You're using an outdated browser. Please upgrade to a modern browser for the best experience.
Industrial Value of Nonconventional Yeasts
Edit
This entry is adapted from the peer-reviewed paper 10.3390/fermentation8110656

Nonconventional yeasts (NCYs) have several advantages over S. cerevisiae from an industrial viewpoint. S. cerevisiae is often directed toward ethanol synthesis (due to its Crabtree-positive effect), restricting product diversification. In contrast, NCYs may have desired metabolic pathways, enabling product profile expansion. The ability to resist various stresses is additional key benefit in industrial bioprocesses.

nonconventional yeast genome editing metabolic engineering

1. Introduction

Petroleum-derived chemical production has detrimental effects on the environment and exhibits industrial noncompatibility associated with cost-effectiveness due to multiple labor-intensive processes [1]. To overcome this problem, biotransformation using microorganisms can be considered an alternative approach [1][2], which has the advantage of rapid growth rate and easy cultivation of microorganisms under laboratory conditions [3]. In particular, eukaryotic yeasts represent robust microbial cell factories owing to their simple structure and ability to grow on various substrates, as well as the relatively simple gene editing techniques used to manipulate their genomes [4].
Saccharomyces cerevisiae is the most widely used eukaryotic platform in bioprocesses [5][6]. S. cerevisiae has long been a model organism for fundamental biological research and industrial applications because of its ease of handling and safety as a generally recognized as safe (GRAS) strain [7]. Moreover, its genetics are well understood, and tools for manipulating it are well established; thus, numerous specialized strains and plasmids are available. However, as a Crabtree-positive organism, the carbon flux in S. cerevisiae is mainly directed toward the ethanol fermentation pathway. This preference for ethanol production often limits its utilization as a host when nonethanol products are to be synthesized. Therefore, other suitable yeasts, known as nonconventional yeasts (NCY), should be considered as alternative hosts.
Approximately 1500 NCY species have been identified to date [8], each of which exhibits unique genetics, physiology, and characteristics. They often have excellent potential for industrial uses that are not feasible with S. cerevisiae. Thus, NCY can be considered as promising eukaryotic hosts alternative to S. cerevisiae to overcome or improve its limitations. Specifically, NCYs that are Crabtree-negative can diversify the profile of industrially useful products. In addition, a much higher capacity of NCYs for pentose phosphate pathway relative to S. cerevisiae is also advantageous feature when synthesizing products using NCYs as the cell factory by increasing the available pool of cofactors and precursors [9]. Moreover, a high tolerance against multiple stress factors, such as heat, low pH, and salt, can extend yeasts’ utility. Advancements in genetic and metabolic engineering technologies will facilitate NCY-based scaled-up bioprocesses [8][10].
Despite their beneficial traits, genetic information and manipulation tools for many NCYs are lacking compared with those for S. cerevisiae [11]. As most organisms have not been thoroughly analyzed for safety and lack genome sequencing data with the limited information on the exact gene loci and protein functions, it is challenging to develop and apply suitable gene editing tools, together with establishing transformation protocols and selectable marker genes [11].
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is an adaptive immune system of bacteria and archaea that protects them from invasion by foreign genetic elements [12]. As a gene editing tool, the CRISPR-Cas system is based on a simple single guide RNA (sgRNA)/DNA hybrid that recognizes specific target DNA, providing a simple-to-design methodology, which has more sophisticated, accurate, and cheaper gene editing capabilities compared with traditional methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [13]. Thus, this revolutionary CRISPR-Cas9 system has been applied to various yeast strains, such as S. cerevisiae, Pichia pastoris, Kluyveromyces marxianus, and Yarrowia lipolytica; among these, most studies have been conducted on S. cerevisiae [14][15]. Although numerous studies have reported the CRISPR-guided metabolic engineering of S. cerevisiae, only few studies have explored the applicability of this system in NCYs [16]. To achieve commercial scale bioproduction using NCYs as cell factories, it is necessary to develop highly efficient and convenient engineered strains [10][17].

