What makes Yarrowia lipolytica well suited for industry?

Selection of the most appropriate microorganism is one of the key aspects for the industrial success of microbial bioprocesses.Yarrowia lipolytica has gained interest as a chassis strain in academia and industry because of its capacity to make products at high yields, use a broad range of substrates, and be genetically amenable.Y. lipolytica has many features that are desired at an industrial scale, such as safety, robustness, efficient and stable genetic modifications, capacity to use a variety of substrates, and ability to grow at very high cell density.To further improve the industrial use of Y. lipolytica, some characteristics must be improved through metabolic engineering, such as the high oxygen requirement, byproduct formation, and excessive foam synthesis. Yarrowia lipolytica possesses natural and engineered traits that make it a good host for the industrial bioproduction of chemicals, fuels, foods, and pharmaceuticals. In recent years, academic and industrial researchers have assessed its potential, developed synthetic biology techniques, improved its features, scaled its processes, and identified its limitations. Both publications and patents related to Y. lipolytica have shown a drastic increase during the past decade. Here, we discuss the characteristics of this yeast that make it suitable for industry and the remaining challenges for its wider use at large scale. We present evidence herein that shows the importance and potential of Y. lipolytica in bioproduction such that it may soon be one of the preferred choices of industry. Yarrowia lipolytica possesses natural and engineered traits that make it a good host for the industrial bioproduction of chemicals, fuels, foods, and pharmaceuticals. In recent years, academic and industrial researchers have assessed its potential, developed synthetic biology techniques, improved its features, scaled its processes, and identified its limitations. Both publications and patents related to Y. lipolytica have shown a drastic increase during the past decade. Here, we discuss the characteristics of this yeast that make it suitable for industry and the remaining challenges for its wider use at large scale. We present evidence herein that shows the importance and potential of Y. lipolytica in bioproduction such that it may soon be one of the preferred choices of industry. Industrial biotechnology (white biotechnology) includes the application of enzymes, cell extracts, or whole microorganisms in industrial processes that lead to the manufacture of a wide variety of products, including fuels, food ingredients, enzymes, materials, and pharmaceuticals [1.Frazzetto G. White biotechnology.EMBO Rep. 2003; 4: 835-837Crossref PubMed Scopus (63) Google Scholar,2.Heux S. et al.White biotechnology: state of the art strategies for the development of biocatalysts for biorefining.Biotechnol. Adv. 2015; 33: 1653-1670Crossref PubMed Scopus (71) Google Scholar]. Compared with chemical processes, industrial biotechnology is often more sustainable and environmentally friendly and enables specificity and reactivity that is difficult to achieve otherwise [3.Soetaert W. Vandamme E. The impact of industrial biotechnology.Biotechnol. J. 2006; 1: 756-769Crossref PubMed Scopus (81) Google Scholar]. In the past two decades, along with advances in metabolic engineering and synthetic biology, industrial biotechnology has delivered products and innovations in the chemical, textile, food, packaging, and healthcare sectors [4.Jullesson D. et al.Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals.Biotechnol. Adv. 2015; 33: 1395-1402Crossref PubMed Scopus (166) Google Scholar]. One of the keys to the success of a bioprocess (see Glossary) is selection of the microorganism. The selection should consider the natural capacity of the host to make the desired products or their intermediates, to consume the preferred substates, and to resist the toxicity of intermediates and/or final products. In addition, it is important to consider the wealth of knowledge on the organism's physiology and metabolism as well as the degree of development of techniques for strain engineering [5.Keasling J.D. Synthetic biology and the development of tools for metabolic engineering.Metab. Eng. 2012; 14: 189-195Crossref PubMed Scopus (340) Google Scholar]. A given bioprocess can also be improved by the optimization of fermentation conditions and evolutionary adaptation [5.Keasling J.D. Synthetic biology and the development of tools for metabolic engineering.Metab. Eng. 2012; 14: 189-195Crossref PubMed Scopus (340) Google Scholar, 6.Sopko R. et al.Mapping pathways and phenotypes by systematic gene overexpression.Mol. Cell. 2006; 21: 319-330Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 7.Förster A.H. Gescher J. Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products.Front. Bioeng. Biotechnol. 2014; 2: 16PubMed Google Scholar]. Y. lipolytica has been often considered a nonconventional yeast due to its distinctive genome structure and its relatively large phylogenetic distance to other yeasts while sharing common properties with higher eukaryotes [8.Barth G. Gaillardin C. Yarrowia lipolytica.in: Wolf K. Nonconventional Yeasts in Biotechnology: A Handbook. Springer-Verlag, 1996: 313-388Crossref Google Scholar]. This yeast was originally isolated from lipid-rich or protein-rich environments such as fermented dairy products (cheese, yogurt), meat, poultry, and also from wastes such as lipid-rich sewage or oil-polluted environments [9.Beopoulos A. et al.Yarrowia lipolytica: a model and a tool to understand the mechanisms implicated in lipid accumulation.Biochimie. 2009; 91: 692-696Crossref PubMed Scopus (216) Google Scholar]. Since its isolation, Y. lipolytica has been used for the production of organic acids and heterologous proteins and for the bioremediation of oil-contaminated soil and water [10.Nicaud J.-M. Yarrowia lipolytica.Yeast. 2012; 29: 409-418Crossref PubMed Scopus (208) Google Scholar,11.Madzak C. Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering.Appl. Microbiol. Biotechnol. 2015; 99: 4559-4577Crossref PubMed Scopus (167) Google Scholar]. Some of the unique characteristics that initially drew the attention of researchers to this yeast are its capacity to accumulate lipids, its dimorphism (with both yeast and pseudo-hyphae forms) [10.Nicaud J.-M. Yarrowia lipolytica.Yeast. 2012; 29: 409-418Crossref PubMed Scopus (208) Google Scholar,12.Abdel-Mawgoud A.M. et al.Metabolic engineering in the host Yarrowia lipolytica.Metab. Eng. 2018; 50: 192-208Crossref PubMed Scopus (126) Google Scholar], and its ability to degrade hydrophobic carbon sources (fatty acids, triglycerides, alkanes, alkenes, etc.) [13.Barth G. Gaillardin C. Physiology and genetics of the dimorphic fungus Yarrowia lipolytica.FEMS Microbiol. Rev. 1997; 19: 219-237Crossref PubMed Google Scholar,14.Lazar Z. et al.Holistic approaches in lipid production by Yarrowia lipolytica.Trends Biotechnol. 2018; 36: 1157-1170Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar]. Since the 1990s, publications involving Y. lipolytica increased rapidly in parallel with the early development of genetic engineering and the availability of the whole-genome sequence of this yeast that encouraged new research groups to work with this microbe. Together with the development of synthetic biology tools for efficient strain manipulation, the number of published articles has grown exponentially, as shown in Figure 1. In parallel with academia, industrial research noticed the potential of this yeast, and the number of patents also increased (Figure 2). We have briefly explored the patent landscape of Y. lipolytica and found 4536 international patents containing the keywords 'Yarrowia lipolytica' (Figure 2). As expected, the number of patents has significantly increased since 2001, alongside the development of engineering tools for this yeast, which reflect the increasing interest in this organism as an industrial host. Examples of the products made in Y. lipolytica and explored by industry are described in Box 1 (see Box 2 for Survey to industry).Box 1Y. lipolytica commercial products in industry(i)Polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3), also known as long-chain omega-3 fatty acids, have a global market valued at US$2.49 billion in 2019 [70.Xie D. et al.Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.Appl. Microbiol. Biotechnol. 2015; 99: 1599-1610Crossref PubMed Scopus (149) Google Scholar]. E.I. DuPont de Nemours and Company developed a metabolic engineering strategy in Y. lipolytica, which expressed heterologous Δ6 desaturase, C18/20 elongase, Δ5 desaturase, and Δ17 desaturase, enabled production of up to 40% EPA [71.Maccol, D.J. et al. DuPont US Holding LLC. Mortierella alpina C16/18 fatty acid elongase. US7470532B2.Google Scholar, 72.Yadav, N.S. et al. EI Du Pont de Nemours and Co. Δ12 desaturases suitable for altering leveles of polyunsaturated fatty acids in oleaginous yeast. US7504259B2.Google Scholar, 73.Zhu Q. et al.Cohen Z. Ratledge C.B.T.-S.C.O. 3 - Metabolic Engineering of an Oleaginous Yeast for the Production of Omega-3 Fatty Acids. 2nd ed. AOCS Press, 2010: 51-73Google Scholar]. Production was further enhanced by eliminating the competitive pathways and introducing several copies of the crucial enzymes, reaching to approximately 25% of dry cell weight and 50% of fatty acid methyl ester [70.Xie D. et al.Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.Appl. Microbiol. Biotechnol. 2015; 99: 1599-1610Crossref PubMed Scopus (149) Google Scholar,74.Hong, S.P. et al. EI Du Pont Nemours and Co. Expression of caleosin in recombinant oleaginous microorganisms to increase oil content therein. WO2012162368A1.Google Scholar]. This strategy enabled DuPont to commercialize EPA produced from Y. lipolytica, New Harvest EPA oil, and Verlasso salmon.(ii)Skotan SA started the production of single-cell protein (edible protein for human and animals [75.Bornscheuer U.T. The fourth wave of biocatalysis is approaching.Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 2018; 376: 1-7Google Scholar]) from waste glycerol and registered the feed product in the EU [15.Groenewald M. et al.Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.Crit. Rev. Microbiol. 2014; 40: 187-206Crossref PubMed Scopus (325) Google Scholar]. Microbial enzymes such as lipases already have their own market valued at US$425 million in 2018 [76.Chandra P. et al.Microbial lipases and their industrial applications: a comprehensive review.Microb. Cell Fact. 2020; 19: 169Crossref PubMed Scopus (315) Google Scholar]. Commercial recombinant enzymes from Y. lipolytica are now available, such as the phospholipase enzyme Lipomod 833L2 by Biocatalysts Ltd., the lipase obtained by LIP2 gene overexpression by Mayoly, or the human acid α-glucosidase OXY2810 by Oxyrane [44.Madzak C. Engineering Yarrowia lipolytica for use in biotechnological applications: a review of major achievements and recent innovations.Mol. Biotechnol. 2018; 60: 621-635Crossref PubMed Scopus (73) Google Scholar,77.Madzak C. Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement.J. Fungi. 2021; 7: 548Crossref PubMed Scopus (42) Google Scholar].(iii)Citric acid is widely used by the food industry as additives, preservatives, anticoagulants, antimicrobial agents, fine chemicals, and so forth [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar]. The global market volume of citric acid is greater than 2 million tons, and its value is estimated to reach US$6.28 billion by 2030 [79.Market Research Future Citric Acid Market Research Report: Information by Form (Anhydrous and Liquid), Function (Acidulant, Antioxidant, Preservative, and Flavouring Agent), Application [Food & Beverages (Beverages; Bakery & Confectionery; Sweet & Savoury Snacks; Soups, Sauces, and Dressings, RTE & RTC Meals, and Others), Pharmaceuticals & Nutraceuticals, Personal Care, and Others] and Region (North America, Europe, Asia-Pacific, and RoW)—Forecast till 2030.2021Google Scholar]. Due to easy cultivation, high conversion rate, and tolerance to high product concentrations, Y. lipolytica has been proposed as an alternative citric acid producer to Aspergillus niger [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar,80.Börekçi B.S. et al.Citric acid production of yeasts: an overview.Eurobiotech J. 2021; 5: 79-91Crossref Scopus (12) Google Scholar]. Several companies, such as DSM, Akad Wissenschaften DDR, and OrganoBalance GmbH, own approximately 40 patents on citric acid production by Y. lipolytica [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar].(iv)Carotenoids have a number of applications in the food processing, animal feed, pharmaceutical, and cosmetics industries. There is already an enormous global market, which is estimated to be US$1.57 billion in 2022 and reach a valuation of US$2.09 billion by 2027 [81.Market Data Forecast Global Carotenoids Market Segmented by Type (Astaxanthin, Canthaxanthin, Lutein, Beta-Carotene, Lycopene, & Zeaxanthin), by Application (Food & Beverages, Dietary Supplements, Animal Feed and Pharmaceuticals and others), By Sources (Natural and Synthetic) and by Regional Analysis (North America, Europe, Asia Pacific, Latin America, and Middle East & Africa) - Global Industry Analysis, Size, Share, Growth, Trends, and Forecast (2022 – 2027).2022Google Scholar]. The production of carotenoids from Y. lipolytica has been described by numerous patents filed by DSM, E.I. DuPont de Nemours and Company, Amyris, and Microbia Inc., among others [82.Royer, J. et al. DSM IP Assets BV. Microbial production of terpenoids. US20180148697A1.Google Scholar, 83.Farrell, C. et al. DSM IP Assets BV. Acetyl transferase and their use for producing carotenoids. US10865392B2.Google Scholar, 84.Sharp, P.L. et al. EI Du Pont de Nemours and Co. Carotenoid production in a recombinant oleaginous yeast. US8846374B2.Google Scholar, 85.Gardner, T.S. et al. Amyris Inc. Production of acetyl-coenzymeA derived isoprenoids. US8603800B2.Google Scholar, 86.Bailey, R.B. et al. DSM IP Assets BV. Production of carotenoids in oleaginous yeast and fungi. US9297031B2.Google Scholar]. Microbia (now part of DSM) brought several carotenoid production strains to pilot scales, and a GRAS self-affirmation has been demonstrated for the β-carotene produced with Y. lipolytica [15.Groenewald M. et al.Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.Crit. Rev. Microbiol. 2014; 40: 187-206Crossref PubMed Scopus (325) Google Scholar].Box 2Survey to industryAlthough the opinion of academic researchers often can be found in publications, those from industry are often more difficult to obtain. We therefore conducted a survey of companies working with Y. lipolytica to find out what they think about this yeast. The survey consisted of several statements or 'ranked questions' and a few 'open questions' where the answer could be typed. The survey was answered by ten companies producing different products at different scales (from lab to pilot and industrial scale).The first 'ranked question' related to the most suitable target products for Y. lipolytica (Figure IA). When considering 'agree' and 'mostly agree' answers, lipids, terpenoids, and proteins were considered the best targets with an agreement of 100%, 80%, and 73%, respectively. Interestingly, the responses to the related 'open question' 'Which products from Y. lipolytica are most promising in the near future?' were very diverse, and lipids, terpenoids, amino acids, proteins, and sugar alcohols were mentioned, which shows the high potential of Y. lipolytica for a broad range of applications as well as the different interests among companies.A second 'ranked question' was set to assess industrial features of Y. lipolytica (Figure IB). Overall, there is a high agreement ('agree' and 'mostly agree') in the fact that Y. lipolytica is good for high cell density cultivation (100%), has a good robustness (100%), engineering tools (70%), and wide substrate utilization (80%), whereas there were less positive answers in regard to downstream processing, byproduct formation, and scaling up, where 30%, 60%, and 40% of the opinions, respectively, were 'neutral' or 'mostly disagree'.The third 'ranked question' investigated the need for further development of fundamental studies, synthetic biology tools, and fermentation knowledge (Figure IC). Ninety percent of the answers agree with the fact that Y. lipolytica still needs more fundamental research and synthetic biology tools. Eighty-two percent of the answers recognized the need of deepening our understanding of Y. lipolytica behavior in bioreactors.These industries use different Y. lipolytica strains, being 40% academic strains and 50% wild-type (nonengineered) strains. In addition, an anonymous response stated that 'Y. lipolytica has a strong body of knowledge, including regulatory approvals for prior products (e.g., is on the short list of microbial chassis that do not need a full safety assessment before manufacture/sale in the EU), which accelerates R & D and manufacturing timelines'.Figure ISurvey of ten companies using Yarrowia lipolytica.Show full caption(A) Agreement or disagreement with the high capacity of Y. lipolytica to produce proteins, lipids, terpenoids, amino acids, and other products. (B) Agreement or disagreement with the presence of desired industrial features in Y. lipolytica. (C) Agreement or disagreement with the research fields that need further study in Y. lipolytica.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (i)Polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3), also known as long-chain omega-3 fatty acids, have a global market valued at US$2.49 billion in 2019 [70.Xie D. et al.Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.Appl. Microbiol. Biotechnol. 2015; 99: 1599-1610Crossref PubMed Scopus (149) Google Scholar]. E.I. DuPont de Nemours and Company developed a metabolic engineering strategy in Y. lipolytica, which expressed heterologous Δ6 desaturase, C18/20 elongase, Δ5 desaturase, and Δ17 desaturase, enabled production of up to 40% EPA [71.Maccol, D.J. et al. DuPont US Holding LLC. Mortierella alpina C16/18 fatty acid elongase. US7470532B2.Google Scholar, 72.Yadav, N.S. et al. EI Du Pont de Nemours and Co. Δ12 desaturases suitable for altering leveles of polyunsaturated fatty acids in oleaginous yeast. US7504259B2.Google Scholar, 73.Zhu Q. et al.Cohen Z. Ratledge C.B.T.-S.C.O. 3 - Metabolic Engineering of an Oleaginous Yeast for the Production of Omega-3 Fatty Acids. 2nd ed. AOCS Press, 2010: 51-73Google Scholar]. Production was further enhanced by eliminating the competitive pathways and introducing several copies of the crucial enzymes, reaching to approximately 25% of dry cell weight and 50% of fatty acid methyl ester [70.Xie D. et al.Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.Appl. Microbiol. Biotechnol. 2015; 99: 1599-1610Crossref PubMed Scopus (149) Google Scholar,74.Hong, S.P. et al. EI Du Pont Nemours and Co. Expression of caleosin in recombinant oleaginous microorganisms to increase oil content therein. WO2012162368A1.Google Scholar]. This strategy enabled DuPont to commercialize EPA produced from Y. lipolytica, New Harvest EPA oil, and Verlasso salmon.(ii)Skotan SA started the production of single-cell protein (edible protein for human and animals [75.Bornscheuer U.T. The fourth wave of biocatalysis is approaching.Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 2018; 376: 1-7Google Scholar]) from waste glycerol and registered the feed product in the EU [15.Groenewald M. et al.Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.Crit. Rev. Microbiol. 2014; 40: 187-206Crossref PubMed Scopus (325) Google Scholar]. Microbial enzymes such as lipases already have their own market valued at US$425 million in 2018 [76.Chandra P. et al.Microbial lipases and their industrial applications: a comprehensive review.Microb. Cell Fact. 2020; 19: 169Crossref PubMed Scopus (315) Google Scholar]. Commercial recombinant enzymes from Y. lipolytica are now available, such as the phospholipase enzyme Lipomod 833L2 by Biocatalysts Ltd., the lipase obtained by LIP2 gene overexpression by Mayoly, or the human acid α-glucosidase OXY2810 by Oxyrane [44.Madzak C. Engineering Yarrowia lipolytica for use in biotechnological applications: a review of major achievements and recent innovations.Mol. Biotechnol. 2018; 60: 621-635Crossref PubMed Scopus (73) Google Scholar,77.Madzak C. Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement.J. Fungi. 2021; 7: 548Crossref PubMed Scopus (42) Google Scholar].(iii)Citric acid is widely used by the food industry as additives, preservatives, anticoagulants, antimicrobial agents, fine chemicals, and so forth [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar]. The global market volume of citric acid is greater than 2 million tons, and its value is estimated to reach US$6.28 billion by 2030 [79.Market Research Future Citric Acid Market Research Report: Information by Form (Anhydrous and Liquid), Function (Acidulant, Antioxidant, Preservative, and Flavouring Agent), Application [Food & Beverages (Beverages; Bakery & Confectionery; Sweet & Savoury Snacks; Soups, Sauces, and Dressings, RTE & RTC Meals, and Others), Pharmaceuticals & Nutraceuticals, Personal Care, and Others] and Region (North America, Europe, Asia-Pacific, and RoW)—Forecast till 2030.2021Google Scholar]. Due to easy cultivation, high conversion rate, and tolerance to high product concentrations, Y. lipolytica has been proposed as an alternative citric acid producer to Aspergillus niger [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar,80.Börekçi B.S. et al.Citric acid production of yeasts: an overview.Eurobiotech J. 2021; 5: 79-91Crossref Scopus (12) Google Scholar]. Several companies, such as DSM, Akad Wissenschaften DDR, and OrganoBalance GmbH, own approximately 40 patents on citric acid production by Y. lipolytica [78.Cavallo E. et al.Yarrowia lipolytica: a model yeast for citric acid production.FEMS Yeast Res. 2017; 17: fox084Crossref Scopus (74) Google Scholar].(iv)Carotenoids have a number of applications in the food processing, animal feed, pharmaceutical, and cosmetics industries. There is already an enormous global market, which is estimated to be US$1.57 billion in 2022 and reach a valuation of US$2.09 billion by 2027 [81.Market Data Forecast Global Carotenoids Market Segmented by Type (Astaxanthin, Canthaxanthin, Lutein, Beta-Carotene, Lycopene, & Zeaxanthin), by Application (Food & Beverages, Dietary Supplements, Animal Feed and Pharmaceuticals and others), By Sources (Natural and Synthetic) and by Regional Analysis (North America, Europe, Asia Pacific, Latin America, and Middle East & Africa) - Global Industry Analysis, Size, Share, Growth, Trends, and Forecast (2022 – 2027).2022Google Scholar]. The production of carotenoids from Y. lipolytica has been described by numerous patents filed by DSM, E.I. DuPont de Nemours and Company, Amyris, and Microbia Inc., among others [82.Royer, J. et al. DSM IP Assets BV. Microbial production of terpenoids. US20180148697A1.Google Scholar, 83.Farrell, C. et al. DSM IP Assets BV. Acetyl transferase and their use for producing carotenoids. US10865392B2.Google Scholar, 84.Sharp, P.L. et al. EI Du Pont de Nemours and Co. Carotenoid production in a recombinant oleaginous yeast. US8846374B2.Google Scholar, 85.Gardner, T.S. et al. Amyris Inc. Production of acetyl-coenzymeA derived isoprenoids. US8603800B2.Google Scholar, 86.Bailey, R.B. et al. DSM IP Assets BV. Production of carotenoids in oleaginous yeast and fungi. US9297031B2.Google Scholar]. Microbia (now part of DSM) brought several carotenoid production strains to pilot scales, and a GRAS self-affirmation has been demonstrated for the β-carotene produced with Y. lipolytica [15.Groenewald M. et al.Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.Crit. Rev. Microbiol. 2014; 40: 187-206Crossref PubMed Scopus (325) Google Scholar]. Although the opinion of academic researchers often can be found in publications, those from industry are often more difficult to obtain. We therefore conducted a survey of companies working with Y. lipolytica to find out what they think about this yeast. The survey consisted of several statements or 'ranked questions' and a few 'open questions' where the answer could be typed. The survey was answered by ten companies producing different products at different scales (from lab to pilot and industrial scale). The first 'ranked question' related to the most suitable target products for Y. lipolytica (Figure IA). When considering 'agree' and 'mostly agree' answers, lipids, terpenoids, and proteins were considered the best targets with an agreement of 100%, 80%, and 73%, respectively. Interestingly, the responses to the related 'open question' 'Which products from Y. lipolytica are most promising in the near future?' were very diverse, and lipids, terpenoids, amino acids, proteins, and sugar alcohols were mentioned, which shows the high potential of Y. lipolytica for a broad range of applications as well as the different interests among companies. A second 'ranked question' was set to assess industrial features of Y. lipolytica (Figure IB). Overall, there is a high agreement ('agree' and 'mostly agree') in the fact that Y. lipolytica is good for high cell density cultivation (100%), has a good robustness (100%), engineering tools (70%), and wide substrate utilization (80%), whereas there were less positive answers in regard to downstream processing, byproduct formation, and scaling up, where 30%, 60%, and 40% of the opinions, respectively, were 'neutral' or 'mostly disagree'. The third 'ranked question' investigated the need for further development of fundamental studies, synthetic biology tools, and fermentation knowledge (Figure IC). Ninety percent of the answers agree with the fact that Y. lipolytica still needs more fundamental research and synthetic biology tools. Eighty-two percent of the answers recognized the need of deepening our understanding of Y. lipolytica behavior in bioreactors. These industries use different Y. lipolytica strains, being 40% academic strains and 50% wild-type (nonengineered) strains. In addition, an anonymous response stated that 'Y. lipolytica has a strong body of knowledge, including regulatory approvals for prior products (e.g., is on the short list of microbial chassis that do not need a full safety assessment before manufacture/sale in the EU), which accelerates R & D and manufacturing timelines'. The success and popularity of Yarrowia research goes beyond academia, and an increasing number of companies are now choosing this yeast as their preferred workhorse, but what makes Y. lipolytica so appealing for bioproduction? Here, we try to answer this question by describing the unique features of Y. lipolytica that make it an ideal fit for industrial biomanufacturing, its current use in academic and industrial research, and the challenges yet to be overcome to make it a better host. Traditionally, wild-type strains of Y. lipolytica have been used to produce lipids, organic acids, and polyols. In the past years, the number of applications of this yeast has greatly expanded, as described in Figure 3. The high capacity to divert flux toward acetyl coenzyme A has turned engineered strains into high producers of terpenes and lipid-derived products. In recent years, the high production of metabolites derived from different pathways (e.g., the shikimate pathway and pentose phosphate pathway) has also been demonstrated. The range of compounds made from these pathways have applications in the production of food, additives, cosmetics, chemicals, fuels, pharmaceuticals, and materials (Figure 3). Safety is one of the critical issues for the implementation of industrial applications, especially for products intended for human consumption. Y. lipolytica is considered as a 'safe-to-use organism', with a granted GRAS (generally regarded as safe) status for the production of citric acid, erythritol, and eicosapentaenoic acid by the US FDA [15.Groenewald M. et