Kluyveromyces marxianus : Current State of Omics Studies, Strain Improvement Strategy and Potential Industrial Implementation

: Bioethanol is considered an excellent alternative to fossil fuels, since it importantly contributes to the reduced consumption of crude oil, and to the alleviation of environmental pollution. Up to now, the baker yeast Saccharomyces cerevisiae is the most common eukaryotic microorganism used in ethanol production. The inability of S. cerevisiae to grow on pentoses, however, hinders its e ﬀ ective growth on plant biomass hydrolysates, which contain large amounts of C 5 and C 12 sugars. The industrial-scale bioprocessing requires high temperature bioreactors, diverse carbon sources, and the high titer production of volatile compounds. These criteria indicate that the search for alternative microbes possessing useful traits that meet the required standards of bioethanol production is necessary. Compared to other yeasts, Kluyveromyces marxianus has several advantages over others, e.g., it could grow on a broad spectrum of substrates (C 5 , C 6 and C 12 sugars); tolerate high temperature, toxins, and a wide range of pH values; and produce volatile short-chain ester. K. marxianus also shows a high ethanol production rate at high temperature and is a Crabtree-negative species. These attributes make K. marxianus promising as an industrial host for the biosynthesis of biofuels and other valuable chemicals.

In summary, the rapid development of Omics technologies helps to gain insight into transcriptomic and proteomic profiles of K. marxianus in response to stress conditions such as high temperature, high ethanol concentration or furfural, phenol inhibitors. Based on the gene expression patterns and/or protein abundance upon these harsh circumstances, best candidate genes could be selected for further detailed study or metabolic engineering to develop industrially relevant phenotypes.

Advanced Techniques in Kluyveromyces marxianus Strain Improvement
K. marxianus can transport various types of sugar, such as glucose [20], lactose [21], fructose [22], galactose [23], xylose [24], cellobiose, and arabinose [8], and organic acids, such as lactic acid [25] and malic acid [26], into the cells. However, the ability of K. marxianus to digest cellobiose was still very poor [8]. To improve the capability of metabolizing cellobiose for K. marxianus KY3, Chang et al. [8] transformed a rumen fungal β-glucosidase gene from Neocallimastix sp. W5 into its genome. Consequently, the transformant K. marxianus KY3-NpaBGS strain was able to use cellobiose better and produced approximately 1 g/L ethanol when growing on YP medium supplemented with 20 g/L cellobiose. In contrast, K. marxianus SSSJ-0, a native kefir yeast strain that possesses β-glucosidase enzyme, could only use cellobiose for cell growth, but was unable to convert cellobiose into ethanol.
Lignocellulosic biomass is an abundant and renewable resource for the production of biofuels and other value-added compounds [27]. Therefore, many efforts have been made to combine the high ethanol yield and the robust lignocellulose degradability into a single host cell for a consolidated bioprocessing (CBP). In this concept, the K. marxianus KY3 was engineered to be an artificial cellulolytic microbe with five cellulase genes including two exoglucanases (from Trichoderma reesei), two endoglucanases (from Aspergillus niger) and one β-glucosidase (from Neocallimastix patriciarum) transformed into the yeast genome using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) technique [28,29]. In addition, to facilitate the import of cellodextrin into the cells, a fungal cellodextrin transporter gene from the red bread mold Neurospora crassa was selected for genetic transformation. Consequently, the ethanol yield of the recombinant K. marxianus KR7 strain in YP medium with 10% (w/v) Avicel as the sole carbon source was~0.6 g/L. These foreign genes functioned properly in the host cell, reflecting via cellulolytic enzyme assay, cellodextrin transport, cellobiose digestion, and ethanol production. Although the conversion of Avicel to ethanol was not that efficient, the PGASO method proved its potential for practical applications, as it could assemble multiple exogenous genes into K. marxianus genome in one single step to facilitate enzyme combinations or to construct de novo desired pathways in K. marxianus host cell [28].
