Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species

Liu, Yanbin; Yap, Sihui Amy; Koh, Chong Mei John; Ji, Lianghui

doi:10.1186/s12934-016-0600-x

Cited by 54 publications

(56 citation statements)

References 40 publications

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“…Promoters P6, P9, P10, P12, P14 and P15 contain introns in their predicted 5ʹ UTRs, a feature often associated with increased promoter strength. Introns can increase transcript levels by affecting the rate of transcription, nuclear export, transcript stability and even mRNA translation So far, there have been 3 reports of relatively constitutive promoters for use in R. toruloides, totaling a set of 8 promoters: GAPDH (also called GPD1), FBA1, PGK1, PGL1, TPI1, LDP1, ACC1 and FAS1 [10,11,13,14]. Since normalization of the reporter genes used to characterize these promoters differs between these studies, it is challenging to compare strength of these promoters with the collection presented here.…”

Section: Discussionmentioning

confidence: 99%

“…A growing branch of synthetic biology involves the pursuit and improvement of standard parts that are reliable, orthogonal and robust for non-conventional hosts [8,9]. A small selection of promoters has already been characterized to modulate expression of heterologous genes in R. toruloides, the majority of them being metabolite-responsive promoters rather than constitutive [7,[10][11][12][13]. While promoters that are responsive to certain metabolites are valuable tools, a toolset of well characterized constitutive promoters remains necessary to explore the full potential of strain engineering.…”

Section: Introductionmentioning

confidence: 99%

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A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

Nora

Wehrs

Kim

et al. 2019

Preprint

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Background: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. Results:Our data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression strength by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) of potential value for genetic engineering of R. toruloides. Conclusions:We present a list of robust and constitutive, native promoters to facilitate genetic engineering of R. toruloides. This set of thoroughly characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

Nora

Wehrs

Kim

et al. 2019

Preprint

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“…While this yeast has been touted for its great bioproduction potential, the sparse genetic manipulation toolkit relative to other organisms such as Y. lipolytica (27) previously hindered engineering efforts (55)(56)(57)(58). The past 4 years have seen various researchers remedy this problem with the development of tools for transforming R. toruloides and efficiently expressing exogenous DNA (15,59). While the toolkit has expanded to include useful promoters (56), drug markers (11), and targeted gene editing methods (18), CRISPR-Cas9 methods for advanced genome engineering have been lacking.…”

Section: Discussionmentioning

confidence: 99%

Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

Otoupal

Ito

Arkin

et al. 2019

mSphere

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Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

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“…While this yeast has been touted for its great bioproduction potential, the sparse genetic manipulation toolkit relative to other organisms such as Y. lipolytica (27) previously hindered engineering efforts (52–55). The past four years have seen various researchers remedy this problem with the development of tools for transforming R. toruloides and efficiently expressing exogenous DNA (15, 56). While the toolkit has expanded to include useful promoters (53), drug markers (11), and targeted gene editing methods (18), CRISPR-Cas9 methods for advanced genome engineering have been lacking.…”

Section: Discussionmentioning

confidence: 99%