Expanding the Knowledge on the Skillful Yeast Cyberlindnera jadinii

Cyberlindnera jadinii is widely used as a source of single-cell protein and is known for its ability to synthesize a great variety of valuable compounds for the food and pharmaceutical industries. Its capacity to produce compounds such as food additives, supplements, and organic acids, among other fine chemicals, has turned it into an attractive microorganism in the biotechnology field. In this review, we performed a robust phylogenetic analysis using the core proteome of C. jadinii and other fungal species, from Asco- to Basidiomycota, to elucidate the evolutionary roots of this species. In addition, we report the evolution of this species nomenclature over-time and the existence of a teleomorph (C. jadinii) and anamorph state (Candida utilis) and summarize the current nomenclature of most common strains. Finally, we highlight relevant traits of its physiology, the solute membrane transporters so far characterized, as well as the molecular tools currently available for its genomic manipulation. The emerging applications of this yeast reinforce its potential in the white biotechnology sector. Nonetheless, it is necessary to expand the knowledge on its metabolism, regulatory networks, and transport mechanisms, as well as to develop more robust genetic manipulation systems and synthetic biology tools to promote the full exploitation of C. jadinii.


Introduction
The future of our society challenges researchers to find novel technologies to address global environmental problems, mitigate ecosystems' damage, and biodiversity losses, as the current model of development based on natural resources exploitation is unsustainable. Exploring microorganisms for the production of platform chemicals constitutes an alternative approach to avoid the use of nonrenewable petrochemical-based derivatives. Developing applications for the industrial sphere using biological systems instead of classical chemical catalysts is the main focus of white biotechnology [1]. In microbialbased industrial processes, several features have to be addressed to obtain robust cell factories capable of achieving superior metabolic performances, such as the optimization of metabolic fluxes, membrane, and transporter engineering, and increased tolerance against harsh industrial conditions and toxic compounds [2]. In addition, specifications like the cost of feedstock, product yield and productivity, and downstream processing have to be taken in account to develop successful industrial approaches [1]. Withal the fact that Saccharomyces cerevisiae is by far the most relevant industrial yeast species, Cyberlindnera jadinii is an example of the so-called non-Saccharomyces yeasts [3] claiming for a place as a relevant contributor to the industrial biotechnological sector. The yeast C. jadinii is able to produce valuable bioproducts being an attractive source of biomass enriched in protein and vitamins. The richness of protein content, around 50% of dry cell weight, and amino acid diversity turn its biomass ideal as a source of protein supplement for 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].   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.

Life Cycle and Genome Organization
Kurtzman and colleagues proposed C. jadinii as the teleomorphic parental species of C. utilis, due to the 85% reassociation rate obtained between genomic DNA of the two yeast species and to the high similarities of ribosomal RNA sequences [20,34]. The formation of ascospores allied with genomic sequencing data confirmed the diploidy of C. jadinii NRRL Y-1542 strain and the identification of MATa and MATα genes allelic locations [20,37]. Ikushima et al. studied the polyploid of several C. utilis strains (NBRC0396, 0619, 0626, 0639, 0988, 1086, and 10707) detecting an overall ploidy switching between 2n and 5n [43]. Later, Kondo and colleagues inferred, through the analysis of C. utilis ATCC 9950 transformants the presence of a diploid state, although some years later, the sequential disruption of the URA3 and PDC1 locus suggested the tetraploidy of this strain [25,43,44]. A fluorescenceactivated cell sorting analysis pointed out a ploidy of 3n to 5n in this latter C. utilis strain, following the aforementioned data by Ikushima et al. [16,43]. Furthermore, a single nucleotide polymorphism analysis suggested that the C. utilis NBRC0988 genome was triploid [16,25,44]. Overall, these results suggested that C. utilis has derived from the parental yeast C. jadinii through triploidization pursuing an unexplained sequence of genetic events [16].
Recently, Krahulec and colleagues determined the C. utilis CCY 39-38-18 genome ploidy through the analysis of the copy number of the maltase gene in deleted mutants, pointing out the tetraploidy of this strain [32]. Despite the existing ploidy variation, the diploid state of C. jadinii impelled its genetic manipulation and subsequent utilization in the biotechnological industry [16,37]. Table 2 summarizes the genetic features of the C. jadinii strains sequenced so far [24]. Although the GC-content is quite similar, the genome size is different among the distinct strains evaluated (Table 2). When compared to S. cerevisiae, C. jadinii has an increased genome size and higher GC-content. The genomic features of C. jadinii strains are different from two closed phylogenetic species Wickerhamomyces ciferrii and Cyberlindnera fabianii ( Figure 1) that, respectively, contain a G-C ratio of 30.4% and 44.4%, genome sizes of 15.9 and 12.3 Mb, and a total of 6702 and 5944 CDS [45,46]. Among the genomic indicators presented here, the genome size and predicted/proteincoding genes seem to be strain dependent, whereas the GC-content is species independent, which is in accordance with the previously reported ploidy variations. Some differences were also detected among C. jadinii strains considering their specific genetic features, namely, the NBRC 0988 strain has 6417 predicted open reading frames (ORFs) comprising 16 unique ORFs [5,10,24], whereas the CBS1600 strain has 5689 ORFs, including 64 unique ORFs [10]. In 2015, Rupp and colleagues revealed a close haploid consensus sequences sizes, 12.7 Mbp for C. jadinii and 12.8 Mbp for C. utilis with an overall sequence identity of 98% [16].

