and 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.

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