Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii

Nurzhan Kuanyshev, Christopher V Rao, Bruce Dien, Yong Su Jin

Research output: Contribution to journalArticlepeer-review

Abstract

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site-specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.

Original languageEnglish (US)
JournalBiotechnology and bioengineering
DOIs
StatePublished - Oct 2020

Keywords

  • CRISPR Cas9
  • lactic acid
  • metabolic engineering
  • Zygosaccharomyces bailii

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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