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Introduction: In lambs, the function of the rumen is incompletely developed at weaning, and the inclusion of yeast cultures in the diet can profoundly influence the morphological and functional development of the rumen. Methods: In this study, the effects of Saccharomyces cerevisiae and Kluyveromyces marxianus (NM) yeast co-cultures on ruminal histomorphology were assessed, and corresponding transcriptomic changes within the rumen epithelium were identified. In total, 24 lambs were grouped into four groups of six lambs including a control (C) group fed a basal diet, and N, M, and NM groups in which lambs were fed the basal diet, respectively, supplemented with Saccharomyces cerevisiae yeast cultures (30 g/d per head), Kluyveromyces marxianus yeast cultures (30 g/d per head), and co-cultures of both yeasts (30 g/d per head), the experiment lasted for 42 d. Results: In morphological analyses, lambs from the NM group presented with significant increases in papilla length, papilla width, and epithelial thickness in the rumen relative to lambs in the C group (p < 0.05). Transcriptomic analyses revealed 202 genes that were differentially expressed between samples from the C and NM groups, with the largest proportion of these genes being associated with the oxidative phosphorylation pathway. In a weighted gene coexpression network analysis, a positive correlation was observed between the MEgreen and MEpurple modules and rumen morphology. Of these modules, the MEgreen module was found to be more closely linked to fatty acid metabolism and oxidative phosphorylation, whereas the MEpurple module was linked to oxidative phosphorylation and fatty acid degradation. Ultimately, these results suggest that dietary supplementation with NM has driven the degradation of fatty acids, the induction of oxidative phosphorylation, the acceleration of lipid metabolism, the production of ATP to sustain ruminal growth, and the maintenance of intracellular NADH/NAD+ homeostasis on weaned lambs and is superior to single yeast fermentation. Discussion: These results thus offer a theoretical foundation for further studies examining the mechanisms through which NM cultures can influence ruminal development in lambs. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) is a valuable amino acid widely used in food, healthcare, and agriculture. GABA bioproduction by budding yeasts has been commonly reported, but related studies using non-conventional yeasts remain limited. In this study, two non-conventional natural yeast strains, namely, Kluyveromyces marxianus JMY140K and Metschnikowia reukaufii JMY075, were identified as promising GABA producers, and M. reukaufii JMY075 was discovered to be a GABA producer. Enhanced GABA production was observed in the two yeast strains under stress conditions, including high temperature and high ethanol and acetic acid levels. In particular, K. marxianus JMY140K showed 7.93 times higher GABA titers under thermal stress than that of the control. External stress conditions significantly influenced the GABA production of these two yeast strains. The culture filtrate of K. marxianus JMY140K also showed promising activities in human skin cells. In addition, K. marxianus JMY140K could also produce GABA using rice straw hydrolysate, which indicated that it has the potential to produce GABA using renewable biomass. Our studies provide insight for further enhancing the GABA production of natural yeasts and promoting its biotechnology applications. [ABSTRACT FROM AUTHOR]

Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO , which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle-school students in Denver, CO, USA, to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. In contrast, we isolated the "dairy yeast," Kluyveromyces lactis , from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state. The outreach activity partnered middle-school, high-school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus. [ABSTRACT FROM AUTHOR]

In the last decade, the production of guinea pig meat in Andean countries has increased due to the growing number of consumers of this meat. Objective: To evaluate the effect of including different doses (0.50, 1.00, and 1.50 mL) of agro-industrial substrates (molasses distillery waste) fermented with lactic acid bacteria and yeasts on productive performance, hematological profile, relative weight changes in digestive tract organs, and changes in the intestinal microbiota in guinea pigs (Cavia porcellus). Materials: A total of 300 guinea pigs, Kuri breed, aged 20 days and weighing 330 g, were distributed into 10 groups of 30 animals each. Ctrl, Control. La, substrate fermented with Lactobacillus acidophilus (8.1 × 107 CFU/mL). Kf, substrate fermented with Kluyveromyces fragilis (7.4 × 106 CFU/mL). La + Kf, substrate fermented with bacteria and yeasts; the evaluated doses were 0.50, 1.00, and 1.50 mL/animal. The indicators evaluated in the study included weight gain, health, hematological profile, relative weight of digestive tract organs, and changes in the intestinal microbiota. Results: The parameters evaluated were toxicity, productive parameters, occurrence of diarrhea and mortality, and blood profile. The results showed a significant increase in the weight of the animals consuming probiotics, especially at higher doses. Additionally, an improvement in the intestinal microbiota was observed, with an increase in beneficial bacteria such as Lactobacillus and a decrease in pathogenic bacteria. Probiotics also influenced the hematological parameters and the weight of digestive tract organs, suggesting a positive effect on the overall health of the animals. Conclusions: Supplementation with probiotics proved to be a promising strategy for improving productive performance and intestinal health in guinea pigs. Supplementation with L. acidophilus and K. fragilis significantly enhances guinea pig growth and modulates the intestinal microbiota. The combination of strains and appropriate doses maximizes benefits. These results promise applications in animal production, requiring further studies to confirm their efficacy in other species and developmental stages. [ABSTRACT FROM AUTHOR]