2. Industrial Value of NCYs

NCYs have several advantages over S. cerevisiae from an industrial viewpoint [18]. S. cerevisiae is often directed toward ethanol synthesis (due to its Crabtree-positive effect), restricting product diversification. In contrast, NCYs may have desired metabolic pathways, enabling product profile expansion. The ability to resist various stresses is a key benefit in industrial bioprocesses. For example, ethanol production from lignocellulose can be enhanced by developing alternative microbial platforms that are highly resistant to inhibitors. NCYs often exhibit strong resistance to various stresses, such as heat, acid, and high sugar concentrations, as environmental adaptations. Another key advantage of NCYs is their ability to utilize a wide range of carbon sources [8][19]. Additionally, many NCYs can exist in both haploid and diploid types like S. cerevisiae, and sexual reproduction is possible. Therefore, NCY strains with a desired ploidy can be developed through mating depending on the purpose. For example, diploid P. pastoris strains having a higher stability than its haploid form were constructed through a well-designed mating process for production of proteins [20], or various auxotrophic K. marxianus libraries were constructed using mating and dissection [21]. In this section, the industrial potentials of five promising NCYs are described (Figure 1).
Fermentation 08 00656 g001
Figure 1. Properties and industrial values of promising NCYs.

2.1. Pichia pastoris

P. pastoris is a methylotrophic yeast with a developed peroxisomal system responsible for compartmentalized methanol metabolism [22]. Its gene expression system, together with its secretory system, is well established, which enables easy genetic manipulation using a publicly available commercial kit. P. pastoris is mainly used for the production of pharmaceutical proteins and other industrial enzymes, because of simple post-translational modifications process [23][24]. In fact, hyperglycosylation of proteins in S. cerevisiae often causes an allergic reaction in human body; therefore, using it as an expression host for producing pharmaceuticals and medicinal proteins is often undesirable. P. pastoris exhibits superior growth rate and cell density compared to S. cerevisiae, and like S. cerevisiae, its protein expression is controlled under strong and tightly regulated methanol inducible promoters (AOX1). Thus, high cell density in addition to a high yield of recombinant proteins, produced either intracellular or extracellular, can be achieved, which may lead to increased biotransformation efficiency of whole cells [23][25].

2.2. Pichia kudriavzevii

P. kudriavzevii is a multi-stress tolerant yeast commonly found in fermented foods and beverages, such as Nuruk, which is a starter used for making Korean traditional alcoholic beverages and various sub-Saharan African indigenous foods [26][27][28][29]. Previous studies have isolated robust strains of P. kudriavzevii that can withstand multiple stress factors, such as high salt concentration, high temperature, and low pH. In particular, there are several advantages of using thermotolerant strains as production hosts for the ethanol industry. For S. cerevisiae, scaled-up ethanol production through simultaneous saccharification and fermentation is normally performed at 30 °C, above which growth and fermentation are repressed. Ethanol production at a high temperature is beneficial for reducing microbial contamination, as well as energy and water costs required to cool the fermentation system [30][31][32]. Indeed, thermotolerant P. kudriavzevii produce more ethanol at a higher temperature (44 °C) compared with S. cerevisiae. Furthermore, considering the information on genome sequence and genetic engineering tools, it is a potent host for various industrial metabolites, such as organic acids (e.g., succinic acid) and bioethanol [30][32][33][34].

2.3. Yarrowia lipolytica

As an oleaginous microorganism that accumulates lipids up to 20% of dry cell weight, Y. lipolytica is used for the industrial production of fatty acid-derived products [35]. Y. lipolytica is a GRAS organism and assimilates hydrophilic (e.g., glucose, glycerol, alcohols, and acetate) and hydrophobic substrates (e.g., fatty acids, triacylglycerols, and alkanes) [36]. With its high protein secretory capacity and lipophilicity, the organism has also been used for the fermentation of waste cooking oil to achieve bioremediation and waste valorization. During this fermentation process, Y. lipolytica produces extracellular lipase, which subsequently generates free fatty acids, utilizable as a carbon source, from waste cooking oil. With the development of metabolic engineering, Y. lipolytica can potentially produce several other metabolites, such as organic acids [37], erythritol [38], and flavonoids [39].

2.4. Ogataea polymorpha

O. polymorpha is a methylotroph, an organism that utilizes C1 compounds, such as methanol, as its sole carbon source, and is one of the most heat-resistant yeasts. Because native S. cerevisiae, the most widely used workhorse for bioethanol production, is incapable of xylose fermentation and engineered S. cerevisiae to possess heterologous xylose metabolic pathway may suffer from high metabolic burden, a benefit of O. polymorpha is its innate ability to metabolize xylose, the second most abundant sugar of lignocellulosic biomass [40]. Thus, high-temperature (i.e., 45–50 °C) ethanol fermentation of lignocellulose hydrolysate mainly consisting of glucose and xylose is possible. As one of a few methylotrophic yeasts, their key enzymes involved in methanol metabolism are strongly induced by methanol present within membrane-bound peroxisomes, which enables a compartmentalized reaction. Based on this expression machinery, O. polymorpha is a useful expression host for producing heterologous and difficult-to-express proteins via establishing expression systems induced by methanol under the control of strong and tightly regulated promoters.