Hungateiclostridium thermocellum cellulosome, nature's largest cellulolytic machinery, accounts for the fastest growth rate of any bacterium on crystalline cellulose [30]. A cellulosomal enzyme contains a type I dockerin, which could interact with the type I cohesin of the central nonenzymatic scaffolding subnit CipA via type I dockerin-type I cohesin interaction. Due to the Lego-like architecture of cellulosome, each scaffolding subunit CipA, with nine type I cohesins on its structure, can carry simultaneously nine different cellolosomal enzymes. In turn, CipA, with its type II dockerin, enables the interaction with one of three surface anchoring proteins SdbA, Orf2p, or OlpB via type II dockerin-type II cohesin modules. Since the anchoring protein OlpB has seven type II cohesins, the interaction between CipA-OlpB can accommodate up to 9 × 7 = 63 cellolosomal enzymes in a single cellulosome complex. Up to now, several research groups have been sought to design cellulosome microbes that can express a full size of cellulosome structure instead of some individual cellulosomal genes called mini-cellulosomes [31][32][33][34][35][36]. Recently, the group of Anandharaj et al. [37] succeeded in developing an engineered K. marxianus host that can express a full size of H. thermocellum cellulosome on its cell surface. The engineered yeast, with its de novo powerful cellulosome, could efficiently degrade Avicel and phosphoric acid-swollen cellulose (PASC) to produce 3.09 g/L and 8.61 g/L of ethanol, respectively. This result could be recorded as the highest ethanol titer of any constructed yeast cellulosome thus far [37].
The thioredoxin/thioredoxin reductase (Trx/TrxR) system is widely present in yeast mitochondria and plays important roles in protecting yeast from ROS damages [16]. In the study of Gao et al. [16], they found that the gene encoding thioredoxin reductase (KmTrxR) in K. marxianus was upregulated under high substrate loading and aerobic conditions. To confirm the protective functions of K. marxianus Trx/TrxR system in other yeasts, two genes KmTRX and KmTrxR were transformed into the S. cerevisiae 280 host cell to create the KmTRX overexpression strain, the KmTrxR overexpression strain, and the double KmTRX-KmTrxR overexpression strain. The results showed that although the overexpression of a single KmTRX gene in S. cerevisiase 280 had adverse effect on the host cell, the overexpression of KmTrxR, in contrast, aided the host cell tolerate to lignocellulose-derived inhibitors, such as acetic and formic acids. Moreover, the double overexpression of two genes KmTRX and KmTrxR, with their synergistic effects, could improve ethanol yield, and shorten the lag phase of S. cerevisiae cell under the inhibitory effects of mixed chemicals such as acetic and formic acids and furfural (FAF) [38]. Additionally, in the study of Gao et al. [16], the KmTPX1 gene, which encodes peroxiredoxin, was found greatly upregulated under aerobic conditions and high inulin concentration. The gene KmTPX1 is homologous to Tsa1p gene in S. cerevisiae, which is involves in redox reactions to remove excess ROS like peroxides, to regulate the concentration of peroxides to protect cells from DNA damage and cell death [39]. Taking advantage of their previous finding, Gao et al. [40] constructed an overexpression vector which contained KmTPX1 gene and transformed it into S. cerevisiae cell. As expected, the overexpression of KmTPX1 in the transformant S. cerevisiae strain helped the yeast tolerate better to both oxidative stress and inhibitory compounds released from the degradation of lignocellulose. Consequently, the enhanced tolerance of S. cerevisiae to oxidative stress and furfural led to the overall higher rates of glucose consumption and ethanol fermentation in the transformant KmTPX1 strain compared with the control.
Based on the stress-related transcription factor (TF) profiles in S. cerevisiae in a prior study [41], Li et al. [42] performed a protein-protein BLAST to determine the stress-related TFs in K. marxianus. Subsequently, they carried out the genetic transformation of exogeneous stress-related TF derived from K. marxianus DMKU3-1042 into S. cerevisiae TSH3 cell to enhance the thermotolerance, growth and ethanol yield of S. cerevisiae TSH3. As a consequence, at elevated temperature (43 • C) and 104.8 g/L glucose, the transformant KmHSF1 and KmMSN2 S. cerevisiae strains yielded the final ethanol concentrations of 27.2 ± 1.4 g/L and 27.6 ± 1.2 g/L, respectively, much higher than the control with 18.9 ± 0.3 g/L ethanol. When looking into details, the transcriptomic profiles of these transgenic S. cerevisiae strains revealed that the KmHsf1 gene improved ethanol production by regulating transporter-related genes in the host cell to limit the excessive ATP consumption and by promoting glucose uptake, whereas the KmMsn2 gene might aid in regulating glucose metabolism and glycolysis/gluconeogenesis. In addition, KmMsn2 promoted the host cell tolerate better to high temperature by regulating genes involved in lipid metabolism, thereby changing membrane fluidity. These above studies exemplify excellently a straightforward procedure from transcriptomic or proteomic studies to the selection of candidate genes for genetic transformation or other technologies for the improvement of microbial biofuel microorganisms.
