Kluyveromyces Marxianus Research Articles

Brewer’s spent grain (BSG), the main lignocellulosic by-product of the beer industry, represents an abundant yet underutilized resource with high potential for valorization. This study presents an integrated biorefinery approach to convert BSG into second-generation (2G) ethanol, bioactive vinasse for plant growth promotion, and fungal biomass as a potential mycoprotein source. The biomass was first subjected to biological delignification using the white-rot fungus Ganoderma lucidum, after which two valorization routes were explored: (i) evaluation of the fungal biomass as a mycoprotein candidate and (ii) alcoholic fermentation for ethanol production. For the latter, three pretreatment strategies were assessed (diluted sulfuric acid and two deep eutectic solvents (DESs) based on choline chloride combined with either glycerol or lactic acid) followed by a one-pot enzymatic saccharification and fermentation using Kluyveromyces marxianus SLP1. The highest ethanol yield on substrate (YP/S) was achieved with [Ch]Cl:lactic acid pretreatment (0.46 g/g, 89.32% of theoretical). Vinasse, recovered after distillation, was characterized for organic acid content and tested on Solanum lycopersicum seed germination, showing promising biostimulant activity. Overall, this work highlights the potential of BSG as a sustainable feedstock within circular economy models, enabling the production of multiple bio-based products from a single residue.

Kluyveromyces marxianus fermenta tion improves tomato sensory profile, functional metabolites, lycopene bioaccessibility, and human fecal microbiota composition.

Dietary supplementation of hydrolyzed yeast from Kluyveromyces fragilis with or without high-dose zinc oxide modulates growth performance and diarrhea incidence of weaned pigs by improving intestinal health

Establishment of succinic acid production in recombinant Kluyveromyces marxianus enhanced by AI-guided enzyme specificity engineering and redox-controlled fermentation optimization

Endogenous pathway dysregulation in Kluyveromyces marxianus under nitrogen limitation drives 2-phenylethyl acetate and 2-phenylethanol overproduction

The economic feasibility of lignocellulosic ethanol production depends on obtaining high yields and productivity. This condition requires extensive research to identify strategies that maximize yield and productivity in lignocellulosic ethanol production. Self-cycling fermentation (SCF) has been shown to increase ethanol productivity. To validate whether the SCF system could enhance productivity during ethanol fermentation, we simulated the process using experimental results obtained from six cycles in a stirred bioreactor and compared the outcomes with separate hydrolysis and fermentation (SHF) and simultaneous saccharification-fermentation (SSF) for ethanol production from wheat straw using Kluyveromyces marxianus SLP1. The volumetric ethanol productivity (Qp) was 1.11g/Lh, 1.13g/Lh, and 0.42g/Lh for SCF, SHF, and SSF, respectively. The system with the highest global productivity (Qg) was SCF with 0.65g/Lh. Moreover, the estimated annual ethanol productivity for SCF (PSCF) improved by 21.7% compared with SHF and by 3.1-fold improvement over SSF. This result confirms that SCF can be an effective strategy for increasing productivity during lignocellulosic ethanol production. However, the SCF system is limited by the physiological states of the yeasts.

The yeast Kluyveromyces marxianus (K. marxianus), characterized by its thermotolerance and rapid growth, is emerging as a promising new platform organism for the production of recombinant proteins. In this study, we constructed an expression vector designed for the efficient expression of exogenous proteins in K. marxianus. Initially, qPCR was employed to assess the expression efficiency of endogenous promoters within the yeast. The PDC1 promoter was selected, and its ability to drive the expression of EGFP was validated. The constructed vector exhibited high stability, maintaining approximately 5.2-fold higher copy numbers than the K. marxianus genome after 72 hours of cultivation without hygromycin selection. Notably, the fluorescence signal intensity of K. marxianus harboring the vector was approximately 15.6-fold higher than that of the wild-type strain at 72 h. Subsequently, the cap gene of porcine circovirus type 3 (PCV3) was integrated into the vector, resulting in the production of soluble PCV3 cap protein. Electron microscopy analysis revealed that the PCV3 cap protein self-assembled into virus-like particles (VLPs). This study successfully established the expression vector and characterized its key elements in K. marxianus, which will facilitate further research on the expression of exogenous proteins in this yeast species. Moreover, the soluble expression of the PCV3 cap protein and its formation of VLPs provide a solid foundation for the future development of PCV3 vaccines.

