Title: 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'-tRNApromoter 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. 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 toolsmore » 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.« less

Authors:

Tran, Vinh G. ^[1]; Cao, Mingfeng ^[1]; Fatma, Zia ^[1]; Song, Xiaofei ^[2]; Zhao, Huimin ^[3]; Mitchell, ed., Aaron P.

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+ Show Author Affiliations

1. Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
2. Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA, Department of Microbiology, Nankai University, Tianjin, China
3. Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA, Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA, Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA, Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Publication Date:

Wed Jun 26 00:00:00 EDT 2019

Research Org.:

Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL: (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1529164

Alternate Identifier(s):

OSTI ID: 1542920

Grant/Contract Number:  

SC0018420

Resource Type:

Journal Article: Published Article

Journal Name:

mSphere

Additional Journal Information:

Journal Name: mSphere Journal Volume: 4 Journal Issue: 3; Journal ID: ISSN 2379-5042

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9, Issatchenkia orientalis, genome editing, metabolic engineering, synthetic biology