Recently, in the study of da Silveira et al. [43], ethanol-tolerant K. marxianus CCT 7735 strains were developed using the Adaptive laboratory evolution (ALE) strategy [44]. Briefly, hundreds of generations of K. marxianus were exposed to 4% (v/v) ethanol, and the trained yeast cells were considered "ethanol tolerant" when a significant increase (>50%) in the specific growth rate was observed. In the evolved ethanol-tolerant K. marxianus ETS4 strain, the intracellular amine/amide compounds and organic acids abundance were higher than those in its parent strain P4 under ethanol stress. The membrane fatty acid and ergosterol, an important sterol in yeast membranes, which is responsible for ethanol tolerance trait [45], were more abundant in the evolved strain ETS4 than in the P4 strain. This phenotype was in accordance with a INDEL mutation in the upstream region of the coding sequence (CDS) detected in the RRI1 gene which is involved in the positive regulation of ergosterol biosynthesis. Likewise, two genes KLMA_10136 and PXA, which are associated with lipid metabolic process, had mutations as follows: INDEL in the upstream region of KLMA_10136 CDS and INDEL in the downstream region of PXA CDS, respectively. Additionally, the accumulations of valine and metabolites of the TCA cycle such as isocitric acid, citric acid, and cis-aconitric acid were recorded only in the ETS4 strain when exposed to ethanol. This might contribute to an increase in ethanol tolerance of the evolved strain.
The TATA-binding protein (TBP) Spt15, one of the components of the general factor RNA polymerase II (RNA Pol II) transcription factor D (TFIID), is the most common target of yeast for global transcription machinery engineering (gTME) technique [46,47]. This technique could induce the global perturbations of the transcriptome through mutagenesis of key proteins that regulate the global transcriptome, thereby improving complex phenotypes quickly and effectively [47]. In the study of Li et al. [48], the SPT15 gene was subjected to error-prone PCR, cloned into an expression vector and, then, pooled recombinant plasmids were transformed into K. marxianus to construct a random mutagenesis library in its cells. The results of mutant screening under 6% (v/v) ethanol stress showed that two mutant strains M2 and M10 demonstrated faster growth rates than others. Regarding ethanol productivity, M2 strain performed better compared with M10 and control strain (i.e., M2 produced 57.29 ± 1.96 g/L ethanol, which was 23.74% and 22.05% higher than those of M10 and the control, respectively). Moreover, the M2 strain also tolerated to high ethanol concentration better than M10 and the control, e.g., its ethanol inhibition concentration (EIC) value was 57 g/L, much higher than that of M10 and the control with 46 and 47 g/L, respectively. As a global transcriptome regulator, a non-synonymous (Non-Syn) mutation (Lys was substituted by Glu 31 ) in the Spt15 gene could influence the expression patterns of hundreds of genes including those involved in the central carbon metabolism, amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway (upregulated) and also ribosome biosynthesis, translation and protein synthesis (downregulated). From this perspective, the gTME method could be used for the improvement of other complex phenotypes, such as furfural tolerance or thermotolerance in K. marxianus.
