Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>

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 engineerin...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Vinh G. Tran, Mingfeng Cao, Zia Fatma, Xiaofei Song, Huimin Zhao
Formato: article
Lenguaje:EN
Publicado: American Society for Microbiology 2019
Materias:
Acceso en línea:https://doaj.org/article/e165b947c0cd40daa4d29acc408185ce
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:e165b947c0cd40daa4d29acc408185ce
record_format dspace
spelling oai:doaj.org-article:e165b947c0cd40daa4d29acc408185ce2021-11-15T15:22:20ZDevelopment of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>10.1128/mSphere.00345-192379-5042https://doaj.org/article/e165b947c0cd40daa4d29acc408185ce2019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00345-19https://doaj.org/toc/2379-5042ABSTRACT 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.Vinh G. TranMingfeng CaoZia FatmaXiaofei SongHuimin ZhaoAmerican Society for MicrobiologyarticleCRISPR/Cas9Issatchenkia orientalisgenome editingmetabolic engineeringsynthetic biologyMicrobiologyQR1-502ENmSphere, Vol 4, Iss 3 (2019)
institution DOAJ
collection DOAJ
language EN
topic CRISPR/Cas9
Issatchenkia orientalis
genome editing
metabolic engineering
synthetic biology
Microbiology
QR1-502
spellingShingle CRISPR/Cas9
Issatchenkia orientalis
genome editing
metabolic engineering
synthetic biology
Microbiology
QR1-502
Vinh G. Tran
Mingfeng Cao
Zia Fatma
Xiaofei Song
Huimin Zhao
Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
description 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.
format article
author Vinh G. Tran
Mingfeng Cao
Zia Fatma
Xiaofei Song
Huimin Zhao
author_facet Vinh G. Tran
Mingfeng Cao
Zia Fatma
Xiaofei Song
Huimin Zhao
author_sort Vinh G. Tran
title Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
title_short Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
title_full Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
title_fullStr Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
title_full_unstemmed Development of a CRISPR/Cas9-Based Tool for Gene Deletion in <italic toggle="yes">Issatchenkia orientalis</italic>
title_sort development of a crispr/cas9-based tool for gene deletion in <italic toggle="yes">issatchenkia orientalis</italic>
publisher American Society for Microbiology
publishDate 2019
url https://doaj.org/article/e165b947c0cd40daa4d29acc408185ce
work_keys_str_mv AT vinhgtran developmentofacrisprcas9basedtoolforgenedeletioninitalictoggleyesissatchenkiaorientalisitalic
AT mingfengcao developmentofacrisprcas9basedtoolforgenedeletioninitalictoggleyesissatchenkiaorientalisitalic
AT ziafatma developmentofacrisprcas9basedtoolforgenedeletioninitalictoggleyesissatchenkiaorientalisitalic
AT xiaofeisong developmentofacrisprcas9basedtoolforgenedeletioninitalictoggleyesissatchenkiaorientalisitalic
AT huiminzhao developmentofacrisprcas9basedtoolforgenedeletioninitalictoggleyesissatchenkiaorientalisitalic
_version_ 1718428040711634944