Marxianus Strains Research Articles

Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology.

Kluyveromyces marxianus is an emerging non-conventional food-grade yeast that is generally isolated from diverse habitats, like kefir grain, fermented dairy products, sugar industry sewage, plants, and sisal leaves. A unique set of beneficial traits, such as fastest growth, thermotolerance, and broad substrate spectrum (i.e., hemi-cellulose hydrolysates, xylose, l-arabinose, d-mannose, galactose, maltose, sugar syrup molasses, cellobiose, and dairy industry) makes this yeast a particularly attractive host for applications in a variety of food and biotechnology industries. In contrast to Saccharomyces cerevisiae, most of the K. marxianus strains are apparently Crabtree-negative or having aerobic-respiring characteristics, and unlikely to endure aerobic alcoholic fermentation. This is a desirable phenotype for the large-scale biosynthesis of products associated with biomass formation because the formation of ethanol as an undesirable byproduct can be evaded under aerobic conditions. Herein, we discuss the current insight into the potential applications of K. marxianus as a robust yeast cell factory to produce various industrially pertinent enzymes, bioethanol, cell proteins, probiotic, fructose, and fructo-oligosaccharides, and vaccines, with excellent natural features. Moreover, the biotechnological improvement and development of new biotechnological tools, particularly CRISPR–Cas9-assisted precise genome editing in K. marxianus are delineated. Lastly, the ongoing challenges, concluding remarks, and future prospects for expanding the scope of K. marxianus utilization in modern biotechnology, food, feed, and pharmaceutical industries are also thoroughly vetted. In conclusion, it is critical to apprehend knowledge gaps around genes, metabolic pathways, key enzymes, and regulation for gaining a complete insight into the mechanism for producing relevant metabolites by K. marxianus.

Isolation and Identification of Lactose-Degrading Yeasts and Characterisation of Their Fermentation-Related Ability to Produce Ethanol

Kefir is traditionally produced by fermenting cow’s milk using kefir grains as a starter culture. As the viability of microbes within kefir grains is limited and preparing the grains for kefir fermentation is laborious, here, a single starter that ferments lactose and produces ethanol is developed. For this purpose, it is important to isolate yeasts that can ferment lactose and subsequently produce alcohol. This study aimed to isolate and identify yeasts from kefir and characterise their ability as single starters to produce kefir. Based on morphological and physiological evaluations, 15 presumptive yeast isolates were obtained, 10 of which grew well on lactose-containing media. Those that were able to grow on lactose using only carbon sources were subjected to molecular identification based on the internal transcribed spacer (ITS) of the 5.8 rDNA using PCR technology. Molecular identification confirmed four isolates—namely, KFA 3, KFA 7, KFA 9 and KFB 1—as belonging to Kluyveromyces marxianus. The batch fermentation data of these strains were fitted on a logistic model to obtain the carrying capacity coefficients and strain performances were compared. The kinetic modelling revealed that KFA 9 had the highest values for the carrying capacity coefficient, biomass yield and product yield, indicating that, among the four K. marxianus strains, this was superior due to its relatively fast growth and good ethanol productivity.

Comparative metabolome and transcriptome analyses of the properties of Kluyveromyces marxianus and Saccharomyces yeasts in apple cider fermentation

This study explored the application of Kluyveromyces marxianus and Saccharomyces cerevisiae (commercial and wild type) in the alcoholic fermentation of Fuji apple juice under static conditions. Metabolome analyses revealed that ethyl esters, including ethyl hexanoate, ethyl decanoate, ethyl octanoate, octanoic acid and decanoic acid, were the dominant components in ciders fermented by the Saccharomyces yeasts. In the K. marxianus ciders, ethyl acetate, hexyl acetate, propyl acetate and acetic acid were the most abundant volatiles, suggesting that the cider fermented by K. marxianus might have a fruitier smell. Transcriptome analyses were adapted to gain insight into the differential metabolite patterns between K. marxianus and S. cerevisiae during cider fermentation. GO and KEGG enrichments revealed that the metabolic pathways of glucose, organic acids and amino acids during cider fermentation were quite different between these two yeasts. The K. marxianus strain exhibited a higher rate of glycolysis and ethanol fermentation than did Saccharomyces yeasts under oxygen-limited conditions. It also reduced the metabolic flux of acetate into acetyl-CoA and then into the TCA cycle, increasing the syntheses of ethyl acetate and relevant esters, which may affect its cell growth under anaerobic conditions but enriched the taste and variety of aromas in apple cider.

