Engineering the oleaginous yeast Yarrowia lipolytica to produce nutraceuticals: From metabolic design to industrial applications (original) (raw)
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Engineering in Life Sciences, 2019
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 th...
Journal of Fungi, 2021
Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in...
Yarrowia lipolytica as a Cell Factory for Oleochemical Biotechnology
Handbook of Hydrocarbon and Lipid Microbiology, 2010
The yeast Y. lipolytica has developed a very efficient mechanism in the degradation and utilization of hydrophobic substrates. In addition, its capacity to accumulate great amounts of lipids places it among the oleaginous yeasts. The completion of the sequence of the Y. lipolytica genome and the existence of genetic tools for gene manipulation allow the use of its metabolic functions for biotechnological applications. Examples are presented demonstrating that wild type and genetically engineered strains of Y. lipolytica can be used for fatty acid bioconversion, substrate valorization, polyunsaturated fatty acids production, oil polluted water bioremediation and single cell oil production. This review also presents the potential uses of this yeast as an alternative production (cell) factory in the oleochemical field.
Metabolic Engineering for Unusual Lipid Production in Yarrowia lipolytica
Microorganisms
Using microorganisms as lipid-production factories holds promise as an alternative method for generating petroleum-based chemicals. The non-conventional yeast Yarrowia lipolytica is an excellent microbial chassis; for example, it can accumulate high levels of lipids and use a broad range of substrates. Furthermore, it is a species for which an array of efficient genetic engineering tools is available. To date, extensive work has been done to metabolically engineer Y. lipolytica to produce usual and unusual lipids. Unusual lipids are scarce in nature but have several useful applications. As a result, they are increasingly becoming the targets of metabolic engineering. Unusual lipids have distinct structures; they can be generated by engineering endogenous lipid synthesis or by introducing heterologous enzymes to alter the functional groups of fatty acids. In this review, we describe current metabolic engineering strategies for improving lipid production and highlight recent researche...
Engineering of a high lipid producing Yarrowia lipolytica strain
Biotechnology for Biofuels, 2016
Background: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways. Results: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h. Conclusions: The yields, productivities, and titers reported in this study may further support the applied goal of costeffective, large-scale microbial lipid production for use as biofuels and biochemicals.
Microbial Biotechnology, 2011
The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high-value biotechnological, nutritional or pharmacological products. Under the EU-sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high-throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid-derived compounds like wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added-value products.
Holistic Approaches in Lipid Production by Yarrowia lipolytica
Trends in Biotechnology, 2018
Concerns about climate change have driven research on the production of lipid-derived biofuels as an alternative and renewable liquid fuel source. Using oleaginous yeasts for lipid synthesis creates the potential for costeffective industrial-scale operations due to their ability to reach high lipid titer, yield, and productivity resulting from their unique metabolism. Yarrowia lipolytica is the model oleaginous yeast, with the best-studied lipid metabolism, the greatest number of genetic tools, and a fully sequenced genome. In this review we highlight multiomics studies that elucidate the mechanisms allowing this yeast to achieve lipid overaccumulation and then present several major metabolic engineering efforts that enhanced the production metrics in Y. lipolytica. Recent achievements that applied novel engineering strategies are emphasized. Lipid Production and Its Metabolism in Yarrowia lipolytica Contemporary society relies heavily on fossil fuels (petroleum, coal, and natural gas) as the source of energy [1]. However, concerns about climate change have prompted research into the development of renewable liquid fuels [2]. These new technologies need to supply fuels in a cost-effective and sustainable manner while contributing to the reduction of greenhouse gases [3]. Biofuels produced from microbes, primarily bioethanol and biodiesel, are such promising alternatives (Box 1). In particular, lipid-derived biodiesels are garnering much attention due to their high energy density, which makes them superior substitutes for diesel fuels and jet fuels compared with other forms of renewable energy. The cost-effective production of biodiesel relies on several important criteria, including lipid content (see Glossary), lipid titer, lipid productivity, and lipid yield. Therefore, oleaginous organisms, which excel at accumulating intracellular lipids, are often chosen as the industrial workhorse. Y. lipolytica, an oleaginous yeast belonging to the Yarrowia clade [4,5], is widely regarded as the model organism for this purpose [6]. Its lipid metabolism and supporting pathways has been studied extensively, it has a plethora of genetic engineering tools, and its genome has been fully sequenced [7-14]. More recently, the ubiquitous genome editing technique CRISPR-Cas9 has also been demonstrated in Y. lipolytica, allowing high-frequency homologous recombination as well as targeted gene insertion and deletion [15,16]. These characteristics enhance the potential of Y. lipolytica in achieving economic biodiesel production at an industrial scale. Highlights Yarrowia lipolytica is a model oleaginous yeast for the production of lipids and lipid-derived biofuels, studies of lipid metabolism, and the biosynthesis of various industrially important metabolites.
Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica
Background: Recent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (H eff /C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process. Results: Adsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration. Conclusion: The nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.
Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica
Metabolic Engineering, 2015
Conversion of carbohydrates to lipids at high yield and productivity is essential for cost-effective production of renewable biodiesel. Although some microorganisms can convert sugars to oils, conversion yields and rates are typically low due primarily to allosteric inhibition of the lipid biosynthetic pathway by saturated fatty acids. By reverse engineering the mammalian cellular obese phenotypes, we identified the delta-9 stearoyl-CoA desaturase (SCD) as a rate limiting step and target for the metabolic engineering of the lipid synthesis pathway in Yarrowia lipolytica. Simultaneous overexpression of SCD, Acetyl-CoA carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) in Y. lipolytica yielded an engineered strain exhibiting highly desirable phenotypes of fast cell growth and lipid overproduction including high carbon to lipid conversion yield (84.7% of theoretical maximal yield), high lipid titers (~55 g/L), and enhanced tolerance to glucose and cellulose-derived sugars. Moreover, the engineered strain shows a threefold growth advantage over the wild type strain. As a result, a maximal lipid productivity of ~1 g/L/h is obtained during the stationary phase. Furthermore, we show that the engineered yeast required cytoskeleton remodeling in eliciting the obesity phenotype. Altogether, our work describes the development of a microbial catalyst with the highest reported lipid yield, titer and productivity to date. This is an important step towards the development of an efficient and cost-effective process for biodiesel production from renewable resources.