Despite several advantageous traits for industrial applications, however, genetic engineering approaches for K. marxianus strain improvement have been still limited since the genome-editing tools and stable heterologous expression systems for this yeast species have not well-established yet [49]. In the study of Löbs et al. [50], CRISPR-Cas9 system, which was adapted from Streptococcus pyogenes, was used to create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes in K. marxianus. The study aimed to investigate the metabolic pathways that are involved in the ethyl acetate and ethanol biosynthesis. In industry, ethyl acetate is used as a solvent and as flavor and fragrance compound and its worldwide demand is~1.7 million tons per year [51]. The data from Löbs et al. report showed that the knockout of KmAtf gene reduced the production of ethyl acetate by 15%, whereas the disruption of KmAdh2 gene almost entirely abolished the production of ethanol, resulting in the accumulation of acetaldehyde. The data obtained from KmADH2 and KmATF knock-out strains indicated the fundamental role of KmAdh2 gene in ethanol production in both aerobic and anaerobic conditions. In regard to ethyl acetate biosynthesis, KmADH2 played a role in providing ethanol as a substrate for the reaction of Atf-catalyzed condensation with acetyl-CoA. Since the disruption of KmAtf gene only reduced a little amount of ethyl acetate, it suggested that probable alternative metabolic routes might take responsibility for the biosynthesis of ethyl acetate in K. marxianus.

Mono-, Co-Culture Systems and Other Fermentation Process Configurations in Bioethanol Production Using K. marxianus
Many fermentation approaches have been widely investigated to improve the productivity of bioethanol, thereby reducing the cost of industrial operation [28,35,37,48, (Table 2). A compatible co-culturing strategy could leverage the useful features from different microbes, thereby improving the productivity relative to monocultures [76]. Since an ideal microbe for consolidated bioprocessing (CBP) still remains to be found, the co-culture of engineered microorganisms, which confer newly advantageous genetic traits on microbes, would be a good approach for biofuels production. As numerous studies have been published, we just took few examples to clarify this concept. In the study of Ho et al. [77], a recombinant cellulosomal Bacillus subtilis which carried eight genes from H. thermocellum, namely one scaffolding protein gene (cipA), one cell-surface anchoring gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ) was cultured with a recombinant K. marxianus KY3-NpaBGs carrying a β-glucosidase gene from rumen fungus in the YP medium supplemented with 20 g/L Napier grass as the sole carbon source. At 42 • C, the dual-microbe co-culturing yielded 3.28 g/L, indicating the potential of K. marxianus as a complementary partner for bioprocessing. In this dual K. marxianus-B. subtilis system, the engineered B. subtilis was responsible for cellulolytic hydrolysis via its complex heterologous cellulosomal enzymes and the engineered K. marxianus, in turn, helps to convert the resultant cellobiose into glucose via secretory β-glucosidase. The study of Guo et al. [54], used cheese whey powder (CWP), a by-product of cheese industry, which contains high concentration of lactose and other essential nutrients for co-culturing S. cerevisiase and K. marxianus. As S. cerevisiase cannot ferment lactose but K. marxianus can, the co-culturing strategy was applied to make use of the carbon source and nutrient availability in CWP to produce ethanol. In addition, the mixed and alginate-immobilized cells produced higher ethanol yield relative to the free cell cultures. To enhance ethanol production and thermotolerance of yeast cells, the immobilized cocultures of K. marxianus DMKU 3-1042 and S. cerevisiae M30 on thin-shell silk cocoons (TSC) and alginate-loofa matrix (ALM) were carried out by Eiadpum et al. [55]. At high temperatures (range of 40-45 • C), both monoculture and coculture performed better than the monoculture of S. cerevisiae in producing ethanol. TSC and ALM functioned as yeast cell carriers and might protect cells from adverse conditions like high concentration of inhibitors or elevated temperatures [78]. On average, TSC-immobilized cell system yielded 16% higher ethanol production than ALM-immobilized cell system. This might be due to the high biocompatibility, high mechanical strength, light weight, high surface area, and proper porous structure of TSC that provided a convenient growth environment for yeast cells to live and to produce ethanol [78]. However, in a mixed culture, the cells-cells interaction between different strains is an important issue that should be taken into consideration. Differences in growth rates, nutrient uptake rates and secreted metabolites might be probable factors that affect cell viability [79]. In addition, killer toxins and extracellular proteases synthesized by yeasts may be another matter of mixed fermentation, as these toxic compounds might function against their coculture partners [80]. As both S. cerevisiae and K. marxianus could produce killer toxins [80], they might exclude each other in specific circumstances. In the study by Lopez et al. [79], the viability loss of K. marxianus was recorded in mixed culture conditions. Moreover, in the direct contact mixed culture, S. cerevisiae was also unfavorably affected. Table 2. Monoculture, co-culture of K. marxianus with other microbes and other fermentation processes for bioethanol production.