Survivability of Kluyveromyces marxianus Isolated From Korean Kefir in a Simulated Gastrointestinal Environment.

Kluyveromyces marxianus accounts for > 90% of the yeast population of kefir, and recently, its probiotic potential has been actively explored with a focus on its health benefits and safety. Herein, the survivability of five kefir-isolated K. marxianus strains (Km A1–A5) in a simulated gastrointestinal (GI) environment was evaluated and compared with those of commercial probiotic yeast, Saccharomyces boulardii MYA-796. To further explore the potential to survive in the host GI tract, biochemical activities, hydrophobicity assay, biofilm formation, auto-aggregation analysis, and phenol tolerance of the strains were assessed. K. marxianus A4 exhibited the best survivability among all tested strains, including the clinically proven probiotic yeast strain S. boulardii MYA-796 (p = 0.014) in the artificial GI tract ranging from pH 2.0 to 7.5. In addition, the five K. marxianus strains and S. boulardii MYA-796 displayed different assimilation of lactose, xylitol, D-sorbitol, and DL-lactate, indicating that K. marxianus metabolized a wide range of substances and, thus, might be more feasible to nourish themselves in the host GI tract for survival. K. marxianus strains showed a greater hydrophobicity of cell surface, abilities to biofilm formation and auto-aggregation, and phenol tolerance than S. boulardii MYA-796, suggesting greater potential for survival in the host GI tract.

Optimization of fermentation for γ-aminobutyric acid (GABA) production by yeast Kluyveromyces marxianus C21 in okara (soybean residue).

γ-Aminobutyric acid (GABA) is a non-protein amino acid with a variety of physiological functions. Recently, yeast Kluyveromyces marxianus strains involved in the catabolism and anabolism of GABA can be used as a microbial platform for GABA production. Okara, rich in nutrients, can be used as a low-cost fermentation substrate for the production of functional materials. This study first proved the advantages of the okara medium to produce GABA by K. marxianus C21 when L-glutamate (L-Glu) or monosodium glutamate (MSG) is the substrate. The highest production of GABA was obtained with 4.31g/L at optimization condition of culture temperature 35°C, fermentation time 60h, and initial pH 4.0. Furthermore, adding peptone significantly increased the GABA production while glucose and vitamin B6 had no positive impact on GABA production. This research provided a powerful new strategy of GABA production by K. marxianus C21 fermentation and is expected to be widely utilized in the functional foods industry to increase GABA content for consumers as a daily supplement as suggested.

Exploring the phenotypic diversity of oenological traits in Kluyveromyces marxianus strains.

The use of non-Saccharomyces yeasts in the winemaking process may have several positive outcomes. Kluyveromyces marxianus has recently been revealed as a promising species for this industry. While the majority of studies mention the use of K. marxianus in various industries including food production (e.g. dairy and cocoa), recent studies have also shown that its aroma and pectinase production make it a suitable yeast for the wine industry. Nevertheless, only particular strain, IWBT Y885, was investigated. In this study, five different K. marxianus strains as well as one protoplast fusant (BF2020) were compared to strain Y885. These comparisons focused on various oenological traits such as fermentation performance, fermentation metabolites, hydrogen sulfide, and pectinase production. Throughout the study, variations were found between the K. marxianus strains investigated. Indeed, although common traits such as high pectinase activity appeared conserved among K. marxianus strains, a fairly large phenotypic diversity was also evident. Using cluster analysis, strain groupings emerged with strains L01, L05, Y885, and BF2020 grouping together. Similarly, strains L02 and L04 grouped together while strain L03 appearing to show the most variation between the strains investigated. Variation between strains was observed regardless of the original source of isolation.

