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After having drawn some industrialists’ attention as early as the 1950s, the non-conventional oleaginous yeast Yarrowia lipolytica has been recognized since several decades, as a powerful host for heterologous protein expression, secretion and surface display. The development of sequencing and genetic engineering tools, combined with an increasing knowledge of its metabolism, have then facilitated the complex engineering of the metabolic pathways of this yeast for various applications. Since nearly two decades, numerous laboratories throughout the world have chosen Y. lipolytica as a chassis for designing microbial cell factories. White biotechnology applications of this yeast include notably single cell oil production, whole cell bioconversion and upgrading of industrial wastes.
A major challenge for our societies is to replace polluting technologies, based on fossil fuels, with clean ones, based on renewable resources. White biotechnology, using microorganisms and their enzymes to manufacture compounds of industrial interest (chemicals, biomaterials, biofuels, pharmaceuticals, feed, food), has an important role to play in this transition. This rapidly developing field aims to design industrial processes more environmentally friendly and making use of agricultural, forest and industrial waste or by-products. Among the microorganisms amenable for such industrial applications, yeasts cells present the cumulated advantages of high growth capacity, easy genetic manipulation and presence of a eukaryotic organisation allowing posttranslational processing, vesicular secretion and subcellular compartmentalization. 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.
2. Main Characteristics
2.1. Natural Habitats
2.3. Physico-Chemical Conditions for Growth
2.4. Ploidy and Morphology
3.1. Carbon Sources
3.2. Secretion Pathway
3.3. Lipid Storage
4. Genomic Organization
5. Engineering Y. lipolytica strains into cell factories
5.1. Brief history of industrial use
The high potential of Y. lipolytica for industrial applications has been exploited since more than 70 years, at first in the fields of biomass and valuable metabolites production, using proprietary wild-type isolates or traditionally improved strains (mutants, strains issued from hybridizations and crossings), as reviewed previously [1,44,45,50]. Notable applications of wild-type strains include the production of single-cell protein (SCP) from crude oil until the oil crisis of the 1970s (Toprina G, for livestock feeding) and, presently, industrial citric acid production (ADM, Chicago, IL, USA), erythritol production (Baolingbao Biology Co., Yucheng, Shandong, China), use of Y. lipolytica biomass as fodder yeast for farm and pet animals (Skotan SA, Chorzów, Poland). The outstanding capacity of Y. lipolytica for degrading hydrocarbons, and especially alkanes, explains that wild-types isolates were frequently found in oil-polluted environments and justifies the use of this yeast in bioremediation projects [44,51,52]. A starter for depolluting wastewaters is commercialized by Artechno (Isnes, Belgium), based on traditionally-obtained highly lipolytic mutants of ATCC 48436 strain.
In the 1980s, the newly developed technics of molecular biology rejuvenated the interest in Y. lipolytica, this time as an expression host for producing heterologous proteins . Metabolic engineering of this yeast ensued rapidly, following the development of transformation methods, shuttle vectors and non-leaky non-reverting auxotrophic strains . As Y. lipolytica started, in the 2000s, to be recognized as a valuable host for recombinant protein production [19,20], the YLEX kit for expression/secretion of heterologous proteins in this yeast was commercialized in 2006 (Yeastern Biotech Co., Taipei, Taiwan), based on a GM derivative of W29 wild-type isolate. Other W29 derivatives have been established as commercial protein production platforms by Protéus (Sequens Group, Ecully, France) and Oxyrane UK (Manchester, UK). With the continuous progress of genetic engineering technics, increasingly complex modifications of Y. lipolytica metabolism, such as the introduction of complete heterologous metabolic pathways, could be performed. Proofs of concept of the use of this yeast as cell factory for the production of valuable compounds or as arming yeast for bioconversion processes are abundantly reported in the scientific literature since a few decades [54,55,56]. However, most of the proposed applications for these GM Y. lipolytica strains remain, until now, only at an exploratory stage and are not developed further to the industrial stage. This matter of fact could be attributed at least in part to social acceptance issues concerning GM microorganisms, especially in the domain of food applications. Until now, only a few commercial or industrial applications of GM Y. lipolytica strains can be reported [1,45]. GM Y. lipolytica cell factories are presently used for industrial production of two kinds of food/feed additives: carotenoids  (DSM, Heerlen, The Netherlands) and polyunsaturated fatty acids (PUFAs)-rich SCOs (DuPont, Wilmington, DE, USA). The technology of PUFAs-rich SCOs production by a heavily engineered Y. lipolytica strain derived from the ATCC 20362 wild-type isolate was more particularly applied to industrial production of ω-3 eicosapentaenoic acid (EPA)-rich products [57,58], such as notably EPA-rich Y. lipolytica biomass marketed (in joint venture with AquaChile, Puerto Montt, Chile) as an ω-3 feed supplement for “harmoniously raised” salmon VerlassoTM.
Another domain of successful applications for GM Y. lipolytica strains is the therapeutic use of recombinant enzymes: several enzyme replacement therapies (ERTs) based on this yeast are now marketed or on the edge to marketing stage [54,55]. The first of these ERTs, developed by Mayoly Spindler (Chatou, France), uses a recombinant extracellular LIP2 lipase  for the treatment of exocrine pancreatic insufficiency (also under Phase 2 clinical trial for two other fat malabsorption diseases, cystic fibrosis and chronic pancreatitis). More recently, Oxyrane (Ghent, Belgium) established a proprietary Y. lipolytica engineering platform able to produce recombinant glycoproteins, with the possibility of added mannose-6-phosphate (M6P) glycan residues , for treatment of different lysosomal storage diseases. The presence of M6P on therapeutic glycoproteins improves their internalization into the patient’s cells and addresses them to lysosomes, their targeted subcellular site of action. A recombinant human acid α-glucosidase produced in Y. lipolytica, OXY2810, is currently marketed for use as ERT in Pompe disease (in which glycogen accumulates in the patient’s tissues) and recombinant glucocerebrosidases are in preclinical testing for treatment of Parkinson’s disease or neuronopathic Gaucher disease [54,55], while other new ERTs are in project.
5.2. Rewiring the metabolism for a bio-based economy
The wide range of engineering tools and strategies now available will contribute to establish Y. lipolytica as a workhorse for a wide range of applications in the very competitive world of white biotechnology. However, for an optimal development of Y. lipolytica cell factories, it is to hope that a future easing of the regulation policy for the new GMOs (especially for gene edited/CRISPR-generated organisms) could allow the relieving of the regulatory constraints that presently limit their use in some of their numerous domains of application. Even though it would be difficult to determine what influence GMO regulations and societal acceptance could have had on the strategic choices of laboratories and companies, we can note that the major food-oriented applications of Y. lipolytica strains (citric acid, erythritol, KGA) have majorly favoured traditionally improved strains. If this tendency was to increase in the future, a more systematic exploration of the natural Y. lipolytica biodiversity for potential applications, leveraged by new mutagenesis technics (ARTP: atmospheric and room temperature plasma), adaptative evolution strategies and high-throughput screening technologies, would constitute a valuable asset. Therefore, Y. lipolytica is in good position to become a biotechnological workhorse, through both traditional and genetic engineering pathways.
The entry is from 10.3390/jof7070548
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