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 (DLlactate), 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.
(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.

Molecular Tools for C. jadinii Manipulation
The genetic manipulation of C. jadinii has enabled the heterologous expression of various genes, resulting in the improvement of metabolic traits targeted for the optimization of exogenous product formation [8,10,44,56]. The establishment of genetic transformation methods allowed the efficient production of enzymes, carotenoids compounds, and organic acids such as L-lactic acid [44,56,59,60]. The development of an integrative transformation vector for the C. jadinii ATCC 9226 and C. utilis ATCC 9256 strains relied on a gene encoding a mutated ribosomal protein L41, conferring cycloheximide resistance as a dominant selection marker [25]. Dominant markers were used for cell transformation of aph, hph, nat, and ble genes, conferring resistance to G418, hygromycin B, nourseothricin, or zeocin, respectively, and the endogenous gene YAP1, conferring cycloheximide resistance [56,[60][61][62]. A multiple gene disruption method based on the Cre-loxP system allowing the reuse of selection markers was developed for C. jadinii NBRC0988 [43]. Auxotrophic ura3 strains were transformed with the expression vectors further integrated in the rDNA locus or in other chromosomal loci (e.g., TDH3). Single-plasmid integrations were reported for the TDH3 and HIS3 loci [16,25,56] as well as multiple plasmid integrations for the rDNA and URA3 loci for the expression of heterologous genes in high copy number (up to 90 copies) [63]. High plasmid stability was observed mainly for integrations into the URA3 and HIS3 loci, in contrast to the integrations in the rDNA, while integrations at the TDH3 locus were reported to be both stable and unstable [56,64,65]. Two chromosomal autonomously replicating sequences (ARS) were uncovered in C. jadinii ATCC 9226 and C. utilis ATCC 9256. Six plasmids harboring these ARS were obtained using a G418-resistance marker. The low copy number plasmids pCARS6 (CuARS1 region) and pCARS7 (CuARS2 region) presented the highest transformation efficiency [26]. A set of promoters were also explored to promote an efficient expression in C. jadinii, the TDH3, homolog of TDH3 from S. cerevisiae, encoding glycerol-3-phosphate dehydrogenase, as well PGK1 and PDC1 promoters from C. jadinii NBRC0988 encoding the phosphoglycerate kinase and pyruvate decarboxylase, respectively [44,64]. Furthermore, Kunigo et al. identified the highly xylose-inducible, glucose-repressed promoters of XDH1 and GXS1 genes, encoding a NAD-xylitol dehydrogenase and glucose/xylose symporter, respectively [56]. Promoters of the genes encoding the plasma membrane ATPase Pma1, Rpl29/Rpl31 ribosomal proteins, and Rpl41, as well as P14/P57 promoters from unknown chromosomal loci were used for the production of valuable products for the food industry, namely, for the secretion of heterologous proteins [25,[66][67][68]. CRISPR-Cas9 has quickly become the preferred targeted genome-editing technology for the genetic manipulation of yeasts [69], being extensively used in S. cerevisiae. The adaptation of the type II CRISPR/Cas system has been successfully used for the genetic manipulation of non-Saccharomyces species, such as Y. lipolytica, K. pastoris, K. lactis, Schizosaccharomyces pombe, and some pathogenic yeast species, such as Candida albicans and Cryptococcus neoformans [70,71]. Recently, a patent application reported the development of a CRISPR/Cas9 system that was applied to knock out and insert exogenous genes in C. jadinii ATCC 22023 [72]. This strategy also uncovered the triploidy of this strain.