A Combined In Vitro and In Vivo Assessment of the Safety of the Yeast Strains Kluyveromyces marxianus A4 and A5 Isolated from Korean Kefir.

Kefir is a traditional fermented milk containing beneficial bacteria and yeasts. Despite Kluyveromyces marxianus, isolated from kefir, gaining increasing attention as a potential probiotic yeast owing to its biological function, Saccharomyces boulardii is the only species considered as a probiotic yeast. We evaluated the safety of K. marxianus strains A4 and A5, isolated from Korean kefir, in comparisonwith that of S. boulardii. Virulence attributes were preliminarily assessed in vitro including their ability of gelatin hydrolysis, pseudohyphae formation, and hemolysis. To evaluate in vivo safety, the strains were challenged in a healthy animal model, four-week-old female BALB/c mice. Mice were orally administered 0.2mL of 0.9% sterilized saline (NC_S; n = 6), S. boulardii ATCC MYA-796 (high concentration, S.b_H; low concentration, S.b_L; n = 6 for each), K. marxianus A4 (high concentration, A4_H; low concentration, A4_L; n = 6 for each), or K. marxianus A5 (high concentration, A5_H; low concentration, A5_L; n = 6 for each) for 2weeks. At study end, body weight, spleen and liver weights, and blood parameters were assessed. K. marxianus A4 and A5 were tested negative for gelatinase and hemolysis. Overall, hematological, plasma biochemical, and cytokine (interleukin-1β and tumor necrosis factor-α) parameters were comparable between the experimental and negative control (NC) groups. Notably, the interleukin-6 level of the A5_H group was significantly lower than that of the NC group (p < 0.05), suggesting anti-inflammatory potential of K. marxianus A5.

Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.

ABSTRACTThere is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.

A Single Vaccination of IBDV Subviral Particles Generated by Kluyveromyces marxianus Efficiently Protects Chickens against Novel Variant and Classical IBDV Strains

Infectious bursal disease (IBD), caused by the infectious bursal disease virus (IBDV), is a highly contagious and immunosuppressive disease in chickens worldwide. The novel variant IBDV (nvIBDV) has been emerging in Chinese chicken farms since 2017, but there are no available vaccines that can provide effective protection. Herein, the capsid protein VP2 from nvIBDV strain FJ-18 was expressed in Kluyveromyces marxianus with the aim to produce nvIBDV subviral particles (SVPs). Two recombinant strains constructed for expression of nvIBDV VP2 (nvVP2) and His-tagged VP2 (nvHVP2) formed two types of nvIBDV subviral particles (SVPs), namely nvVP2-SVPs and nvHVP2-SVPs. TEM scans showed that both SVPs were about 25 nm in diameter, but there was a large portion of nvVP2-SVPs showing non-spherical particles. Molecular dynamics simulations indicate that an N-terminal His tag strengthened the interaction of the nvHVP2 monomer and contributed to the assembly of SVPs. Vaccination of chicks with the nvHVP2-SVPs provided 100% protection against novel variant IBDV infection when challenged with the FJ-18 strain, as well as the classical strain BC6/85. By contrast, vaccination with the nvVP2-SVPs only provided 60% protection against their parent FJ-18 strain, suggesting that the stable conformation of subviral particles posed a great impact on their protective efficacy. Our results showed that the nvHVP2-SVPs produced by the recombinant K. marxianus strain is an ideal vaccine candidate for IBDV eradication.

Establishment of Kluyveromyces marxianus as a Microbial Cell Factory for Lignocellulosic Processes: Production of High Value Furan Derivatives.

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans’ inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1.

Characterization of Yeasts Isolated from Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization.

Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

Diploid genome assembly of Kluyveromyces marxianus NRRL Y-50883 (SLP1).

