2. Ecology, Taxonomy, and Evolution

The natural environment of Cyberlindnera jadinii is still an open question. It is thought that it may be associated with the decomposition of plant material in nature, as it is able to assimilate pentoses and tolerate lignocellulosic by-products [3], displays great fermentative ability, and is copiotrophic [13]. The current laboratory strains were isolated from distinct environments, namely, from the pus of a woman abscess (CBS 1600/NRRL Y-1542), a cow with mastitis (CBS 4885/NRRL Y-6756), a yeast deposit from a distillery (CBS 567), yeast cell factories (CBS 621), and flowers (CBS 2160) [5,6,7,8,9,12]. The extensive nomenclature revisions of this species are well described in “The tortuous history of Candida utilis” by Barnet [14]. In 1926, this yeast was isolated from several German yeast factories, which had been cultivated without a systematic name during the time of World War I for food and fodder [14]. It was first named Torula utilis being later referred to as Torulopsis utilis (1934). The “food yeast” was also designated as Saccharomyces jadinii (1932), Hansenula jadinii (1951), Candida utilis (1952), Pichia jadinii (1984), and Lindnera jadinii (2008) [12,14,15,16]. From the aforementioned, C. utilis was the nomenclature most commonly used, having almost 1000 published papers in PubMed (results available at: https://pubmed.ncbi.nlm.nih.gov/?term=%22candida+utilis%22; Accessed 16 October 2020). The Candida genus comprised species that form pseudohyphae or true hyphae with blastoconidia, among other standard characteristics [17,18,19]. In the classification system implemented in 1952 by Lodder and Kreger-van Rij, the Candida genus included yeasts that produce only simple pseudohyphae [14]. At that time, the majority of the isolates was renamed as C. utilis [18]. Later, the C. utilis was established as an asexual state of a known ascosporogenous yeast, Hansenula jadinii, as it was found to share some similarities between phenotypic traits [20]. In 1984, even though concurring with a publication of an extensive chapter of the genus Hansenula, Kurtzman moved most of the Hansenula species to the Pichia genus, due to their “deoxyribonucleic acid relatedness.” Thus, C. utilis was renamed Pichia jadinii. A quarter of a century later, this yeast species was again renamed as Lindnera jadinii based on analyses of nucleotide sequence divergence in the genes coding for large and small-subunit rRNAs [12]. Species integrated into the Lindnera differ considerably in ascospore morphology ranging from spherical to hat-shaped or Saturn-shaped spores. In addition, this clade includes both hetero- and homothallic species and physiological features as fermenting glucose and assimilating a variety of sugars, polyols, and other carbon sources are defining characteristics of the Lindnera genus [12]. Finally, 1 year later, the genus Lindnera was replaced by Cyberlindnera, as the later homonym defined a validly published plant genus [15]. This substitution occurred in 21 new species, including Cyberlindnera jadinii [15]. In summary, any of the aforementioned nomenclatures reported in the literature may refer to the same organism since C. utilis is the anamorph state and C. jadinii the teleomorph state [14]. The anamorph represents the asexual stage of a fungus contrasting with the teleomorph form that defines the sexual stage of the same fungus [21]. The primary name of a species relies on the sexual state or teleomorph, but a second valid name may rely on the asexual state or anamorph [22]. However, this should only happen when teleomorphs have not been found for a specific species or it is not clear if a particular teleomorph is the same species as a particular anamorph. Accordingly, since 2013, the International Botanical Congress states that the system for allowing separate names for the anamorph state should end [23]. The new International Code of Nomenclature for algae, fungi, and plants, the Melbourne Code, supports the directive that fungal species or higher taxon should be assigned with a single valid name. Accordingly, anamorph yeast genera like Candida should be revised to turn the genus consistent with phylogenetic affinities [19]. Notwithstanding, the reclassification of several C. utilis as C. jadinii in several culture type collections is still confusing, reaching a point where the same strain is designated as C. utilis and C jadinii in different culture type collections. This aspect, together with the previous nomenclatures used in research papers, leads to unnecessary misunderstandings. To clarify this, Table 1 presents the alternative designations of the main laboratory strains.