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 AD∆, 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.

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.

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].

Membrane Transporters Characterized in C. jadinii
The production of biocompounds in high yields requires the optimization of several processes, including membrane transport of solutes to improve the entrance of substrates in the cell, exchange of products within cell organelles, and the efflux of metabolites to the extracellular medium, increasing the cell's tolerance to toxic final products, and decreasing downstream processing costs [109]. In C. jadinii, several plasma transporters were physiologically or genetically characterized (Figure 3).
In this yeast, copper (Cu 2+ ) transport is biphasic, energy-dependent, and relatively specific. Uptake is inhibited completely by 2,4-dinitrophenol (DNP), but carbonyl cyanide m-chlorophenylhydrazone (CCCP) had relatively little effect ( Figure 3A) [33,110]. The uptake follows a Michaelis-Menten kinetic with mean values for K t = 3.1 µM and V max , 0.5 nmol min −1 per mg (dry wt.) and has an optimal pH between 5 and 5.5. No exchange of K + for Cu 2+ could be detected during Cu 2+ uptake, and Cu 2+ efflux from preloaded cells was not observed [33].
The zinc uptake-system described in C. jadinii is energy-dependent and apparently unidirectional as no exchange occurs between intracellular accumulated 65 Zn and cold external Zn 2+ ( Figure 3C) [112]. This transporter exhibits a high-affinity for Zn 2+ (K m = 0.36 µM) with a V max of 2.2 nmol min −1 per mg dry wt. of cells. The regulation of zinc homeostasis occurs either by altering the levels of a cytoplasmic zinc-sequestering macromolecule or by inhibition of zinc efflux through a membrane carrier [112][113][114].
The saturable and unidirectional sulfate transporter ( Figure 3D) is pH-, temperature-, and energy-dependent with a K m of 1.43 mM, being competitively inhibited by molybdate, selenate, thiosulfate, chromate, and sulfite [115,116]. The activity of this sulfate transporter is controlled by the pool of external sulfur compounds as well as by the mitochondrial metabolism [116].
The proton-symporter for nitrate ( Figure 3E) is repressed by ammonium [117][118][119]. Ali and Hipkin [118] reported that the addition of 1, 2, or 10 mM ammonium resulted in an inhibition of nitrate uptake close to 30%. Studies using 3,3 -dipropylthiadicarbocyanine, a fluorogenic probe used to detect and measure alterations in transmembrane potential, indicated that the proton-linked uptake of nitrate, amino acids, or glucose during energy metabolism tended to depolarize the plasma membrane of C. jadinii cells [117].
Thirty ABC transporters ( Figure 3H) from different transporter subfamilies were found by homology search on the genome of the strain NBRC0988 [85]. The expression of the C. albicans Pdr1 homolog CjCdr1 in the S. cerevisiae AD∆ strain conferred resistance to geneticin (75 µg), micafungin (40 µg), and nystatin (500 µg). These two proteins present similar substrate specificity, although CjCdr1 is more resistant to Rhodamine 6G [85]. The C. jadinii aquaglyceroporin CjFps1 ( Figure 3I) shares 38% of identity with the S. cerevisiae Fps1. The heterologous expression of CjFPS1 in a glycerol-consuming S. cerevisiae wildtype strain (CBS 6412-13A) promoted cellular growth improvement on glycerol as the sole carbon source [124].
Two different systems were found for glucose transport in C. jadinii, a proton-symporter and a facilitated diffusion ( Figure 3J) [125][126][127], however, the respective genes remain unidentified. The high-affinity proton-symporter presents a K m of 15 µM glucose, displays a stoichiometry of 1:1, and is partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol, and glycerol. This transporter is repressed by high glucose concentration but is induced by glucose up to 0.7 mM [125], a behavior also found for the maltose-uptake system, indicating that both systems share a common glucose control pathway [125,128]. Barnett and Sims (1976) proposed that the differences in glucose vs. maltose affinity can be due to allosteric mechanisms associated with a multimeric transporter or to the hysteretic behavior of a monomeric transporter [126], conditions that have not yet been solved as the correspondent genes(s) remain unidentified. The facilitated diffusion mechanism ( Figure 3J-II) is found in cells growing on glucose at concentrations higher than 10 mM and presents complex kinetics of glucose transport whose K m oscillates between 2 and 70 mM [125,128]. It was also reported that uric acid enters C. jadinii cells by the glucose-dependent active transport [129].
The low-and the high-affinity systems for D-xylose transport ( Figure 3K) with K m values of 67.6 ± 3.2 and 1.9 ± 1.2 mM, respectively, act as proton-symporters with distinct modes of regulation. The starvation of glucose-grown cells decreases the K m value of the low-affinity system (K m = 10.5 ± 2.6 mM) [130]. The high-affinity system found during starvation requires protein synthesis and is inactive when cells are exposed to glucose, through a process independent of protein synthesis. Glucose and acetic acid inhibited both the high-and low-affinity xylose transport systems [130].
Four mediated transport systems for organic acids are described in C. jadinii ( Figure 3L) [29][30][31]. The monocarboxylate proton symport, shared by the L-and D-lactate (K m 0.06 mM), pyruvate (K m 0.03 mM), propionate (K m 0.05 mM), and acetate (K m 0.1 mM) ( Figure 3L-II) is active over a pH range of 3.0-6.0, with an optimum of activity at pH 5.0 [29]. The dicarboxylate-proton symporter ( Figure 3L-III) is shared by L-malate (K m 4.0 ± 0.5 µM), succinate (K m 0.03 ± 0.01 mM), fumarate, oxaloacetate, and α-ketoglutarate [30,31,131]. The tricarboxylate-proton symporter ( Figure 3L-IV) presents a K m of 0.056 mM for citrate and is competitively inhibited by isocitric acid, while aconitic, tricarballylic, trimesic, and hemimellitic acids did not affect citrate uptake [30]. All these carboxylic acid transporters are inducible by the respective substrates, being subjected to glucose repression as well as by acid concentrations higher than 3% (w/v) [31]. The facilitated diffusion for the undissociated form of the acids ( Figure 3L-I), which is likely to operate as a general organic permease, is active at pH below 5.0. It accepts mono-, di-, and tricarboxylates as well as glycine and glutamic acid [31,132]. The following kinetic parameters were obtained at pH 3.0: (a) V max 0.516 nmol of malic acid s −1 per mg (dry wt. of cells) and K m 1.529 ± 0.024 mM malic acid, (b) V max 0.585 nmol of succinic acid s −1 per mg (dry wt. of cells) and K m 1.789 ± 0.089 succinic acid, and (c) V max of 1.14 nmol of s −1 per mg (dry wt. of cells) and K m of 0.59 mM citric acid [29][30][31]. Despite being functionally characterized for several years, the genes encoding these proteins remain to be identified [133]. Recently, our group identified six genes homologous to ScATO1 and six genes homologous to ScJEN1 (unpublished results). In S. cerevisiae, these two genes encode distinct monocarboxylate transporters [134,135] and its homologs, present in bacteria, archaea and eukaryotes, are able to transport mono-and dicarboxylates [136][137][138][139][140][141][142]. The expression of membrane transporters allied to metabolic engineering tools is crucial to develop new and more efficient strains to produce bio-based compounds. Thus, increased knowledge of transporter proteins will enable the development of improved cell factories [109,143,144].