Canastra cheese, an artisanal cheese produced in Serra da Canastra—Brazil, has great cultural importance. Furthermore, this cheese has nutritional and sensory attributes that make it of great economic importance. Its microbiota is composed of different bacteria and yeasts. Some yeasts already isolated by our research group have been characterized as potential probiotics. Probiotic microorganisms have garnered scientific interest, as improvements in the physical, chemical and sensory characteristics of food products have been reported when these microorganisms are used. In this context, the objective of this work was to evaluate Kluyveromyces lactis and Torulaspora delbrueckii, which were previously isolated from Canastra cheese, as autochthonous starter cultures. Canastra cheese was produced under three different conditions: (1) cheese with "Pingo" (natural starter), (2) cheese with "Pingo" + yeast mixed culture, and (3) cheese with only mixed yeast culture. The results showed that the mixed yeast inoculum significantly influenced the lactic acid bacteria population. Yeast populations remained at around 106 CFU/g after 45 days of maturation. Furthermore, cheeses containing the yeast mixed with inoculum had an initial lactose content reduced by 92.80% compared to cheese produced with "Pingo" (87.70%). The antioxidant activity, evaluated using the ABTS method, showed that cheeses containing the mixed yeast culture had higher percentages of antioxidant activity at 45 days of maturation. The texture profile of the cheeses changed over time. In general, the cheese containing the yeast mixed culture and "Pingo" and the cheese containing "Pingo" had the lowest hardness at 30 days of maturation (5245 and 5404 N, respectively). Among the volatile compounds, 3-methylbutyl octanoate, phenethyl butyrate, phenethyl propionate, isobutyl butanoate and pentyl propionate were found only in cheeses produced with yeast mixed culture. The obtained results demonstrated that the use of autochthones probiotic cultures could improve the cheese characteristics without negatively impacting the traditional physicochemical attributes of Canasta cheese. [ABSTRACT FROM AUTHOR]

One strategy for adding unique characteristics and flavors to improve coffee quality is the selection of starter microorganisms. Here, we aimed to evaluate the effect of Saccharomyces cerevisiae LNFCA11 and Kluyveromyces lactis B10 as starter cultures on the quality of four different wet-fermented coffee varieties. Microbiological, molecular, and chemical analyses were carried out to identify yeast, bacteria, volatile compounds, carbohydrates and bioactive compounds in coffee. Sensory analysis was performed by Q-graders certified in coffee. Starter yeasts affected bioactive and volatile compounds as well as sensory descriptors in the coffee varieties. S. cerevisiae CA11 allowed a higher content of trigonelline and chlorogenic acid in MGS Paraíso 2 (P2) and Catuai Amarelo IAC62 (CA62) varieties. K. lactis B10 fermentation resulted in higher chlorogenic acid only on the P2 cultivar and MGS Catucaí Pioneira (CP). In addition, 5-methyl-2-furfuryl alcohol and n-hexadecanoic acid were produced exclusively by yeast fermentation compared to spontaneous fermentation. The coffee cultivars P2 presented more complex sensory descriptors and the attributes of aroma, acidity, and balance when fermented with S. cerevisiae CA11. Sensory descriptors such as lemongrass, citrus, and lemon with honey were related to K. lactis B10. Starter cultures allowed the coffees to be classified as specialty coffees. The fermentation showed that the choice of starter yeast depends on the desired sensory descriptors in the final product. [ABSTRACT FROM AUTHOR]

