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

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.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

“…To overcome this limitation, we examined if a genome-editing tool developed for Zygosaccharomyces bailii can be utilized for introducing genetic perturbations into various I. orientalis strains (SD108, IO21, IO45, and IO46).…”

“…All strains and plasmids are described in Table 1 and primers used in this study are presented in Table S1. To construct plasmids for Cas9-based genome editing, the pCast plasmid 26 was used as a backbone vector and linearized with an AarI restriction enzyme. Each guide RNA targeting sequence in the pCast_ADE2, pCast_IS2, and pCast_IS3 plasmids was generated with a 20 bp targeting sequence of ADE2, intergenic sequence 2 (IS2, located at chromosome 1: 2284520−2284539), and intergenic sequence 3 (IS3, located at chromosome 3: 883681− 883700) (Table S2) with a sticky end overhang of AarI.…”

“…Therefore, to conduct metabolic engineering with newly isolated I. orientalis strains exhibiting high tolerance against low pH or high temperatures, the isolated I. orientalis strains need to be mutated and selected in 5-fluoroorotic acid (FOA) containing media, or URA3 needs to be disrupted in advance. 18,20 To overcome this limitation, we examined if a genomeediting tool developed for Zygosaccharomyces bailii 26 can be utilized for introducing genetic perturbations into various I. orientalis strains (SD108, IO21, IO45, and IO46). First, we found that the pCast plasmid containing a PAN-ARS derived from K. lactis 40 and a dominant drug resistance gene (cloNAT) was transformable into the I. orientalis strains with decent transformation efficiencies.…”