Kluyveromyces marxianus is regarded as an emerging host platform for synthesizing extracellular proteins owing to its capability to grow on an array of cheap feedstocks, including whey, molasses, and spent sulphite liquor (Fonseca et al., 2008). It is also capable of growing on various polysaccharides, such as inulin and pectin (Hensing et al., 1994). K. marxianus exhibits inherent ability to produce enzymes because all complex materials stated earlier can be extracellularly hydrolyzed to monomers (Chi et al., 2011). The K. marxianus growth on inulin and sucrose occurs via the catalytic action of extracellular enzymes, mainly inulinase (Rouwenhorst et al., 1988). The degradation of lactose by β-galactosidases excretion is promising because of simultaneous synthesis of biologically active ingredients, biomass, and enzyme biocatalysts of industrial importance (Fonseca et al., 2008; Chi et al., 2011). A recent summary of different enzyme productions by K. marxianus strains is depicted in Table 1. Figure 2 shows the schematic illustrations of uptake mechanism and metabolism of lactose in K. marxianus.

Fermentative production of bioethanol at elevated temperatures has gained attention because the rapid fermentation rate at high temperature is likely to minimize the cooling cost, circumvent the contamination risk, and enables simultaneous saccharification and fermentation (Nonklang et al., 2008). The optimal K. marxianus growth at a high temperature is particularly alluring to facilitate cooling during the extensive level fermentations for which transferring heat is a restrictive factor. Current industrial production of ethanol mainly employs S. cerevisiae due to its superior production titer and tolerability to high ethanol concentrations (about 120 g/L). Nevertheless, the optimal temperature of this yeast is relatively low (25–30°C) (Qiu and Jiang, 2017). Therefore, much interest has recently been geared to explore yeast species that are capable of producing ethanol at higher temperatures, and K. marxianus appears a robust isolate for this purpose (Figure 2) (Madeira-Jr and Gombert, 2018). Table 2 gives a recent summary of ethanol production by various K. marxianus strains. K. marxianus species are thermotolerant and can grow at 47 (Nonklang et al., 2008; da Silva et al., 2018), 49 (Hughes et al., 1984), and even 52°C (Banat et al., 1992). It is able to produce ethanol at temperature above 40°C (Madeira-Jr and Gombert, 2018), and assimilate a wide range of cheap substrate, including molasses, whey permeates (Hang et al., 2003; Ozmihci and Kargi, 2007; Martínez et al., 2017), and corn silage juice to synthesize ethanol. Given all these features, K. marxianus is appreciated as a prospective alternative to S. cerevisiae for commercial scale ethanol production.

Production of ethanol from non-food substrate is favorably suggested for avoiding criticism with food supply. Nutrients enriched whey can be considered a non-food low-cost substrate for producing ethanol by Kluyveromyces spp. Tesfaw et al. (2021) achieved the optimized ethanol production from crude whey using a newly isolated K. marxianus ETP87 strain. The non-sterilized and sterilized whey were utilized as feedstocks to assess the K. marxianus ETP87 competition with lactic acid and other microflora. The use of sterilized whey resulted in a slightly higher amount of ethanol than non-sterilized counterpart. Likewise, a substantially higher ethanol titer was achieved from whey without pH adjustment, and yeast accumulated the maximum ethanol between 30 and 35°C. It was capable of producing high level of ethanol until 45°C, and ethanol titer was drastically reduced when temperature was increased at 50°C. Inclusion of ammonium sulfate and peptone stimulated the ethanol production, while yeast extract and urea have negative effect on the ethanol productivity. Based on a circular economy concept, Tinôco et al. (2021) produced cellulosic ethanol by K. marxianus CCT 7735. Following acid–base pretreatment and hydrolysis at 50°C for 3 days, the saccharified sweet sorghum was subjected to ethanol production at three different temperatures (37, 42, and 45°C). Precisely, 17.83 g/L ethanol was produced after 24 h at 42°C with a corresponding yield of 2769.8 L/ha_sorghum, which is double compared to corn straw.