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Yeasts are attractive hosts for the production of heterologous products due to their genetic tractability and relative ease of growth. While the baker's yeast Saccharomyces cerevisiae is a powerful workhorse of the biotechnology industry, the species has metabolic limitations and it is critical that we develop alternative platforms that will facilitate the development of bioprocesses that rely on sustainable feedstocks. In this study, we used synthetic biology tools to construct coumaric acid–producing strains of Kluyveromyces marxianus, a yeast whose physiological traits render it attractive for biotechnology applications. Coumaric acid is a building block in the synthesis of many different families of aromatics and is a key precursor for the synthesis of complect phenylpropanoid molecules, including many flavours and aromas. The starting point for this work was a K. marxianus chassis strain that has increased flux towards the synthesis of tyrosine and phenylalanine, the aromatic amino acids that can serve as starting points for coumaric acid synthesis. Following principles of synthetic biology, a modular approach was taken to identify the best solution to different metabolic possibilities and these were then combined in different ways. For the first step, it was established that the route from phenylalanine was superior to that from tyrosine and the combined overexpression of PlPAL , AtC4H and AtCPR1 delivered the highest yield of coumaric acid. Next, it was established that while Pdc5 and Aro10 both had phenylpyruvate decarboxylase activity, inactivation of ARO10 was sufficient to prevent flux loss in the pathway. Since phenylalanine is the starting point, efforts were made to improve efficiency of its production. It was found that glutamate was a preferred nitrogen source for coumaric acid production, and this knowledge was used to engineer a strain that overexpressed S. cerevisiae GDH1 and delivered higher yields of coumaric acid. Ultimately, this strategy led to the development of strains that has yields of up to 48 mg coumaric acid /g glucose. Strains were evaluated in bioreactors to investigate the effects of different process parameters. These analyses indicated that engineered strains face some redox balance challenges and further work will be required overcome these to develop strains that can perform well under industrial conditions. • The yeast Kluyveromyces marxianus was engineered to produce the aromatic precursor molecule coumaric acid. • The highest yield obtained was 48 mg coumaric acid per g glucose. • Engineering the GS/GOGAT pathway to increase intracellular glutamate increased coumaric acid titres. [ABSTRACT FROM AUTHOR]

Global concern about pathogenic resistance to antibiotics is prompting interest in probiotics as a strategy to prevent or inhibit infections. Fermented beverages are promising sources of probiotic yeasts. This study aimed to evaluate the antagonistic effects of Kluyveromyces marxianus, Wickerhamomyces anomalus, and Pichia manshurica strains from kefir and wine against Salmonella enterica serovar Enteritidis in intestinal epithelial cells. The ability of these yeasts to adhere to Caco-2/TC-7 cells was evaluated, as well as their influence on the ability of Salmonella to associate and invade these cells. The behavior of the pathogen was analyzed by (a) incubation of enterocytes with yeast before adding Salmonella, (b) co-incubation of Salmonella with yeast before contact with the enterocytes, and (c) incubation of Salmonella with yeast metabolites before contact with enterocytes. All yeast strains demonstrated adherence to Caco-2/TC-7 cells (33–100%) and effectively inhibited Salmonella invasion. Among the treatments, co-culture showed the greatest effect, reducing Salmonella association and invasion by more than 50%. Additionally, these yeasts modulated the epithelial immune response, significantly decreasing CCL20-driven luminescence by 60–81% (p < 0.0001). These results highlight the potential of yeasts from fermented beverages as probiotics to counteract Salmonella infections, offering a promising alternative in the fight against antibiotic resistance. [ABSTRACT FROM AUTHOR]

In this study, the most appropriate form of the specific growth rate of the yeast Kluyweromyces marxianus var lactis MC5 was determined by a multi-criteria decision-making method (PROMETHEE II). Then, based on experimental data from a total of six batch processes, the simultaneous estimation of the kinetic parameters for four batch processes was made. The remaining two experiments were used to test the model. The parameter estimation problem was designed as a multi-objective optimisation problem. The method of scalarisation of the weighted sum was used. In this way, the multi-objective optimisation problem was converted to a single-objective one. The direct search method was used to solve the problem of obtaining the Pareto solution. In order to test the resulting solution, two batches of experimental data were additionally used. The obtained results showed that the model satisfactorily described the test data. The validated kinetic model can be used for optimal control of batch and fed-batch fermentation. [ABSTRACT FROM AUTHOR]