The yeast Kluyveromyces marxianus SLP1 has the potential for application in biotechnological processes because it can metabolize several sugars and produce high-value metabolites. K. marxianus SLP1 is a thermotolerant yeast isolated from the mezcal process, and it is tolerant to several cell growth inhibitors such as saponins, furan aldehydes, weak acids, and phenolics compounds. The genomic differences between dairy and nondairy strains related to K. marxianus variability are a focus of research attention, particularly the pathways leading this species toward polyploidy. We report the diploid genome assembly of K. marxianus SLP1 nonlactide strain into 32 contigs to reach a size of ∼12 Mb (N50 = 1.3 Mb) and a ∼39% GC content. Genome size is consistent with the k-mer frequency results. Genome annotation by Funannotate estimated 5000 genes in haplotype A and 4910 in haplotype B. The enriched annotated genes by ontology show differences between alleles in biological processes and cellular component. The analysis of variants related to DMKU3 and between haplotypes shows changes in LAC12 and INU1, which we hypothesize can impact carbon source performance. This report presents the first polyploid K. marxianus strain recovered from nonlactic fermenting medium.

PSX-B-14 Properties of Kluyveromyces marxianus strains isolated from natural biotopes

Abstract Kluyveromyces marxianus yeast is often isolated from dairy products and used as probiotic agents in food. As a producer-yeast they must be genetically stable, have simple nutrient requirements, be effective in hostile conditions, grow on inexpensive nutrient media and be easy to store and distribute. Nineteen strains of K. marxianus isolated from natural biotopes were tested for their ability to grow on various carbon sources. For this, the YPD medium (1% yeast extract, 2% peptone, 2% dextrose, pH 5.5) was used with the addition of 20 g/L of one of the following carbon sources: glucose, sucrose, lactose, maltose, mannitol, arabinose, xylose, and galactose. The cultivation was carried out for 1–3 days. All isolates were also tested for their ability to grow in YPD broth at different temperatures (4, 27, 37, 40, 42, 46 and 48 °C) and pH (5; 3 and 2). K. marxianus strains showed widespread use of the substrate and high resistance to elevated incubation temperatures. All tested strains grew at 27 and 37 °C and one strain grew at 46 °C. None of the strains showed active growth at pH 2 and all cultures grew actively at pH 3. At the same time five strains showed growth at pH 5.0. All strains fermented galactose, 16 - xylose, 9 - glucose, 7 - lactose, 12 - maltose, 11 - arabinose, 5 - sucrose, 13 - mannitol. It should be noted that all strains in this study have showed fully restored growth after 2 hours’ incubation at pH 2. It indicates that they could survive when passing through the gastric passage into the intestine. These properties make the use of K. marxianus and its metabolites indispensable in agriculture as a feed additive. This work was supported by the Ministry of Science and Higher Education of Russia.

Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

Augmented ethanol production from alkali-assisted hydrothermal pretreated cassava peel waste

ABSTRACT Cassava peel is a by-product produced at large amounts in starch-based agro industries and could be a major feedstock for bioethanol in most of the Asian countries. In this work, we aimed to evaluate two fermentation strategies for bioethanol production from alkali-assisted hydrothermal pretreated cassava peel by thermotolerant Kluyveromyces marxianus MTCC 4139 strain. Initially, milled cassava peel was subjected to alkali-assisted hydrothermal pretreatment at different autoclaving temperatures (110°C − 130°C) and residence times (15 − 30 min) in the presence of different NaOH concentrations (0.25%−1.0% w/v NaOH). On subsequent enzymatic hydrolysis of pretreated cassava peel (1% NaOH at 120°C for 30 min) with different optimal enzyme loadings (α-amylase, glucoamylase and cellulase) showed maximum 670.58 ± 10.13 mg/g reducing sugar yield with 81.25% ± 3.20% saccharification efficiency. Furthermore, SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation) of cassava hydrolyzates were studied for improved ethanol yield and fermentation efficiencies. SSF of hydrothermal pretreated cassava hydrolyzate with K. marxianus strain showed higher ethanol yield (0.44 g/g) with 86.11% fermentation efficiency as compared to SHF process (0.41 g/g; 80.24% fermentation efficiency).

A biorefinery concept for the production of fuel ethanol, probiotic yeast, and whey protein from a by-product of the cheese industry.

