Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis

Abstract

The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1′-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals. IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.

Genome-scale metabolic reconstruction of the non-model yeast Issatchenkia orientalis SD108 and its application to organic acids production

“…The strain I. orientalis SD108, which was used as a host for succinic acid overproduction, was found to be tolerant to high levels of succinic acid, itaconic acid, adipic acid, and acetic acid ( Xiao et al., 2014 ), making it an attractive alternative organism for the production of industrially-relevant organic acids. Although it is not as well-studied as the model yeast Saccharomyces cerevisiae , new genetic tools have been developed ( Tran et al., 2019 ; Cao et al., 2020 ). Advances in domesticating non-model microorganisms, including yeasts, have recently been reviewed ( Fatma et al., 2020 ).…”

Recent advances in domesticating non‐model microorganisms

“…7,77,78 The organism natively favors HR for DNA repair, and recently an episomal plasmid as well as a CRISPR/ Cas9-based gene-editing tool have been reported. 55 For CRISPR/Cas9-based gene editing in I. orientalis, SpCas9 was expressed from an ARS plasmid using pTEF1 and the gRNA was expressed from the same plasmid, which also contained a homology donor with 50 bp arms containing an 8-bp frame shift mutation for the targeted gene. A variety of promoters were tested for gRNA expression: tRNA Ser , tRNA Leu , the 5S rRNA, 5S rRNA-tRNA Leu , RPR1, and RPR1'-tRNA Leu (RPR1 0 is the RPR1 RNA promoter extended slightly into the RPR1 gene).…”

“…RPR1 0 -tRNA Leu enabled the highest deletion efficiency of ADE2, 97.0 ± 1%, and enabled 100% deletion of LEU2, HIS3, and TRP1. 55 In a comparison of CRISPR-mediated gene deletion efficiency with and without a homology donor for DNA repair, the homology donor proved essential for successful deletion of ADE2, with 93% of the colonies receiving the donor having the gene knocked out, and 0% of those which received no donor. This indicates that I. orientalis relies on HR for DNA repair.…”

“…This indicates that I. orientalis relies on HR for DNA repair. 55 Based on the significant genetic similarity between I. orientalis and S. cerevisiae, the S. cerevisiae ARS from the commonly used plasmid pRS415 was tested in I. orientalis. It was found to confer reasonable stability, with 60% of cells expressing GFP after 24 hours, and 55% after 5 days.…”

“…It was found to confer reasonable stability, with 60% of cells expressing GFP after 24 hours, and 55% after 5 days. 55 The S. cerevisiae centromere from the same plasmid was also tested but did not confer improved plasmid maintenance, indicating that this sequence is not conserved between the two species. Furthermore, in a recent work, centromeric sequences were identified using GC 3 scanning followed by sequence alignment to find a conserved 800-bp motif within each chromosome's centromere.…”