C. jadinii belongs to the phylum Ascomycota, subphylum Saccharomycotina. The members of this subphylum constitute a monophyletic group of ascomycetes that are well defined by ultrastructural and DNA characteristics [13]. These include lower amounts of chitin in overall polysaccharide composition at cell walls, being unable to stain with diazonium blue, low content of guanine and cytosine (G + C < 50%) at nuclear DNA, and presence of continuous holoblastic bud formation with wall layers. C. jadinii belongs to the Saccharomycetes class, Saccharomycetidae family, Saccharomycetales order, and the Cyberlindnera genus. However, a comprehensive phylogenetic analysis and evolutionary relationship are still missing for this species [36,37,38]. Aiming at filling this gap, we performed a robust phylogeny reconstruction [36,39,40,41]. As can be depicted in Figure 1, this yeast localizes in the Phaffomycetaceae clade together with Cyberlindnera fabianii and Wickerhamomyces ciferri. The nearest neighbors belong to the Saccharomycetaceae family, which includes a clade with S. cerevisiae/Torulaspora delbrueckii species and another clade with Eremothecium gossypii (former Ashbya gossypii), Kluyveromyces lactis/marxianus, and Lachancea species. Despite the previous genus nomenclature adopted for C. jadinii (Candida and Pichia), it is phylogenetically distant from the Debaryomycetaceae and Pichiaceae families that include the Candida species, except C. glabrata, the Pichia kudriavzevii, and Ogataea species. Komagataella phaffi is as expected included in the Pichiaceae clade, together with Komagataella pastoris [36,40]. The Trichomonascaceae/ Dipodascaceae clade, formerly known as the Yarrowia clade, includes now the Sugiyamaella lignohabitans species together with Yarrowia lipolytica, and is the most distant yeast clade, except for the Schizosaccharomycetaceae that clusters with all the Basiodiomycota. The filamentous fungi Neurospora crassa and Fusarium graminearum are in different clades as members of the Sordariaceae and Nectriaceae clades, respectively [40]. In addition, in the Sordariaceae clade, the phylogenetic position of Thermothelomyces thermophila species (Myceliophthora thermophila) was uncovered [39,42].

Figure 1. Evolutionary relationship of Cyberlindnera jadinii, a member of the Phaffomycetaceae clade. The phylogenetic reconstruction was obtained using the following parameters: maximum likelihood in IQ-TREE (http://www.iqtree.org), the model of amino acid evolution JTT (Jones-Taylor-Thornton), and four gamma-distributed rates. Homologues were detected for 1567 proteins across the proteome of 77 selected fungal species from NCBI. The 1567 set of proteins were aligned and then concatenated in order to use in the phylogenetic analysis. These proteins offer a clear high-resolution evolutionary view of the different species, as they are essential proteins beyond the specific biology of the different yeasts. Bootstrapping provided values of 100% for all the nodes. Yeast and fungi families are highlighted with different colors and shades. The phylogenetic relationships reflect evolutionary ancestries, independently of adaptations and overall gene contents within the various species. All families with more than one representative species in the analyses formed monophyletic groups.

3. Morphology and Physiology

The C. jadinii microscopic view provided by Kurtzman et al. (2011) has shown the diversity of cell shapes and sizes [20,34] after 10–30 days at 25 °C in 5% Malt Extract Agar media. The cell patterns of C. jadinii CBS 1600 varied from ellipsoidal to elongated occurring in single cells or in pairs. Some pseudohyphae forms were also detected with diameter balanced between (2.5–8.0) and (4.1–11.2) µm.

Figure 2 shows C. jadinii DSM 2361 strain cultivated on YPD or Malt Extract Agar media for 3 (A and C) and 12 days (B and D) at 30 °C. The colonies are white, round, with a smooth texture, an entire margin, and a convex elevation trait (C and D). Cells present an ellipsoidal to elongated form, with a diameter between 5 and 7.5 µm (A and B). Yeast cell morphology can be tightly influenced by the environment. These modifications can affect the fermentation performance by inducing rheological changes that can influence mass and heat transfer alterations in the bioreactor [49]. However, in a study performed by Pinheiro et al. (2014), the CBS 621 strain cultured in a pressurized-environment triggered with 12 bar air pressure presented no significant differences in cell size and shape [35]. C. jadinii is a homothallic species and forming hat-shaped ascospores that can be present in a number of one to four in unconjugated deliquescent asci [34]. Cyberlindnera species can assimilate several compounds, namely, sugars and organic acids. The robust fermentation characteristics of C. jadinii allow growth in a diversity of substrates from biomass-derived wastes, including hardwood hydrolysates from the pulp industry, being able to assimilate glucose, arabinose, sucrose, raffinose, and D-xylose [8,9,10,50]. As previously mentioned, C. jadinii is a Crabtree-negative yeast, reaching higher cell yields under aerobic conditions [51,52,53] than Crabtree-positive species. The Crabtree-negative effect favors the respiration process over fermentation, enabling the development of phenotypes relevant for protein production [54]. This species has a high tolerance to elevated temperatures, being able to grow in a broad spectrum of temperatures from 19 to 37 °C [37] and to tolerate long-term mild acid pHs (~3.5) [55]. Another relevant property is the ability to release proteins to the extracellular medium [56]. Significant lipase and protease content were achieved using a wild C. jadinii strain isolated from spoiled soybean oil, using solid-state fermentation [57,58]. C. jadinii assimilates alcohols, acetaldehyde, organic acids, namely, monocarboxylates (DL-lactate), dicarboxylates (succinate), and tricarboxylates (citrate), sugar acids (D-gluconate), and various nitrogen sources comprising nitrate, ammonium hydroxide, as well as amino acids [8,10,12,37]. A set of metabolic advantages, as the high metabolic flux in TCA cycle, the great amino acid synthesis ability, and strong protein secretion turns C. jadinii a yet underexplored host for bioprocesses. An incomplete understanding of genetics, metabolism, and cellular physiology combined with a lack of advanced molecular tools for genome edition and metabolic engineering manipulation of C. jadinii hampered its development for cell factory utilization.