Strain
Growth Condition

Studies of Crabtree Effect in K. marxianus
Crabtree effect is the repression of respiration in aerobic glucose excess conditions and this effect is believed to play roles in a competition mechanism as it allows yeasts to growth rapidly and produce ethanol in such conditions [81]. This evolution feature promotes the rapid use of glucose and the production of ethanol, an antimicrobial chemical, resulting in the advantage of Crabtree-positive species over other microorganisms in its ecological niche. However, in the context of microbial production of biofuels and chemicals, this feature also hinders yields when the Crabtree-positive yeasts would be used as cell factory platforms to produce other chemicals than ethanol [82]. Consequently, it is of interest in abolishing this effect in Crabtree-positive species. The disruption of genes encoding puruvate decarboxylase in S. cerevisiae completely eliminated the Crabtree effect, however, it caused the growth deficiency of mutant strains in excess glucose condition [83]. Recently, Dai et al. [81] succeeded in turning the Crabtree-positive property of S. cerevisiae into Crabtree-negative by using systematic engineering. In the study of Sakihama et al. [84], under anaerobic condition and 5.5 g/L glucose, the Crabtree-negative species K. marxianus had significantly increased metabolite abundances in glycolysis (e.g., phosphoenol pyruvate, isocitrate, 2-ketoglutarate, succinate, malate, and fumarate), especially pyruvate with 4.5-fold higher than that in aerobic condition. Furthermore, under anaerobic condition, the transcript abundances of genes involved in glycolysis in K. marxianus were higher than those in aerobic condition, in accordance with their metabolic profiles. In contrast, the pool sizes of metabolites in aerobic condition (e.g., fructose 6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, acetyl-CoA, isocitrate, fumarate, fructose 1,6-bisphosphate, glucose 6-phosphate, and dihydroxyacetone phosphate) in S. cerevisiae, a Crabtree-positive yeast, were higher than those under anaerobic condition, suggesting the incline of these metabolites toward glycolytic route in the presence of oxygen. Concerning biomass production, under aeration, K. marxianus cell density was 2.9-fold higher than that of itself in anaerobic condition and 2.2-fold higher than that of S. cerevisiae in aerobic cultivation. The cell density of S. cerevisiae in aerobic culture (OD 600~5 4), however, was not much higher than that in the anaerobic culture (OD 600~4 6), suggesting a slow growth rate when the Crabtree effect occurred. Regarding ethanol and acetate productions, under aerobic conditions, S. cerevisiae reached an ethanol titer of 22.1 g/L and acetate titer of 1.3 g/L, while K. marxianus only produced 5 g/L ethanol and 0.5 g/L acetate in the same circumstances. These data were consistent with the previous study of Wardrop et al. [85], as they also found that K. marxianus yielded higher biomass than S. cerevisiae but produced a lower ethanol concentration (0.4 g/L vs. 6 g/L of S. cerevisiae) in glucose pulse treatment (sudden increased glucose from 1 g/L to 50 g/L). In addition, the oxygen uptake in S. cerevisiae immediately declined after glucose upshift, whereas the increase in oxygen uptake in such circumstances was recorded in K. marxianus, indicating the maintenance of respiratory activity in the Crabtree-negative yeast. These data indicate the fundamental differences between Crabtree-negative and Crabtree-positive species in aerobic culture with high concentration of glucose

Conclusions
The non-conventional yeast K. marxianus has been proved to be a promising eukaryotic microbe for bioethanol production and other food and environmental applications. Although having several useful traits that are suitable for bioethanol production at an industrial scale, its genetic drawbacks, such as the sensitivity to high concentration of ethanol or the incapability of growing on polysaccharides should be improved to meet the demands of industrial fermenting yeast strains. In addition, despite a lot of efforts having been deployed for constructing a robust K. marxianus strain appropriate for CBP, the ethanol production of these current engineered strains was still modest. Up to now, the highest ethanol concentration produced by an engineered K. marxianus is only 8.61 g/L, too low for any practical consideration.