Conclusions and Future Perspectives
As biotechnological applications expand, it becomes necessary to explore novel expression hosts as more efficient and robust microbial cell factories are demanded [123][124][125]. Over the years, Cyberlindnera jadinii has been widely explored as a source of single-cell protein, having the ability to produce vitamins (e.g., biotin), organic acids (e.g., glutamate), and proteins (e.g., enzymes). The capacity to utilize and degrade a great variety of carbon sources and its natural ability to produce significant compounds make C. jadinii an attractive microorganism for industrial applications [5,7,8,10]. Additionally, several features turn this yeast an ideal platform for biotechnological processes, like the higher level of tolerance to changes occurring during growth and multiplication conditions [5,55]. However, C. jadinii is still lagging behind when compared to other non-Saccharomyces yeasts, mainly due to the inexistence of extensive knowledge on its metabolism, regulatory networks, and transport mechanisms. The genomic characterization of several strains is also necessary to reveal the genetic features underlying the existing interspecies variability, particularly between its teleomorph and anamorph state. The potential of this yeast as a therapeutic agent is due to its already known antagonistic effects on human pathogens and utilization as a probiotic agent. Its protein secretion system is another attractive feature for the heterologous expression of soluble proteins. Nonetheless, only with the improvement of advanced genetic manipulation systems and the development of synthetic biology tools will the full exploitation of C. jadinii biotechnological potential be achieved. These and other advances will certainly allow C. jadinii to become a robust microbial cell factory in an expanding era of metabolite bioproduction.