Functional integration of multiple genes into the genome of the oleaginous yeast<i>Rhodosporidium toruloides</i>

Lin, Xinping; Wang, Yanan; Zhang, Sufang; Zhu, Zhiwei; Zhou, Yongjin J.; Yang, Fan; Sun, Weili; Zhao, Zongbao K.

doi:10.1111/1567-1364.12140

Cited by 100 publications

(116 citation statements)

References 35 publications

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“…To delineate the molecular basis of microbial oleaginity, we have conducted differential proteomics study and defined the dynamic nature of lipid droplet proteomes of this yeast (Liu et al ., ; Zhu et al ., ). We (Lin et al ., ) and others (Liu et al ., ) successfully developed an Agrobacterium ‐mediated transformation (ATMT) protocol for integration of genes into the R. toruloides genome and obtained a number of transformants with different antibiotic resistant phenotype. It should also be noted that the transformation of R. toruloides was reported using a protoplast transformation technique (Tully and Gilbert, ).…”

Section: Introductionmentioning

confidence: 97%

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

Wang

Lin

Zhang

et al. 2016

Yeast

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The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.

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Section: Introductionmentioning

confidence: 97%

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

Wang

Lin

Zhang

et al. 2016

Yeast

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“…Due to its high lipid productivity, R. toruloides has been explored extensively for production of triglyceridebased biodiesel, including production of cellulosic fuels. 18 Numerous genetic tools exist for engineering R. toruloides, 19,20 and strains have now been engineered for production of both monoterpene and sesquiterpene biofuel precursors 13 as well as C16-C18 fatty alcohols.…”

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confidence: 99%

Demonstrating a separation-free process coupling ionic liquid pretreatment, saccharification, and fermentation with Rhodosporidium toruloides to produce advanced biofuels

et al. 2018

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Achieving low cost and high efficiency lignocellulose deconstruction is a critical step towards widespread adoption of lignocellulosic biofuels. Certain ionic liquid (IL)-based pretreatment processes effectively reduce recalcitrance of lignocellulose to enzymatic degradation but require either costly separations following pretreatment or novel IL compatible processes to mitigate downstream toxicity. Here we demonstrate at benchtop and pilot bioreactor scales a separation-free, intensified process for IL pretreatment, saccharification, and fermentation of sorghum biomass to produce the sesquiterpene bisabolene, a precursor to the renewable diesel and jet fuel bisabolane. The deconstruction process employs the IL cholinium lysinate ([Ch][Lys]), followed by enzymatic saccharification with the commercial enzyme cocktails Cellic CTec2 and HTec2. Glucose yields above 80% and xylose yields above 60% are observed at all scales tested. Unfiltered hydrolysate is fermented directly by Rhodosporidium toruloides -with glucose, xylose, acetate and lactate fully consumed during fermentation at all scales tested. Bisabolene titers improved with scale from 1.3 g L −1 in 30 mL shake flasks to 2.2 g L −1 in 20 L fermentation. The combined process enables conversion of saccharified IL-pretreated biomass directly to advanced biofuels with no separations or washing, minimal additions to facilitate fermentation, no loss of performance due to IL toxicity, and simplified fuel recovery via phase separation. This study is the first to demonstrate a separation-free IL based process for conversion of biomass to an advanced biofuel and is the first to demonstrate full consumption of glucose, xylose, acetate, and lactic acid in the presence of [Ch][Lys].

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“…To generate gene editing in a target locus, the specific sgRNA plasmid was transformed into Cas9‐expressing R. toruloides strain through the AMT method . Colonies were selected on YEPD agar plates supplemented with hygromycin.…”

Section: Methodsmentioning

confidence: 99%

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

Jiao

Zhang

Liu

et al. 2019

Biotechnology Journal

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The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon‐optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single‐guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9‐expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome‐editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

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Functional integration of multiple genes into the genome of the oleaginous yeastRhodosporidium toruloides

Cited by 100 publications

References 35 publications

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

Demonstrating a separation-free process coupling ionic liquid pretreatment, saccharification, and fermentation with Rhodosporidium toruloides to produce advanced biofuels

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

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