Ethyl acetate is at present exclusively produced from fossil resources. Microbial synthesis of this ester from sugar‐rich waste as an alternative is an aerobic process. Ethyl acetate is highly volatile and therefore stripped with the exhaust gas from the bioreactor which enables in situ product recovery. Previous research on microbial formation of ethyl acetate has focused on the kinetics of ester synthesis and in part on the ester stripping, while the separation of the ester from the exhaust gas has hardly been investigated. A mixed matrix membrane was developed consisting of Silikalite‐1 embedded in polydimethylsiloxane which was installed in a radial–symmetrical membrane module. Evaluation of the separation of ethyl acetate was based on the analysis of the composition of the feed and retentate gas by mass spectrometry. The separation efficiency of the membrane was first tested with varied flows of artificial exhaust gas, containing defined amounts of ethyl acetate. A model for describing the separation process was parametrized by the measured data and used to design a real separation experiment. Ethyl acetate produced from delactosed whey permeate by Kluyveromyces marxianus DSM 5422 in a stirred bioreactor gassed with 0.5 vvm air was successfully separated from the exhaust gas by membranes; 93.6% of the stripped ester was separated. Liquid ethyl acetate was recovered by cooling the permeate gas to ‒78°C, whereby 99.75% of the condensed organic compounds were ethyl acetate. This study demonstrates for the first time that microbially produced and stripped ethyl acetate can be effectively separated from the exhaust gas of bioreactors by membrane technology to obtain the ester in high yield and purity. [ABSTRACT FROM AUTHOR]

Cocoa fermentation plays a key role in defining chocolate's flavor, with yeasts being central to this process. This study aimed to explore intraspecific genetic diversity of major indigenous yeasts (i.e., Saccharomyces cerevisiae and Pichia kudriavzevii), and their potential interaction in the cocoa pulp environment. Their metabolic intraspecific diversity was characterized in synthetic cocoa pulp medium. Then, Saccharomyces cerevisiae, Pichia kudriavzevii, and other strains were introduced to each other to evaluate their potential negative interaction. Interesting strain associations were selected to further explore their interaction in synthetic cocoa pulp medium as well as real fresh cocoa pulp. From a fermentation campaign in Ivory Coast, a set of Saccharomyces (S.) cerevisiae and Pichia (P.) kudriavzevii strains were isolated from batches classified according to their chocolate quality (i.e., standard, intermediate, or premium chocolate). Less abundant species (i.e., Torulaspora franciscae, Kluyveromyces marxianus) were also isolated and tested for their potential negative interactions with S. cerevisiae and P. kudriavzevii. A set of strains were selected and cultured in single and in co-culture in a minimal cocoa pulp synthetic medium and in fresh cocoa pulp to highlight potential positive and/or negative interactions regarding fermentative aroma profile (i.e., higher alcohols, acetate esters, medium-chain fatty acids, and ethyl esters). The results highlighted the dominance of S. cerevisiae in fermentation kinetics and medium- to long-chain ester production, contrasted with P. kudriavzevii's efficiency in short-chain ester synthesis. Intraspecific aroma profile variations can be pointed out. The co-cultures of P. kudriavzevii and S. cerevisiae strains isolated from the premium chocolate batch had a positive impact on the fermented pulp aroma profile. Negative interactions were observed with Torulaspora franciscae, which eliminated P. kudriavzevii's aroma expression. Finally, the comparison of the data obtained for the minimal cocoa pulp synthetic medium compared to the cocoa pulp allowed us to draw conclusions about the use of synthetic media for studying cocoa fermentation. These findings emphasize the complex microbial interactions in cocoa fermentation that could shape future cocoa bean aroma. [ABSTRACT FROM AUTHOR]

Background: Whey fermentation could produce bioactive substances with immunomodulatory effects, metabolic syndrome modulation, and antioxidant properties, thereby imparting functional characteristics to products and facilitating the development of novel foods with health-promoting potential. Methods: A non-targeted metabolomics approach using liquid chromatography–mass spectrometry (LC-MS) was employed to investigate changes in the metabolite profiles of whey fermented by Kluyveromyces marxianus strain KM812 over varying fermentation durations. Results: The findings demonstrated a progressive enrichment of metabolites over time. A total of 151 differential metabolites were identified and categorized primarily into amino acids, peptides, and analogues, fatty acids and conjugates, and carbohydrates and conjugates, as well as benzoic acids and derivatives. The highest relative content of whey metabolites was observed at 48 h of fermentation, with a cumulative increase of 1.45-fold, 1.49-fold, 3.39-fold, and 1.24-fold for peptides and amino acids, peptides, and analogues, fatty acids and conjugates, and carbohydrates and conjugates, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed associations with 23 specific metabolites and delineated 9 metabolic pathways, predominantly involved in amino acid and lipid metabolism. Conclusions: Based on the above, KM812 could effectively degrade macromolecular substances in whey into small molecules such as L-isoleucine, ornithine, betaine, α-linolenic acid, and palmitoleic acid, thereby influencing the nutritional and functional properties of whey. In-depth analysis of the metabolic products in KM812-fermented whey could provide a theoretical basis for the development of functional foods derived from small molecules in the future. [ABSTRACT FROM AUTHOR]