Figure 2. C. jadinii DSM 2361 morphological traits. (A) Microscopic photographs of C. jadinii cells after 3 days (A) and 12 days (B) of growth at 30 °C on yeast extract-peptone-dextrose media. Scale bars are 5.0 µm. Macro-morphological features of C. jadinii after 3 days of growth in YPD (C) and Malt Extract Agar (D) media, at 30 °C.

4. Emerging Biotechnological Applications

4.1. Therapeutic Applications

Being an edible yeast, C. jadinii has the potential to target the gastrointestinal tract in humans and animals [10,55]. C. jadinii’s robust growth characteristics including insensitivity to low pH and temperatures up to 40 °C, allow its transit in the gastrointestinal tract without losing viability [55,73]. Additionally, like other food constituents, intact/partially degraded C. jadinii cells may adhere to M cells in the small intestine. Upon this, they are translocated to antigen-presenting cells of Peyer’s plaques or to other lymphoid tissue connected with the gastrointestinal tract [74,75]. The ingestion of engineered C. jadinii cells, carrying a myelin oligodendrocyte glycoprotein antigen on its surface, promoted tolerance to self-antigens in a mouse model of the autoimmune multiple sclerosis (MS) disease [76]. C. jadinii cells expressing the immunodominant MOG_35–55 epitope of a myelin protein on their surface, fused with the native fungal Gas1 cell wall protein, prevented the typical MS symptoms in this animal model [76]. The cell surface display of antigens by C. jadinii seems to modulate immune responses, either by suppression to combat autoimmune disease or through immune stimulation, enabling the creation of edible vaccines [73,76,77]. C. jadinii cells were also applied as probiotic agents against fungal infections (particularly oral candidiasis) as an antagonist to the relevant human fungal pathogens Candida albicans (strains SC5312, 10341, and GDH2346), Aspergillus sp., and Fusarium sp. [73]. The unidentified toxins secreted by C. jadinii act as antagonistic compounds conditioning the growth, systemic invasion, and disease caused by these fungal pathogens. Buerth et al. [10] reported the role of C. jadinii and also W. farinosa as antagonistic agents against C. albicans by inhibiting its growth and morphogenesis, proposing their exploitation for the formulation of new prebiotic compounds and strategies to tackle candidiasis. C. jadinii was also described to secrete heterologous proteins into the growth medium, including lipase B from Candida antarctica (CalB) [56]. The signal sequence of the enzyme invertase, one of the most predominant proteins of the C. jadinii secretome, allowed high secretion levels of recombinant CalB [5]. Lipases have a great potential application in substitution therapies, where metabolic deficiency is overcome by external administration of these enzymes in diseased conditions [78]. Furthermore, lipase activity can go from activation of tumor necrosis factor, having a relevant role over the treatment of malignant tumors, to the treatment of gastrointestinal disturbances, digestive allergies, or dyspepsias [78]. In addition, CalB is involved in enzymatic resolutions, desymmetrization, and aminolysis events with application in not only pharmaceutical but also biotech industry, having a role in polymer production [78,79]. C. jadinii is also able to synthesize (R)-phenylacetylcarbinol (L-PAC), the pharmaceutical precursor for L-ephedrine and pseudoephedrine, relevant compounds used in the treatment of nasal congestion [80,81,82,83]. The function of 30 ATP-binding cassette transporters (ABC) transporters was studied by the amplification of predicted C. jadinii gDNA ORFs. The function of putative multidrug efflux pumps was evaluated by heterologous expression in S. cerevisiae ADΔ, a strain disrupted in seven of its major multidrug efflux pumps: Pdr5p, Pdr10p, Pdr15p, Snq2p, Pdr11p, Ycf1p, and Yor1p [84]. This strategy uncovered the mechanism of action of CjCdr1, C. jadinii’s closest homolog of the multidrug efflux pump C. albicans Cdr1. The characterization of C. jadinii multidrug efflux systems can turn C. jadinii into an appealing host for the development of novel antimicrobial agents [85], as it is imperative to understand the structure, function, and expression of multidrug efflux pumps in order to develop optimal novel antimicrobial agents.

