Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production.
Ontology highlight
ABSTRACT:
SUBMITTER: Saez-Saez J
PROVIDER: S-EPMC7672257 | biostudies-literature | 2020 Nov
REPOSITORIES: biostudies-literature
Publications
Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production.
Sáez-Sáez Javier J Wang Guokun G Marella Eko Roy ER Sudarsan Suresh S Cernuda Pastor Marc M Borodina Irina I
Metabolic engineering 20200818
Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway pro ...[more]
Similar Datasets
Project description:Nervonic acid benefits the treatment of neurological diseases and the health of brain. In this study, we employed the oleaginous yeast Yarrowia lipolytica to overproduce nervonic acid oil by systematic metabolic engineering. First, the production of nervonic acid was dramatically improved by iterative expression of the genes ecoding β-ketoacyl-CoA synthase CgKCS, fatty acid elongase gELOVL6 and desaturase MaOLE2. Second, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of glycerol-3-phosphate acyltransferases and diacylglycerol acyltransferases from Malania oleifera in endoplasmic reticulum (ER). Third, overexpression of a newly identified ER structure regulator gene YlINO2 led to a 39.3% increase in lipid production. Fourth, disruption of the AMP-activated S/T protein kinase gene SNF1 increased the ratio of nervonic acid to lignoceric acid by 61.6%. Next, pilot-scale fermentation using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).
| S-EPMC10630375 | biostudies-literature
Project description:BackgroundYarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.ResultsWe constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.ConclusionsYarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.
| S-EPMC6927232 | biostudies-literature
Project description:BackgroundSugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica.ResultsY. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production.ConclusionsWe demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.
| S-EPMC8466642 | biostudies-literature
Project description:BackgroundLimonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene.ResultsTo construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica. Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW.ConclusionsThis is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.
| S-EPMC5057495 | biostudies-literature
Project description:Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push-pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.
| S-EPMC6119263 | biostudies-literature
Project description:The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.
| S-EPMC7418586 | biostudies-literature
Project description:The non-conventional oleaginous yeast Yarrowia lipolytica is able to utilize both hydrophilic and hydrophobic carbon sources as substrates and convert them into value-added bioproducts such as organic acids, extracellular proteins, wax esters, long-chain diacids, fatty acid ethyl esters, carotenoids and omega-3 fatty acids. Metabolic pathway analysis and previous research results show that hydrophobic substrates are potentially more preferred by Y. lipolytica than hydrophilic substrates to make high-value products at higher productivity, titer, rate, and yield. Hence, Y. lipolytica is becoming an efficient and promising biomanufacturing platform due to its capabilities in biosynthesis of extracellular lipases and directly converting the extracellular triacylglycerol oils and fats into high-value products. It is believed that the cell size and morphology of the Y. lipolytica is related to the cell growth, nutrient uptake, and product formation. Dimorphic Y. lipolytica demonstrates the yeast-to-hypha transition in response to the extracellular environments and genetic background. Yeast-to-hyphal transition regulating genes, such as YlBEM1, YlMHY1 and YlZNC1 and so forth, have been identified to involve as major transcriptional factors that control morphology transition in Y. lipolytica. The connection of the cell polarization including cell cycle and the dimorphic transition with the cell size and morphology in Y. lipolytica adapting to new growth are reviewed and discussed. This review also summarizes the general and advanced genetic tools that are used to build a Y. lipolytica biomanufacturing platform.
| S-EPMC6999540 | biostudies-literature
Project description:Here, we report the genome sequence of the oleaginous yeast Yarrowia lipolytica H222. De novo genome assembly shows three main chromosomal rearrangements compared to that of strain E150/CLIB122. This genomic resource will help integrate intraspecies diversity into synthetic biology projects that utilize Yarrowia as a biotechnological chassis for value-added chemical productions.
| S-EPMC6346196 | biostudies-literature
Project description:Oleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genomewide tools enabling functional genomics. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the Y. lipolytica genome. Over 535 thousand independent insertions were identified by next-generation sequencing. Poisson analysis of insertion density classified ~22% of genes as essential. As expected, most essential genes not only have homologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe, but the majority of those are also essential. As an obligate aerobe, Y. lipolytica has significantly more respiration - related genes that are classified as essential than do S. cerevisiae and S. pombe. The findings provide insights into biosynthetic pathways, compartmentalization of enzymes, and distinct functions of paralogs. Contributions of nonessential genes to fitness were determined in log growth cultures with glucose and glycerol carbon sources. Fluorescence-activated cell sorting identified mutants in which lipid accumulation is increased. Biological contributions of genes to growth were used to evaluate two recent genome-scale models Y. lipolytica metabolism. This study is the first functional genomic analysis of an oleaginous yeast and provides an important resource for modeling and bioengineering of Y. lipolytica.
2018-12-06 | GSE90993 | GEO
Project description:BackgroundLimonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly.ResultsIn this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization.ConclusionsThis study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.
| S-EPMC6781337 | biostudies-literature