2.5. Kluyveromyces marxianus

K. marxianus is a GRAS and thermotolerant ethanol-producing species that can grow at temperatures up to 52 °C [41][42][43], enabling high-temperature ethanol fermentation. As a Crabtree-negative yeast, this species is also advantageous for synthesizing non-ethanol products. Like K. lactis [44], K. marxianus has the unique ability to assimilate lactose, which is not feasible with S. cerevisiae and other yeasts [44][45]. Owing to its high capacity to grow on a broad spectrum of cheap carbon sources, such as xylose, arabinose, galactose, lactose, pectin, inulin, hemicellulose hydrolysate, cheese whey, and molasses, this species is an excellent microbial source of enzymes, bioethanol, and food ingredients [46] for commercial-scale applications [47][48][49]. Additionally, K. marxianus can produce fructose and fructooligosaccharides, which are industrially pertinent foods and pharmaceutical ingredients through inulinase secretion [50]. Recently, K. marxianus has been proposed as a probiotic yeast due to its beneficial roles in the gut [46].

References

  1. Li, W.; Shen, X.; Wang, J.; Sun, X.; Yuan, Q. Engineering microorganisms for the biosynthesis of dicarboxylic acids. Biotechnol. Adv. 2021, 48, 107710.
  2. Karim, N.; Shishir, M.R.I.; Gowd, V.; Chen, W. Hesperidin-an emerging bioactive compound against metabolic diseases and its potential biosynthesis pathway in microorganism. Food Rev. Int. 2021.
  3. Srinivasan, P.; Smolke, C.D. Biosynthesis of medicinal tropane alkaloids in yeast. Nature 2020, 585, 614–619.
  4. Raschmanová, H.; Weninger, A.; Glieder, A.; Kovar, K.; Vogl, T. Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects. Biotechnol. Adv. 2018, 36, 641–665.
  5. Parapouli, M.; Vasileiadis, A.; Afendra, A.-S.; Hatziloukas, E. Saccharomyces cerevisiae and its industrial applications. AIMS Microbiol. 2020, 6, 1.
  6. Rainha, J.; Gomes, D.; Rodrigues, L.R.; Rodrigues, J.L. Synthetic biology approaches to engineer Saccharomyces cerevisiae towards the industrial production of valuable polyphenolic compounds. Life 2020, 10, 56.
  7. Rainha, J.; Rodrigues, J.L.; Rodrigues, L.R. CRISPR-Cas9: A powerful tool to efficiently engineer Saccharomyces cerevisiae. Life 2020, 11, 13.
  8. Kręgiel, D.; Pawlikowska, E.; Antolak, H. Nonconventional yeasts in fermentation processes: Potentialities and limitations. In Old Yeasts: New Questions; IntechOpen: London, UK, 2017; p. 87.
  9. Bertels, L.-K.; Fernández Murillo, L.; Heinisch, J.J. The pentose phosphate pathway in yeasts-More than a poor cousin of glycolysis. Biomolecules 2021, 11, 725.
  10. Patra, P.; Das, M.; Kundu, P.; Ghosh, A. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnol. Adv. 2021, 47, 107695.
  11. Jensen, M.K.; Keasling, J.D. Recent applications of synthetic biology tools for yeast metabolic engineering. FEMS Yeast Res. 2015, 15, 1–10.
  12. Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D.A.; Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007, 315, 1709–1712.
  13. Walsh, R.M.; Hochedlinger, K. A variant CRISPR-Cas9 system adds versatility to genome engineering. Proc. Natl. Acad. Sci. USA 2013, 110, 15514–15515.
  14. Levi, O.; Arava, Y. Expanding the CRISPR/Cas9 toolbox for gene engineering in S. cerevisiae. Curr. Microbiol. 2020, 77, 468–478.
  15. Mitsui, R.; Yamada, R.; Ogino, H. CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals. World J. Microbiol. Biotechnol. 2019, 35, 111.