In a recent study, Madeira-Jr and Gombert (2018) demonstrated that the production of bioethanol at elevated temperatures (∼48°C) by K. marxianus NCYC 3396 using sugarcane can reduce cooling costs, risk of contamination, use of antibiotics, water usages, consumption of H₂SO₄ in cell reprocessing, and the use of energy in distillation, which ultimately reduce the bioethanol production cost in Brazilian biorefinery. Using K. marxianus, they achieved a yield of 0.40 g ethanol/g glucose at 48°C that was similar to S. cerevisiae CEN.PK113-7D at 37°C. Though the ethanol titer (0.43 g/g glucose) was comparable by K. marxianus K213 and S. cerevisiae using glucose at 30°C, S. cerevisiae lost its ethanol production ability when temperature raised to 45°C, while K. marxianus K213 retained the same conversion efficacy (0.43 g/g glucose) at this temperature (Yan et al., 2015). Murari et al. (2019) also corroborated that temperature (32.5–35°C) was the most influential parameter to produce ethanol from cheese whey by K. marxianus URM7404 following pH (4.8–5.3) and lactose concentration (61–65 g/L).

Raw starch-degrading enzyme (RSDE) is being employed in the ethanolic fermentation bioprocess for hydrolyzing starchy materials, such as cassava chips and cassava starch (Lomthong et al., 2015; Lomthong et al., 2016). Using RSDE reduces processing costs and energy consumption because the hydrolysis of starch granules is carried out below the gelatinization temperature than traditional starch degradation that occurs at 80–100°C (Cinelli et al., 2015; Lomthong et al., 2018). In a recent study, Lomthong et al. (2016) produced RSDE by Laceyella sacchari LP175 and applied to hydrolyze cassava chips at 50°C. The resulting sugar syrup was used as a fermentative substrate for bioethanol production. The saccharification and hydrolysis of cassava chips was carried out at lower temperature without heating-fostered ethanol fermentation by K. marxianus DMKU-KS07 at 42°C (Trakarnpaiboon et al., 2017). Similarly, molasses is enriched with high sucrose contents and other nutrients, which are conducive for microbial growth to produce bioethanol. Arshad et al. (2017) utilized molasse substrate for very high gravity (VHG) bioethanol by S. cerevisiae, yielding the highest ethanol titer to be 122 g/L. In contrast to other feedstocks, molasses present the benefits of bioethanol production without any pretreatment and saccharification (Chotineeranat et al., 2010). Nevertheless, a high concentration of metal ions impedes the activity of microbial enzymes and might lower ethanol yield (Chotineeranat et al., 2010; Wattanagonniyom et al., 2017). Under non-sterile conditions, Lomthong et al. (2021) evaluated the improvement of VHG bioethanol titer using molasses and cassava chip co-substrate hydrolysates by K. marxianus DMKU-KS07 at 42°C. Results showed a high bioethanol concentration of 118 g/L with ethanol yield (YP/S) at 0.44 g EtOH/g total sugar and productivity of 2.19 g/L/h by simultaneous saccharification and fermentation at 42°C for 12 h.