This study was to determine the spoilage microbiota of palm wine stored at room temperature in order to define appropriate strategies for shelf-life extension. For this purpose, during the storage of palm wine samples, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) counts were performed. Then, species were identified by PCR-RFLP followed by sequencing and the overall bacterial community was determined by NGS (Next Generation Sequencing). The results showed that yeast, LAB and AAB counts decreased in the palm wines collected from producers after 24-48h, while they remained constant in samples collected from resellers. Furthermore, the stored palm wine microbiota was composed of several yeast species namely Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Candida parapsilosis and Brettanomyces bruxellensis. LAB species were Enterococcus durans, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Fructilactobacillus durionis, Lactobacillus parafarraginis and Lactiplantibacillus pentosus. For AAB, the species were Acetobacter oryzoeni and Acetobacter pasteurianus. The species whose proportions increased steadily during storage were B. bruxellensis, C. parapsilosis, Lc. paracasei, L. fermentum and A. oryzoeni. The NGS method confirmed that Lactobacillus was predominant during storage, followed by Zymomonas and Acetobacter. Knowledge of the microorganisms present during the spoilage of palm wine and their dynamics will enable the development of preservation methods specifically targeting the microorganisms identified.

Introduction: Cutaneous candidiasis is an infection caused by the yeast, Candida albicans or other Candida-related species. Recently, Candida infections of the skin have become more prevalent and could be attributed to an increase in the number of immunocompromised individuals. Kluyveromyces marxianus (formerly Candida kefyr) is a rare yeast and a less common cause of cutaneous infection in humans, although it is usually associated with invasive candidiasis, especially among immunocompromised patients. Case summary: A case of cutaneous candidiasis on a 49-year-old adult male patient was evaluated. Laboratory examinations were conducted, including morphological identification, gram staining and molecular identification using Polymerase Chain Reaction (PCR), and gene sequencing. An antifungal susceptibility test was carried out using conventional drugs. Morphological identification revealed the presence of yeast as the most predominant isolate, while molecular and gene sequencing identified the isolate as Kluveromyces marxianus. Antifungal susceptibility test against ketoconazole, fluconazole and Amphotericin B showed a zone of inhibition with mean (± SD) of 38.5 (±0.5mm), 24.5 (±0.5mm) and 0 (±0.0mm), respectively. The patient was treated with both oral and topical ketoconazole but showed no complete therapeutic clearance of the lesion after four months of therapy. Conclusion: This study revealed an in vitro and in vivo ketoconazole sensitivity disparity in a case of Kluyveromyces marxianus, a cutaneous non- Candida albicans species. Based on this finding, further study of this pathogen is imminent to mitigate the health implications, especially in developing countries like Nigeria, for effective patient management.

ABSTRACTKefir and koumiss are both traditional alcoholic fermented milk drinks made from the fermentation of various probiotics and yeasts. The complex microbial composition of kefir and koumiss has hindered the expansion of their consumption in global markets. In this study, we first performed a metagenomic analysis of fermented food to explore the core strains of kefir and koumiss. Five strains (Lactococcus lactis, Lactobacillus helveticus, Lentilactobacillus kefiri, Lactobacillus kefiranofaciens, and Kluyveromyces marxianus) were identified as the core strains shared by both kefir and koumiss. In contrast to the widespread distribution of L. lactis, L. kefiri and L. kefiranofaciens were primarily restricted to kefir and koumiss and certain fermented cheeses. These five strains were subsequently selected for synthetic kefir (sKefir) fermentation. Metagenomic sequencing and metabolomic analyses revealed that, compared with natural kefir, sKefir exhibited a more balanced microbial composition while also harboring metabolites with analogous functional profiles. Functionally, sKefir alleviated DSS‐induced intestinal barrier damage and intestinal inflammation by promoting the expression of gut barrier‐related genes, such as occludin (OCLN). The microbial interaction analysis revealed mutually synergistic growth‐promoting effects among these five strains. In addition, horizontal gene transfer (HGT) was also observed between L. kefiri and L. kefiranofaciens. Our work clarified the core microbial strains of kefir and koumiss through investigation of their evolutionary histories and explored their potential for artificial synthesis.