  16. Geijer, C.; Ledesma-Amaro, R.; Tomás-Pejó, E. Unraveling the potential of non-conventional yeasts in biotechnology. FEMS Yeast Res. 2022, 22, foab071.
  17. Cai, P.; Gao, J.; Zhou, Y. CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications. Microb. Cell Fact. 2019, 18, 63.
  18. Rebello, S.; Abraham, A.; Madhavan, A.; Sindhu, R.; Binod, P.; Karthika Bahuleyan, A.; Aneesh, E.M.; Pandey, A. Non-conventional yeast cell factories for sustainable bioprocesses. FEMS Microbiol. Lett. 2018, 365, fny222.
  19. Mukherjee, V.; Radecka, D.; Aerts, G.; Verstrepen, K.J.; Lievens, B.; Thevelein, J.M. Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation. Biotechnol. Biofuels 2017, 10, 216.
  20. Chen, M.-T.; Lin, S.; Shandil, I.; Andrews, D.; Stadheim, T.A.; Choi, B.-K. Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production. Microb. Cell Fact. 2012, 11, 91.
  21. Yarimizu, T.; Nonklang, S.; Nakamura, J.; Tokuda, S.; Nakagawa, T.; Lorreungsil, S.; Sutthikhumpha, S.; Pukahuta, C.; Kitagawa, T.; Nakamura, M. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae. Yeast 2013, 30, 485–500.
  22. Kao, Y.-T.; Gonzalez, K.L.; Bartel, B. Peroxisome function, biogenesis, and dynamics in plants. Plant Physiol. 2018, 176, 162–177.
  23. Ahmad, M.; Hirz, M.; Pichler, H.; Schwab, H. Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 2014, 98, 5301–5317.
  24. Kurtzman, C.P. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J. Ind. Microbiol. Biotechnol. 2009, 36, 1435–1438.
  25. Baghban, R.; Farajnia, S.; Ghasemi, Y.; Mortazavi, M.; Zarghami, N.; Samadi, N. New developments in Pichia pastoris expression system, review and update. Curr. Pharm. Biotechnol. 2018, 19, 451–467.
  26. Murata, Y.; Nwuche, C.O.; Nweze, J.E.; Ndubuisi, I.A.; Ogbonna, J.C. Potentials of multi-stress tolerant yeasts, Saccharomyces cerevisiae and Pichia kudriavzevii for fuel ethanol production from industrial cassava wastes. Process Biochem. 2021, 111, 305–314.
  27. Greppi, A.; Saubade, F.; Botta, C.; Humblot, C.; Guyot, J.P.; Cocolin, L. Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food. Food Microbiol. 2017, 62, 169–177.
  28. Choi, D.H.; Park, E.H.; Kim, M.D. Isolation of thermotolerant yeast Pichia kudriavzevii from nuruk. Food Sci. Biotechnol. 2017, 26, 1357–1362.
  29. Johansen, P.G.; Owusu-Kwarteng, J.; Parkouda, C.; Padonou, S.W.; Jespersen, L. Occurrence and importance of yeasts in indigenous fermented food and beverages produced in sub-Saharan Africa. Front. Microbiol. 2019, 10, 1789.
  30. Sun, W.; Vila-Santa, A.; Liu, N.; Prozorov, T.; Xie, D.; Faria, N.T.; Ferreira, F.C.; Mira, N.P.; Shao, Z. Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production. Metab. Eng. Commun. 2020, 10, e00124.
  31. Li, C.; Liu, Q.; Wang, Y.; Yang, X.; Chen, S.; Zhao, Y.; Wu, Y.; Li, L. Salt stress improves thermotolerance and high-temperature bioethanol production of multi-stress-tolerant Pichia kudriavzevii by stimulating intracellular metabolism and inhibiting oxidative damage. Biotechnol. Biofuels 2021, 14, 222.
  32. Isono, N.; Hayakawa, H.; Usami, A.; Mishima, T.; Hisamatsu, M. A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions. J. Biosci. Bioeng. 2012, 113, 76–78.
  33. Chan, G.F.; Gan, H.M.; Ling, H.L.; Rashid, N.A.A. Genome sequence of Pichia kudriavzevii M12, a potential producer of bioethanol and phytase. Eukaryot. Cell 2012, 11, 1300–1301.
  34. Xi, Y.; Zhan, T.; Xu, H.; Chen, J.; Bi, C.; Fan, F.; Zhang, X. Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzevii and their applications in succinic acid production. Microb. Biotechnol. 2021, 14, 1130–1147.