Single-cell proteins (SCPs) are referred to as dietary single-cell microorganisms, whose protein extracts or biomass are originated from mixed or pure bacterial cultures, yeasts, mushrooms, or microalgae. These microorganisms can be utilized as food ingredients, dietary supplements, or high protein foods for animal and human consumption (Ritala et al., 2017). Thus, extensive biosynthesis of microbial biomasses could be useful to replace animal- and plant-derived proteins for food or feed owing to high-protein contents, fast proliferation rate, and the aptitude to assimilate a wide spectrum of carbon containing waste materials (Karimi et al., 2018; Karim et al., 2020). Given a shorter microbial life cycle, SCPs can be attained rapidly than that of the proteins produced from agricultural sources. Amid the SCP producers, yeasts have appeared as emerging cell factories due to tiny particle size, large protein content, facile handling, and comparatively minimum production costs. K. marxianus is a promising candidate with considerable potential for SCP biosynthesis and thus extensively employed as a feed organism in many countries. Compared to S. cerevisiae, K. marxianus displays a high specific growth efficiency and highest biomass yield in aerobic continuous fermentation. Yadav et al. (2014a) utilized K. marxianus GQ 506972 to produce SCP with a simultaneous COD elimination of cheese whey. They achieved 6 g/L of biomass along with over 50% removal of COD at pH 3.5 and 40°C after 36 h of batch fermentation. Increase in the inoculum level led to increased biomass production of 15 g/L and 80% removal of COD. The same authors also appraised the efficiency of the mixed culture system for obtaining high-quality SCP and enhancing the COD removal in continuous and batch fermentations of whey under cultivation conditions of pH 3.5 and 40°C (Yadav et al., 2014b). The coculture (C. krusei and K. marxianus) gives rise to 19% greater biomass yield and 33% higher productivity with concurrent 8.8% elimination of COD than the single culture in the batch system. The SCP produced from coculture contained essential amino acids and a desired protein content, indicating a mixed culture prodigious approach for producing SCP and wastewater treatment (Yadav et al., 2014a; 2014b). Yadav et al. (2016) produced food-grade SCPs at pH 6.5 and 30°C using a coculture system of S. cerevisiae and K. marxianus. In comparison to single culture (84%), the mixed culture resulted in 92% of total whey proteins under the optimal conditions. Many microorganisms including K. marxianus, C. kefyr, and S. cerevisiae have been grown in the food wastes via SSF. Among these, K. marxianus contains the maximum concentration of fat and protein, and thus might be utilized to enrich livestock feed (Aggelopoulos et al., 2014).

Probiotics are the living microorganisms that confer health benefits on the host by administrating inadequate amounts (Hill et al., 2014). These include Lactobacillus, Clostridia, Bifidobacterium, Enterococcus, Faecalibacterium, and Propinonibacterium (Altieri, 2016). Although S. cerevisiae var. boulardii has also been considered a probiotic since many years (Moré and Swidsinski, 2015), growing interest is diverted in recent years to explore the probiotic potential of non-conventional yeasts. K. marxianus is regarded as a promising probiotic due to its modification capacity in the adhesion, cell immunity, and human gut microbiota. In addition, it also possesses anti-inflammatory, antioxidant, and hypocholesterolemic features (Xie et al., 2015; Cho et al., 2018). It might be able to survive in the digestive tract reaching the intestines to serve as prebiotics due to acid and bile tolerance present in the gastrointestinal environment. K. marxianus isolated from kefir presented cholesterol-lowering capability, reducing 30% of cholesterol even greater than K. lactis ATCC 34440 and S. cerevisiae ATCC 6037 (Cho et al., 2018). K. marxianus AS4, obtained from dairy products, that is, cheese and yogurts, has shown excellent resistance to acidic pH, high bile salts, and superior antimicrobial activity. It also revealed outstanding anticancer activity against gastrointestinal tumor cells due to the secretion of biologically active metabolites, downregulating Bcl-2 gene, upregulating a pro-apoptotic protein, and BAD and CASP9 gene expression system (Saber et al., 2017). Among various yeast strains with probiotic efficiency, K. marxianus JYC2614 showed the best tolerance to bile salt and acid, as well as good cell surface hydrophobicity and satisfactory auto-aggregation (Hsiung et al., 2021). Recently, Goktas et al. (2021) determined the probiotic properties of yeast species isolated from kefir samples and compared with a commercial probiotic yeast S. boulardii. In contrast to S. boulardii, Kefir isolates, K. marxianus and S. cerevisiae, showed hydrophobicity, high auto-aggregation, and antioxidant potentialities. They also demonstrated potent antifungal activities against some mold species. Motey et al. (2020) assessed the probiotic characteristics of K. marxianus and S. cerevisiae, which were isolated and identified from the West African milk products nunu, lait caillé, and a cereal-based product mawè. Isolates were capable of growing in bile salts with the highest specific growth rate (μ_max) of 0.58–1.50 hour⁻¹. Adhesion of yeast to Caco-2 cells was found to be strain-specific varying between 8.0 and 36.2%. At the gastrointestinal pH environment, S. cerevisiae and K. marxianus also maintained the pH_i homeostasis and increased transepithelial electrical resistance across the Caco-2 monolayers, indicating their appreciable probiotic efficiency in West African fermented products. In another work, K. marxianus PTCC = 5189 was utilized for the production of probiotic yoghurt, which was then contaminated with Penicillium notatum PTCC = 5074 and Aspergillus parasiticus PTCC = 5018. The contaminants and changes in the count of probiotic yeasts during the storage at 4°C at 28 days were assessed and subjected to comparison with the control. Because of K. marxianus, the numbers of yoghurt adulterating molds were significantly reduced. K. marxianus population was satisfactory after 28 days of storage for providing a probiotic product with 7.35 log CFU/gr (p < 0.01), indicating its usefulness to produce probiotic yoghurt on an industrial scale (Oroojzadeh, 2020).