4.2. Bioproduction of Valuable Compounds Using Cost-Effective Carbon Sources

Recombinant C. jadinii strains were developed for the production of a variety of compounds from food supplements such as vitamins (biotin) [86], carotenoids (lycopene, β-carotene, and astaxanthin) [60,68], to proteins (α-amylase, monellin) [64], antioxidant glutathione [87], polysaccharides (glucomannan) [88,89], organic acids (L-lactic acid) [44,59], and ribonucleic acids [5,24]. Additionally, the production of secreted enzymes such as invertase and phospholipase B (NBRC 1086 strain) [27,28] was also explored. Cells of C. jadinii DSM 2361 were successfully engineered for the secretion of Penicillium simplicissimum xylanase (PsXynA) to the culture medium [65] allowing cells to grow on xylan as the sole carbon source. Cells expressing the xylose reductase from Candida shehatae and the native xylitol dehydrogenase, in combination with further multiple site-directed mutations in coenzyme binding sites, resulted in the highest titer of 17.4 g/L of ethanol from 50 g/L of xylose in 20 h [90]. Organic acids present in industrial waste streams (e.g., acetic acid, propionic, or butyric acid) have been demonstrated to be suitable substrates for biomass production, reaching biomass yields varying from 30% to 40% in batch cultures, while in continuous cultures, an average of 44–55% was achieved [91,92]. Despite its already important role as an industrial microorganism, further developments are still necessary to fully explore the biotechnological potential of this yeast.

4.3. Industrial Applications—A Patent-View

In recent years, the applications of C. jadinii were extended to cosmetic and health care products and to the chemical-process industry for the production of chiral chemicals, as well as for agriculture and wine making. In this last application, C. jadinii yeast was used in the production of loquat wine, being introduced after S. cerevisiae fermentation to reduce acid content and enhance the aroma [93,94]. It is also used in another fermentation process, for the production of an alcohol-free fruit wine rich in lovastatin [95]. In the cosmetic industry, a β-D-glucan polysaccharide produced by C. jadinii was applied in formulations of several products, i.e., body lotions [96], sunscreen cream [97], facial cleanser [98], toner [99], eye cream [100], shampoo [101], body wash [102], and hand-care cream [103]. This bioproduct is mainly added for its properties as a moisturizing agent and for conferring oxidation and radiation resistance. C. jadinii was also used for the efficient production of a recombinant uricase, active in humans and with greater stability and/or activity than naturally occurring enzymes [104]. This enzyme can be used for the treatment of hyperuricemia-related diseases or other human pathological symptoms. Considering the chemical industry, the bioproduction of methyl fluorophenyl methyl propionate was achieved with a developed reduction method using C. jadinii as a biocatalyst [105]. The obtained chemical, (2S,3S)-3-(4-fluorophenyl)-3-hydroxy-2-methyl methyl propionate, is reported to be produced in high yield and with a high level of the enantiomeric excess rate. The wide applicability of this chiral building-block chemical can go from the synthesis of chiral drugs, fine chemicals to pesticides [105]. In agriculture, a consortium of strains that include C. jadinii was incorporated in an organomineral granular fertilizer containing among other components fulvic acid, and a natural mineral component (activated natural siliceous zeolite-containing rock). Its properties allow the reduction in the amount of fertilizer introduced in the soil with a prolonging action improved [106]. As C. jadinii is capable of efficiently converting a cadmium form from contaminated soil, it is now being proposed for soil bioremediation [107]. In addition, a microbial soil conditioner for lithified soil was also developed involving a C. jadinii strain along with Bacillus megaterium, Bacillus subtilis, Rhodopseudomonas palustris, and Azotobacter chroococcum species. The full interaction among the aforementioned strains was claimed to improve the soil ecological environment and alter the soil lithiation event, thereby contributing for the purpose of turning the soil suitable for farming [108].