Agroindustrial by-products and residues can be transformed into valuable compounds in biorefineries. Here, we present a new concept: production of fuel ethanol, whey protein, and probiotic yeast from cheese whey. An initial screening under industrially relevant conditions, involving thirty Kluyveromyces marxianus strains, was carried out using spot assays to evaluate their capacity to grow on cheese whey or on whey permeate (100 g lactose/L), under aerobic or anaerobic conditions, in the absence or presence of 5% ethanol, at pH 5.8 or pH 2.5. The four best growing K. marxianus strains were selected and further evaluated in a miniaturized industrial fermentation process using reconstituted whey permeate (100 g lactose/L) with cell recycling (involving sulfuric acid treatment). After five consecutive fermentation cycles, the ethanol yield on sugar reached 90% of the theoretical maximum in the best cases, with 90% cell viability. Cells harvested at this point displayed probiotic properties such as the capacity to survive the passage through the gastrointestinal tract and capacity to modulate the innate immune response of intestinal epithelium, both in vitro. Furthermore, the CIDCA 9121 strain was able to protect against histopathological damage in an animal model of acute colitis. Our findings demonstrate that K. marxianus CIDCA 9121 is capable of efficiently fermenting the lactose present in whey permeate to ethanol and that the remaining yeast biomass has probiotic properties, enabling an integrated process for the obtainment of whey protein (WP), fuel ethanol, and probiotics from cheese whey.Key points• K. marxianus-selected strains ferment whey permeate with 90% ethanol yield.• Industrial fermentation conditions do not affect selected yeast probiotic capacity.• Whey permeate, fuel ethanol, and probiotic biomass can be obtained in a biorefinery.

Technology valuation of cellulosic ethanol production by Kluyveromyces marxianus CCT 7735 from sweet sorghum bagasse at elevated temperatures

Technology valuation of cellulosic ethanol production by Kluyveromyces marxianus CCT 7735 based on the concepts of circular economy was investigated. After an acid-base pretreatment followed by saccharification at 50 °C for 72 h, the sweet sorghum hydrolysate was used to produce ethanol at three temperatures: 37, 42, and 45 °C. About 17.83 g/L of cellulosic ethanol was reached at 42 °C in 24 h, with a yield of 2769.8 L/hasorghum, almost double that obtained for corn straw. This was the best cellulosic ethanol production from sweet sorghum bagasse achieved by K. marxianus strain and, therefore, it was used in the technology valuation. This analysis was carried out through the application of the profit equation, in which the revenue corresponded to ethanol sales in the global market in 2020, and the costs were defined by the activity-based costing method. An integration between 1G and 2G ethanol production, without mixing material streams, was considered to improve the technology valuation analysis. As a result, the equivalent to 17.2% of the profit obtained with the sugarcane juice ethanol was achieved for the sweet sorghum bagasse. Therefore, the technology valuation indicated the potential of sweet sorghum bagasse in the biorefinery ethanol production.

Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Virulence related traits in yeast species associated with food; Debaryomyces hansenii, Kluyveromyces marxianus, and Wickerhamomyces anomalus

There is an increase in the number of human infections associated with yeast species that are also known to be involved in processing foods such as Saccharomyces cerevisiae, Debaryomyces hansenii, Wickerhamomyces anomalus, or Kluyveromyces marxianus. Clinical strains of S. cerevisiae have been well studied over the years and much is known regarding their virulence. However, very little is known about the virulent potential of strains of D. hansenii, W. anomalus, or K. marxianus. In this study, several factors related to virulence in a collection of clinical and food/environmental isolated strains of each species were determined. Physiological factors were looked at as well as parameters related to infection in an epithelial cell culture model and mortality in the invertebrate model Galleria mellonella. The results revealed that all of the evaluated K. marxianus strains (except for one strain) are positive in most physiological virulent factors, and also in the epithelial cell culture parameters. Wickerhamomyces anomalus strains produce 10 times higher levels of mortality in G. mellonella besides showed intermediate levels according to virulent factors. In the case of D. hansenii, all the tested strains are positive, but only in a single and different virulent parameter without a common pattern. Thus, our study reveals that some strains of K. marxianus and W. anomalus, could be a potential risk for human health and that the food/environmental strains could be the origin of infections.