Expanding Toolbox for Genes Expression of <i>Yarrowia lipolytica</i> to Include Novel Inducible, Repressible, and Hybrid Promoters

Xiong, Xiaochao; Chen, Shulin

doi:10.1021/acssynbio.0c00243

Cited by 34 publications

(35 citation statements)

References 36 publications

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“…In metabolic engineering, studies have shown that the yield of the target product can be increased by replacing promoters of pathway genes with stronger ones [ 28 , 29 ]. Therefore, to demonstrate the application of our promoter library for optimizing metabolic pathways, we employed our hybrid promoters in the heterologous isoamyl alcohol pathway of Po1g BAA.…”

Section: Resultsmentioning

confidence: 99%

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Liu

et al. 2021

Biotechnol Biofuels

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Background In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

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Section: Resultsmentioning

confidence: 99%

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Liu

et al. 2021

Biotechnol Biofuels

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“…Cloning of essential genetic elements such as constitutive, inducible, targeted, and repressible promoters is being carried out in R. toruloides and Y. lipolytica ( Nora et al, 2019 ). Besides, engineering of tandem copies pertinent to upstream activation sequences (UASs) has been modified to strengthen the hybrid promoter for higher expressions in Y. lipolytica ( Blazeck et al, 2011 ; Xiong and Chen, 2020 ). Genome editing mediated by clustered regularly interspersed short palindromic repeats (CRISPR) along with Cas protein has been studied in Y. lipolytica and R. toruloides , which can be optimized to improve the efficiency of lipid accumulation by adopting multiplex genome engineering with low off-target effects ( Jiao et al, 2019 ; Otoupal et al, 2019 ; Schultz et al, 2019 ; Abdel-Mawgoud and Stephanopoulos, 2020 ; Yang et al, 2020 ).…”

Section: Metabolic Engineering Approaches For Enhanced Lipid Synthesis and Biorefinery Productsmentioning

confidence: 99%

Biodiesel Production From Lignocellulosic Biomass Using Oleaginous Microbes: Prospects for Integrated Biofuel Production

et al. 2021

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Biodiesel is an eco-friendly, renewable, and potential liquid biofuel mitigating greenhouse gas emissions. Biodiesel has been produced initially from vegetable oils, non-edible oils, and waste oils. However, these feedstocks have several disadvantages such as requirement of land and labor and remain expensive. Similarly, in reference to waste oils, the feedstock content is succinct in supply and unable to meet the demand. Recent studies demonstrated utilization of lignocellulosic substrates for biodiesel production using oleaginous microorganisms. These microbes accumulate higher lipid content under stress conditions, whose lipid composition is similar to vegetable oils. In this paper, feedstocks used for biodiesel production such as vegetable oils, non-edible oils, oleaginous microalgae, fungi, yeast, and bacteria have been illustrated. Thereafter, steps enumerated in biodiesel production from lignocellulosic substrates through pretreatment, saccharification and oleaginous microbe-mediated fermentation, lipid extraction, transesterification, and purification of biodiesel are discussed. Besides, the importance of metabolic engineering in ensuring biofuels and biorefinery and a brief note on integration of liquid biofuels have been included that have significant importance in terms of circular economy aspects.

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“…In metabolic engineering, studies have shown that the yield of the target product can be increased by replacing promoters of pathway genes with stronger ones [28,29]. Therefore, to demonstrate the application of our promoter library for optimizing metabolic pathways, we employed our hybrid promoters in the heterologous isoamyl alcohol pathway of Po1g BAA.…”

Section: Application Of the Hybrid Promoter Library To Improve The Isoamyl Alcohol Biosynthesis Pathwaymentioning

confidence: 99%

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Zhao

Liu

et al. 2021

Preprint

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Background: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. We also report for the first time that upstream activation sequences (UAS) of Saccharomyces cerevisiae promoters can be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.Conclusion: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

show abstract

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters

Cited by 34 publications

References 36 publications

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Biodiesel Production From Lignocellulosic Biomass Using Oleaginous Microbes: Prospects for Integrated Biofuel Production

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

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