Wang et al. (2017) studied the impacts of K. marxianus addition on intestinal structure, immune responses, and growth attributes in broiler chickens. Experimental results revealed that increasing supplementation of K. marxianus resulted in reduced feed conversion efficiency but a linear improvement in relative thymus weight, increased serum IgG, and lysozyme levels. Better feed conversion rate and immune function along with intestinal structure in broilers by K. marxianus supplementation might be ascribed to the better structure of ileal microbes.

Fructose is a potential alternate sweetener to sucrose owing to its high sweeting capacity (1.5–2 times than sucrose) and could increase the absorption of iron in children. Fructo-oligosaccharides (FOS) are considered a prospective source of dietary fiber with bifidogenic effects. They are utilized as food ingredients owing to their health beneficial properties such as maintaining a good balance in the intestinal flora and promoting the multiplication of bifidobacterial community in the intestine. Recently, they have granted the “GRAS” status by the FDA (Flores-Gallegos et al., 2015). Both FOS and fructose can be produced through the enzymatic degradation of inulin; however, the synthesis was typically carried out at an elevated temperature (around 60°C) because of inadequate solubility of inulin at room temperature. Therefore, it is of great significance to characterize and isolate thermotolerant inulinases for catalyzing the hydrolysis of inulin at higher temperatures (Flores-Gallegos et al., 2015). Inulin is regarded as a good feedstock for producing inulo-oligosaccharides by endoinulinase-mediated catalytic reactions, but lack of solubility in cold water or slight solubility (only 5%) in water at 55°C remains a major challenge for its hydrolysis. This fact necessitates the exploration of inulinase that are capable of working at higher temperatures for industrial applicability. In this perspective, K. marxianus can be considered a robust candidate for FOS production. Struyf et al. (2017) developed a yeast-based approach by an inulinase-producing K. marxianus strain that resulted in over 90% fructan reduction in the final product, whereas only 56% fructan was reduced in the case of S. cerevisiae strain as a control. In order to develop a high-performing endo-inulinase for FOS biosynthesis from inulin, an inulin-binding module (IBM) was integrated into C- or N- terminal of the enzyme. The C-terminal fusion one (Eninu-IBM) with high activity, thermal stability, and inulin binding efficiency was used to catalyze inulin break down at high-temperature in a 10-L bioreactor. Results obtained high purity FOS (91.4%) with FOS titer, productivity, and yield of 717.3 g/L, 358.6 g/L/h and 0.912 gFOS/g inulin, respectively (Mao et al., 2019). Risso et al. (2012) investigated the FOS production by carrier-supported inulinase from K. marxianus NRRL Y-7571 in aqueous and aqueous-organic systems using sucrose as a substrate. In the case of an aqueous–organic system, the utmost yield of FOS (18.2 ± S0.9 wt%) was achieved at optimal condition of pH 5.0, 40°C, aqueous/organic ratio (25/75 wt%), enzyme activity (4 U/ml), and sucrose concentration of 60% (w/w), whereas the highest yield of FOS was achieved to be 14.6 ± 0.9 wt% for the aqueous system.