Two immunomodulatory signature peptides (PFPEVFG and SPAQILQW) were obtained from casein hydrolysates produced by Kluyveromyces marxianus JY-1 in our previous studies with the aid of virtual screening. However, their mechanisms for improving the composition of the gut microbiota in immunosuppressed mice remain unknown. The aim of this study was to reveal the mechanisms of immunomodulation and improvement of gut microbiota composition mediated by the casein peptides PFPEVFG and SPAQILQW through a cyclophosphamide (Cy)-induced immunosuppression and intestinal mucosal injury mouse model. The casein peptides PFPEVFG and SPAQILQW reversed Cy-induced intestinal structural damage and could correct for Cy-induced intestinal flora disorders. Importantly, fecal microbiota transplantation (FMT) confirmed that the casein peptides PFPEVFG and SPAQILQW attenuate immunosuppression by targeting participation in the dynamic balance of the gut microbiota. These results indicated that the casein peptides PFPEVFG and SPAQILQW have the potential to be used as natural medicines for the treatment of immunosuppression.

Green synthesis of silver iodide nanoparticles from Kluyveromyces marxianus M59 beta-glucan: characterization and anticancer activity

Aldo-keto reductase (AKR) is an important biocatalyst for the synthesis of chiral alcohols; however, its inability to catalyze bulky substrates severely limits its industrial applications. Previously, the T23V/Q213A mutant of AKR from Kluyveromyces marxianus (KmAKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer-assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high-throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate-binding pocket, thereby influencing the substrate specificity of KmAKR.

Synthetic microbial communities: A new strategy to enhance texture, taste, and flavor attributes of slightly fermented dried bonito.

Sequential fermentation with Kluyveromyces marxianus and yogurt starter cultures produces stirred fermented milk with reduced lactose and enhanced natural flavor

The increasing interest in renewable feedstocks for biochemicals production includes the valorization of cheese whey (CW), a by-product of the dairy industry. CW contains a high concentration of lactose, accounting for most of its organic load, making it a major environmental pollutant if untreated. A valorization approach of CW is based on the separation of valuable proteins by ultrafiltration, and the obtainment of a lactose-rich cheese whey permeate (CWP). The conversion of lactose in CWP into bioethanol is considered a sustainable solution for the valorization of this waste. However, lactose is not metabolized by the yeast species Saccharomyces cerevisiae, namely the most industrially used microorganism, to produce bioethanol. Differently, the non-conventional yeast Kluyveromyces marxianus shows high growth rates on a broad range of industrially relevant substrates, including lactose. This study provides a comprehensive physiological characterization of three K. marxianus strains (DSM 5422, DSM 7239, and DSM 5572) to evaluate their potential for lactose hydrolysis and fermentation in order to produce advanced bioethanol from CWP. Microplate growth tests, β-galactosidase activity assays, and flask fermentations were carried out to identify optimal strains for efficient CWP valorization, advancing the applications of K. marxianus in industrial biotechnology. The optimal conditions for lactose utilization and ethanol production were identified as 130 g/L of lactose at 42 °C in semi-synthetic media (SSM). Even though strain DSM 7239 showed the highest β-galactosidase activity of 27.8 ± 0.9 U mg−1, strains DSM 5422 and 5572 were identified as the best performing strains at shake flask experiments in terms of ethanol yield on the substrate, with 0.48 ± 0.03 g/g and 0.50 ± 0.03 g/g, respectively, after 1 day of fermentation.

