Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica
TL;DR: Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products and provides a sustainable source of EPA.
Abstract: Metabolic engineering of the oleaginous yeast Yarrowia lipolytica greatly enhances yields of omega-3 eicosapentaenoic acid.
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TL;DR: D dietary n − 3 in fish feeds can be defined by three levels; the minimum level required to satisfy EFA requirements and thus prevent nutritional pathologies, that required to sustain maximum growth and optimum health in fish being fed modern high-energy diets, and the balance between different PUFA and LC-PUFA, which far exceeds the biological requirements of the fish itself.
524 citations
Cites background from "Production of omega-3 eicosapentaen..."
...Metabolic engineering of the oleaginous yeast Yarrowia lipolytica has resulted in a strain that produced EPA at 15 % of dry weight (Xue et al., 2013)....
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TL;DR: A thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability is reported, which advances fundamental understanding of lipogenesis, and non-canonical environmental and intracellular stimuli are demonstrated and uncouple lipogenesis from nitrogen starvation.
Abstract: Bio-based production of oils and lipids could potentially provide a sustainable fuel alternative to petroleum. Here, the authors show that Yarrowia lipolytica’s metabolism can be rewired to saturate cells with upwards of 90% lipid content and significantly increase lipid production.
480 citations
TL;DR: It is shown that redox engineering could enable commercialization of microbial carbohydrate-based lipid production via synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA.
Abstract: Microbial factories have been engineered to produce lipids from carbohydrate feedstocks for production of biofuels and oleochemicals. However, even the best yields obtained to date are insufficient for commercial lipid production. To maximize the capture of electrons generated from substrate catabolism and thus increase substrate-to-product yields, we engineered 13 strains of Yarrowia lipolytica with synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. A quantitative model was established and identified the yield of the lipid pathway as a crucial determinant of overall process yield. The best engineered strain achieved a productivity of 1.2 g/L/h and a process yield of 0.27 g-fatty acid methyl esters/g-glucose, which constitutes a 25% improvement over previously engineered yeast strains. Oxygen requirements of our highest producer were reduced owing to decreased NADH oxidization by aerobic respiration. We show that redox engineering could enable commercialization of microbial carbohydrate-based lipid production.
319 citations
TL;DR: The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica, a valuable microbial host for chemical production.
Abstract: The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR–Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III–tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1′–tRNAGly promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8–TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor pla...
265 citations
TL;DR: The most important efforts target the efficiency of the oleaginous machinery, via overexpression of key-enzymes involved in lipid biosynthesis, as well as the minimization of lipid degradation, by repressing genes involved in the β-oxidation pathway.
238 citations
References
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TL;DR: The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated and made particularly accessible to gene cloning and genetic engineering techniques.
Abstract: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.
3,547 citations
TL;DR: Important insights into the mechanisms of inflammatory responses, pain, and fever have been gleaned from the current understanding of eicosanoid biology.
Abstract: Prostaglandins and leukotrienes are potent eicosanoid lipid mediators derived from phospholipase-released arachidonic acid that are involved in numerous homeostatic biological functions and inflammation. They are generated by cyclooxygenase isozymes and 5-lipoxygenase, respectively, and their biosynthesis and actions are blocked by clinically relevant nonsteroidal anti-inflammatory drugs, the newer generation coxibs (selective inhibitors of cyclooxygenase-2), and leukotriene modifiers. The prime mode of prostaglandin and leukotriene action is through specific G protein-coupled receptors, many of which have been cloned recently, thus enabling specific receptor agonist and antagonist development. Important insights into the mechanisms of inflammatory responses, pain, and fever have been gleaned from our current understanding of eicosanoid biology.
3,505 citations
TL;DR: Testing the hypothesis that long-term use of eicosapentaenoic acid (EPA) is effective for prevention of major coronary events in hypercholesterolaemic patients in Japan who consume a large amount of fish found it to be a promising treatment.
2,269 citations
TL;DR: Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.
Abstract: Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.
1,604 citations
TL;DR: In this article, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids and are catalyzed by polyketide synthases (PKSs).
Abstract: Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules. Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids. These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism. Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics. It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes.
691 citations