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Industrial Value of Nonconventional Yeasts
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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].

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Subjects: Biotechnology & Applied Microbiology
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Jongbeom Park
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In Jung Kim
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Soo Rin Kim
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Update Date: 12 Dec 2022
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