Various yeasts, including C. kefyr, Debaryomyces antarelli, and K. fragilis or K. marxianus have been used to produce inulinases (Beluche et al., 1980; Negoro and Kito, 1973; Holyavka et al., 2016). Among these, K. marxianus is preferred for fructose biosynthesis because of their growth on fructans, such as inulin; thus, inulinases can hydrolyze plant-originated fructans (Chi et al., 2011). A considerable amount of inulin is present in the tubers of various plants, such as dahlia, yacon, chicory, and Jerusalem artichoke. Biosynthesis of fructose by enzyme-assisted hydrolysis of plant extract-derived oligo- and polysaccharides by K. marxianus-immobilized inulinases could be a gainful strategy for large scale production of sugar (Holyavka et al., 2016). Singh et al. (2016) applied duolite A568-immobilized extracellular exoinulinase from K. marxianus YS-1 for the production of high-fructose syrup. The catalytic action of biocatalyst generated 39.2 and 40.2 g/L of fructose in 4 h using raw and pure inulins, respectively. In addition, inulinases secreted by K. marxianus using xylose medium is another appealing way for producing elevated titer of fructose syrup (Hoshida et al., 2018).

Porcine circovirus (PCV), belongs to the genus Circovirus, family Circoviridae, is among the smallest animal virus consisting of a single-stranded circular DNA (1.7–2 kb). Three genotypes of PCV include PCV1, PCV2, and PCV3. Among these, PCV1 was initially identified and characterized in infected PK15 cells (Tischer et al., 1974). This virus was considered non-pathogenic with widespread infection in all swine-producing areas (Allan et al., 2012). PCV2 is a pervasive kind of virus with considerable pathogenicity and closely related to the postweaning multisystemic wasting syndrome (PMWS), as well as other porcine circovirus diseases (PCVDs), such as respiratory disorder, enteric disease, reproductive failure, porcine dermatitis, and nephropathy syndrome (PDNS), resulting in massive economic losses in the swine industries (Chae 2005; Gillespie et al., 2009; Cruz et al., 2018). PCV3 is a new PCV genotype, which was initially recognized from piglets with cardiac pathology and multisystemic inflammation in the United States (Phan et al., 2016).

Vaccination is regarded as the best strategy for reducing the death rate and improving the cultivation of porcine circovirus disease-affected pig populations (Fachinger et al., 2008). PCV2 virus-like particles (VLPs) are a kind of subunit vaccine, which imitate PCV2 virus morphology and exhibit advantageous features over the inactivated vaccine and live-attenuated vaccines for their safety, non-replicating, and particulate structure (Wu et al., 2012). A number of host platforms, such as insect–baculovirus, E. coli, P. pastoris, S. cerevisiae, and silkworm pupae, have been explored and attempted to express CAP protein for obtaining PCV2 VLPs (Wu et al., 2012; Tu et al., 2013; Nainys et al., 2014; Masuda et al., 2018); however, the cost-effective production remain a major hindrance. Duan et al. (2019) reported PCV2 VLPs preparation by expressing PCV2d CAP protein in K. marxianus with a high yield of 1.91 g/L in a 5-L bioreactor. The yield obtained significantly surpassed the PCV2 VLPs achieved by E. coli, P. pastoris, and baculovirus–insect cell. The PCV2 VLPs was capable of inducing a high titer of anti-PCV2 IgG antibodies in mice serum together with decreasing the virus titers in spleens and livers of the tested mice. Spontaneously assembly of CAP protein into VLPs showed the prospects of cost-effective preparation of PCV2 vaccines. Recently, Yang et al. (2021) used K. marxianus to evaluate the expression and downstream processing of PPV VLPs. The VP2 protein from Kresse strain was subjected to expression following optimization by the codon bias of K. marxianus. After 48-h fermentation, the PPV VLPs yield reached 2.5 g/L in a 5-L fermenter. Two strategies, including cation exchange chromatography with Sephacryl^® S-500 HR chromatography and anion exchange chromatography following cross-flow diafiltration were used for the purification of VLPs from the supernatants. Administration of mice with purified PPV VLPs (95% purity) resulted in the induction of high titers of IgG antibodies in sera, showing hemagglutination inhibitions on both guinea and swine pig erythrocytes. Cytokine’s detection and spleen lymphocyte proliferation suggest K. marxianus-induced PPV VLPs triggered the humoral and immune immunity responses in mice.