  35. Blazeck, J.; Hill, A.; Liu, L.; Knight, R.; Miller, J.; Pan, A.; Otoupal, P.; Alper, H.S. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat. Commun. 2014, 5, 3131.
  36. Liu, H.H.; Ji, X.J.; Huang, H. Biotechnological applications of Yarrowia lipolytica: Past, present and future. Biotechnol. Adv. 2015, 33, 1522–1546.
  37. Cavallo, E.; Charreau, H.; Cerrutti, P.; Foresti, M.L. Yarrowia lipolytica: A model yeast for citric acid production. FEMS Yeast Res. 2017, 17, fox084.
  38. Wang, N.; Chi, P.; Zou, Y.; Xu, Y.; Xu, S.; Bilal, M.; Fickers, P.; Cheng, H. Metabolic engineering of Yarrowia lipolytica for thermoresistance and enhanced erythritol productivity. Biotechnol. Biofuels 2020, 13, 176.
  39. Muhammad, A.; Feng, X.; Rasool, A.; Sun, W.; Li, C. Production of plant natural products through engineered Yarrowia lipolytica. Biotechnol. Adv. 2020, 43, 107555.
  40. Kim, O.C.; Suwannarangsee, S.; Oh, D.B.; Kim, S.; Seo, J.W.; Kim, C.H.; Kang, H.A.; Kim, J.Y.; Kwon, O. Transcriptome analysis of xylose metabolism in the thermotolerant methylotrophic yeast Hansenula polymorpha. Bioprocess Biosyst. Eng. 2013, 36, 1509–1518.
  41. Choudhary, J.; Singh, S.; Nain, L. Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass. Electron. J. Biotechnol. 2016, 21, 82–92.
  42. Fonseca, G.G.; Gombert, A.K.; Heinzle, E.; Wittmann, C. Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res. 2007, 7, 422–435.
  43. Banat, I.M.; Nigam, P.; Marchant, R. Isolation of thermotolerant, fermentative yeasts growing at 52 °C and producing ethanol at 45 °C and 50 °C. World J. Microbiol. Biotechnol. 1992, 8, 259–263.
  44. Álvarez-Cao, M.E.; Rico-Díaz, A.; Cerdán, M.E.; Becerra, M.; González-Siso, M.I. Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase. Microb. Cell Fact. 2018, 17, 137.
  45. Nurcholis, M.; Lertwattanasakul, N.; Rodrussamee, N.; Kosaka, T.; Murata, M.; Yamada, M. Integration of comprehensive data and biotechnological tools for industrial applications of Kluyveromyces marxianus. Appl. Microbiol. Biotechnol. 2020, 104, 475–488.
  46. Bilal, M.; Ji, L.; Xu, Y.; Xu, S.; Lin, Y.; Iqbal, H.M.N.; Cheng, H. Bioprospecting Kluyveromyces marxianus as a robust host for industrial biotechnology. Front. Bioeng. Biotechnol. 2022, 10, 851768.
  47. Ortiz-Merino, R.A.; Varela, J.A.; Coughlan, A.Y.; Hoshida, H.; Da Silveira, W.B.; Wilde, C.; Kuijpers, N.G.A.; Geertman, J.M.; Wolfe, K.H.; Morrissey, J.P. Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates. Front. Genet. 2018, 9, 94.
  48. Rajkumar, A.S.; Morrissey, J.P. Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products. Microb. Cell Fact. 2020, 19, 207.
  49. Cernak, P.; Estrela, R.; Poddar, S.; Skerker, J.M.; Cheng, Y.F.; Carlson, A.K.; Chen, B.; Glynn, V.M.; Furlan, M.; Ryan, O.W.; et al. Engineering Kluyveromyces marxianus as a robust synthetic biology platform host. mBio 2018, 9, e01410–e01418.
  50. Mao, W.; Han, Y.; Wang, X.; Zhao, X.; Chi, Z.; Chi, Z.; Liu, G. A new engineered endo-inulinase with improved activity and thermostability: Application in the production of prebiotic fructo-oligosaccharides from inulin. Food Chem. 2019, 294, 293–301.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
Upload a video for this entry
Information
Subjects: Biotechnology & Applied Microbiology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Jongbeom Park
,
In Jung Kim
,
Soo Rin Kim
View Times: 698
Revisions: 2 times (View History)
Update Date: 12 Dec 2022
Table of Contents
Academic Video Service
Feedback