Removal of safranin-O dye from wastewater samples using Kluyveromyces marxianus biomass: Experimental research and theoretical analysis

Microbiological studies were carried out on specimens of Harmonia axyridis obtained from different regions of Bulgaria (Sofia, Sandanski, Kresna, and Haskovo). It has been established that ladybugs of this species can be carriers of conditionally pathogenic bacteria and fungi for animals and humans, such as Es¬cherichia coli, Citrobacter freundii, Citrobacter amalonaticus, Proteus mirabilis, Hafnia alvei 2, Serratia ficaria, Pseudomonas alcaligenes, Pseudomonas cepacia, Staphylococcus sciuri, Clostridium perfringens, Actinomyces naeslundii, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Penicillium sp., and Aspergillus sp. Of greatest importance in this aspect are the bacteria Escherichia coli, P. mirabilis, and C. perfringens. Their carriage is an indicator of contact of these insects with faecal-contaminated materials and of a significant risk of transmission of other dangerous pathogenic microorganisms. Some of the mi¬crobial species are useful for them and for other insects. H. axyridis are also carriers of bacteria and fungi pathogenic to insects, the most important of which are Beauveria bassiana and Cephalosporium lecanii.

Background:Candida species are opportunistic pathogens that can cause local or systemic invasive infections in patients hospitalized in intensive care units (ICUs). The present study aimed to rapidly and precisely identify Candida species, especially Candida auris, from patients hospitalized in high-risk units using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique.Materials and Methods:In this study, 136 clinical samples were prospectively collected from different organs of patients hospitalized in high-risk units recruited from three tertiary care training hospitals in Isfahan. Direct microscopic analysis using potassium hydroxide 10% followed by culturing on Sabouraud dextrose agar and CHROMagar culture media was primarily used to screen Candida species. Molecular detection of clinical isolates was mainly done using the PCR-RFLP method. Then, all the isolates were subjected to confirmatory diagnosis again through amplification of the C. auris-specific PCR.Results:Candida albicans was the most prevalent isolate (48.8%), followed by Candida glabrata (17.6%), Candida tropicalis (16.1%), Candida parapsilosis (6.6%), Candida krusei (5.8%), Candida famata (4.4%), Candida kefyr (2.9%), and Candida guilliermondii (1.4%). Therefore, the prevalence of non-albicans Candida species was higher than C. albicans isolates. No C. auris isolates were identified among 136 patients. However, according to the positive control, C. auris had no restriction enzyme cleavage site for MspI, and its PCR and digestion products were the same size (401 bp).Conclusions:The simple and reliable PCR-RFLP assay used in the study has the potential to identify rare yeasts such as C. auris. It can reduce turnaround times and costs if applied in developing countries.

The yeast Kluyveromyces marxianus, a yeast known for its ability to ferment ethanol at high temperatures, can utilize various sugars including cellobiose, lactose and xylose. This study focused on improving cellobiose utilization by identifying and engineering a cellobiose transporter in K. marxianus. To assess cellobiose utilization capabilities, K. marxianus strains were grown in a cellobiose medium. The strains showed various growth levels, for example, the NCYC2791 strain grew well, while the DMKU3-1042 strain did not. This difference provided a basis for identifying a cellobiose transporter. Thirteen transporter candidate genes from the NCYC2791 genome were expressed in DMKU3-1042. As a result, KmSTL1 overexpression enhanced cell growth in a cellobiose medium. In addition, its disruption in NCYC2791 caused growth defects. To confirm its function, KmSTL1 was co-expressed with a β-glucosidase gene in Saccharomyces cerevisiae EBY.VW1000, which only uptake maltose. This engineered strain grew in cellobiose medium, indicating that KmSTL1 encodes a cellobiose transporter. Expression of GFP-fused KmStl1p in K. marxianus revealed that KmStl1p localized on cell membrane under cellobiose conditions, but was degraded in glucose conditions, suggesting that the transporter is regulated by available sugars. By individually disrupting seven α-arrestin genes in K. marxianus, KmRog3p was identified as a major ubiquitination mediator for KmStl1p degradation. Deletion analysis of KmStl1p revealed that its C-terminus is crucial for recognition by KmRog3p. Furthermore, expressing KmStl1p C-terminus mutants enhanced cellobiose assimilation in both K. marxianus and S. cerevisiae. These findings demonstrate that engineering KmStl1p is an effective strategy to improve cellobiose utilization in yeasts.