Heterologous production of α-farnesene in metabolically engineered strains of Yarrowia lipolytica

Yang, Xia; Nambou, Komi; Wei, Liujing; Hua, Qiang

doi:10.1016/j.biortech.2016.06.028

Cited by 127 publications

(93 citation statements)

References 35 publications

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“…In metabolic engineering, balancing the expression level of multiple genes is crucial for increasing the productivity of biosynthetic pathways and subsequently for sustainable production of valuable products [ 57 , 67 ]. As an attractive candidate for industrial biotechnology applications, Y. lipolytica has been widely used for production of oleochemicals [ 8 , 14 , 70 , 71 ], biofuels [ 8 , 16 , 72 , 73 ] and acetyl CoA-derived metabolites [ 9 , 10 , 11 , 23 , 74 ]. But the library of available tools is not as developed as that of other yeasts such as Saccharomyces cerevisiae [ 34 , 35 , 54 ], especially for multiplex gene repression simultaneously [ 59 , 75 ].…”

Section: Resultsmentioning

confidence: 99%

Gene repression via multiplex gRNA strategy in Y. lipolytica

et al. 2018

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BackgroundThe oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest.ResultsIn this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized.ConclusionTaken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0909-8) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Gene repression via multiplex gRNA strategy in Y. lipolytica

et al. 2018

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“…In E. coli , the heterologous expression of α-farnesene synthase from fruits made production of α-farnesene in bacterial ( Zhu et al., 2014 ). Recently, the feasibility of producing α-farnesene in metabolically engineered Y. lipolytica was demonstrated for the first time ( Yang et al., 2016 ).…”

Section: Discussionmentioning

confidence: 99%

MdMYC2 and MdERF3 Positively Co-Regulate α-Farnesene Biosynthesis in Apple

et al. 2020

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α-Farnesene, a sesquiterpene volatile compound plays an important role in plant defense and is known to be associated with insect attraction and with superficial scald of apple and pear fruits during cold storage. But the mechanism whereby transcription factors regulate apple α-farnesene biosynthesis has not been clarified. Here, we report that two transcription factors, MdMYC2 and MdERF3 regulated α-farnesene biosynthesis in apple fruit. Dual-luciferase assays and Y1H assays indicated that MdMYC2 and MdERF3 effectively trans-activated the MdAFS promoter. EMSAs showed that MdERF3 directly binds the DRE motif in the MdAFS promoter. Subsequently, overexpression of MdMYC2 and MdERF3 in apple calli markedly activated the transcript levels of MdHMGR2 and MdAFS . Furthermore, transient overexpression of MdMYC2 and MdERF3 in apple fruit significantly increased MdAFS expression and hence, α-farnesene production. These results indicate that MdMYC2 and MdERF3 are positive regulators of α-farnesene biosynthesis and have important value in genetic engineering of α-farnesene production.

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“…Safe, genetically tractable, and industrially robust, Yarrowia lipolytica is becoming a preferred platform for metabolic engineering [ 3 – 6 ] to produce acetyl-CoA-derived molecules such as usual and unusual fatty acids [ 7 – 10 ], fatty alcohols [ 11 ], fatty esters [ 12 ], beta carotenoids [ 13 ], alkanes [ 14 ], and terpenes [ 15 ]. Lipid accumulation is induced by nutrient limitation in the presence of excess carbon and involves fatty acyl-CoA synthesis via a type I fatty acid synthase (FAS), modification of chain length and degree of desaturation by elongases and desaturases, and incorporation into triacylglyceride (TAG) via a series of enzymatic steps (reviewed in [ 16 ]): glycerol-3-phosphate acyltransferase (GPAT) attaches the first fatty acid onto the glycerol backbone to produce lysophosphatidic acid (LPA); lysophosphatidic acid acyltransferase (LPAT) attaches a second fatty acid to produce phosphatidic acid (PA); PA is dephosphorylated by phosphatidate phosphatase (PAP) to produce diacylglycerol (DAG); diacylglycerol acyltransferase (DGAT) activities add a final fatty acid to produce TAGs.…”

Section: Introductionmentioning

confidence: 99%

High-oleate yeast oil without polyunsaturated fatty acids

Tsakraklides¹,

Kamineni²,

Consiglio³

et al. 2018

Biotechnol Biofuels

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BackgroundOleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements.ResultsWe sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content.ConclusionsHigh-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1131-y) contains supplementary material, which is available to authorized users.

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Heterologous production of α-farnesene in metabolically engineered strains of Yarrowia lipolytica

Cited by 127 publications

References 35 publications

Gene repression via multiplex gRNA strategy in Y. lipolytica

Gene repression via multiplex gRNA strategy in Y. lipolytica

MdMYC2 and MdERF3 Positively Co-Regulate α-Farnesene Biosynthesis in Apple

High-oleate yeast oil without polyunsaturated fatty acids

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