In a world where climate change and sustainability are at the forefront, innovative solutions are more pressing than ever. In the production process of distillates, the critical stage is the cooking of agave for thermal hydrolysis of fructans. This stage determines the efficiency of the process, incurs the highest production cost, and has a detrimental impact on the environment. In this study, we investigate the saccharification of agave fructans in fresh juice using fructanases produced in situ by Kluyveromyces marxianus as an alternative to traditional agave cooking. furthermore, we explore both sequential and simultaneous alcoholic fermentation processes using a native mixed culture to improve the overall yield and obtain a distillate with enjoyable sensory characteristics. The results show that saccharification and simultaneous fermentation allow for obtaining a fermented product containing an average of 7% alcohol. Gas chromatography and mass spectrometry analysis shows that the distillate is essentially an ethanol – water mixture with a broad profile and high concentration of volatile compounds. The composition of the distilled product provides a well-balanced sensory experience that is highly valued by consumers. Hence, for the first time, a novel, efficient, low-cost, and sustainable process for producing agave distillates has been successfully designed. [ABSTRACT FROM AUTHOR]

High-temperature fermentation (HTF) of ethanol can reduce costs of cooling, sterilization, and related equipment compared to the costs of general ethanol fermentation. To realize HTF, however, there are various issues to be considered, such as the fermentation temperature upper limit for ethanol-producing thermotolerant yeast, the size of a fermenter that does not require cooling, and the effective temperature for suppressing microbial contamination. This study focused on these issues and also on downstream processes that exploit the advantages of HTF at temperatures exceeding 40 °C. The permissible size of a fermenter without cooling was estimated by simulating heat generation and heat dissipation. Fermentation productivity at high temperatures when using the thermotolerant yeast Kluyveromyces marxianus and the inhibitory effect of high temperatures on the growth of contaminant microorganisms were examined. After fermentation, the recovery and concentration of ethanol were performed by reduced-pressure distillation (RPD) and membrane separation. These experiments demonstrate that efficient HTF can reduce the amount of saccharifying enzymes in simultaneous saccharification and fermentation and can shorten the transition time from the saccharification step to the fermentation step in separate saccharification and fermentation, that RPD at fermentation temperatures enables a smooth connection to the HTF step and can be performed with a relatively weak vacuum, and that membrane separation can reduce the running cost compared to the cost of general distillation on a compact scale. [ABSTRACT FROM AUTHOR]

Using PEI as an aggregating agent with β-galactosidase from Kluyveromyces lactis was investigated with no solid support. PEI, a cationic polymer initiated instant aggregation with the enzyme in solution. The factors affecting the aggregation such as PEI to enzyme ratio, glutar aldehyde for cross-linking and pH were investigated. Aggregation and sedimentation as well as the residual activity of aggregates were effectively dependent on the PEI to enzyme ratios. Easily precipitating aggregates at the ratio of 1/8, PEI enzyme aggregates were able to contain all enzyme in the complexation and displayed 60% of initial lactase activity. The PEI aggregation of enzyme led to enhancements in chemical and physical characteristics compered to free enzyme. The soluble enzyme showed a narrow optimum at about pH 7 while pH optimum of the aggregates extended one pH unit toward the alkaline range. Upon overnight incubation at 40 °C, aggregated enzyme displayed 30% greater stability on average at all pHs tested. Although the free enzyme showed the highest activity at 40°C, it rapidly lost 50% of its activity at 50°C. In contrast, the aggregated enzyme retained full activity at 50°C and 70% activity at 65°C. With markedly enhanced thermal stability, the half-life of the aggregated enzyme increased from 76 hours to 254 hours at 40°C. Overall, the simple and rapid aggregation of PEI with the enzyme led to instant and intense clustering, resulting in higher thermal and pH stabilities. This method may potentially offer efficient and cost-effective catalysis in lactase conversion processes. [ABSTRACT FROM AUTHOR]

Abstract Background Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy’s Apulia region, designed to process 539 m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. Results The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model’s dependence on the price or credit associated with cheese-whey handling. Conclusions This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

Protein glycosylation is a fundamental modification crucial for numerous intra- and extracellular functions in all eukaryotes. The phosphorylated dolichol (Dol-P) is utilized in N-linked protein glycosylation and other glycosylation pathways. Dolichol kinase (DK) plays a key role in catalyzing the phosphorylation of dolichol. The glycosylation patterns in the Kluyveromyces lactis DK mutant revealed that the yeast well tolerated a minor deficiency in Dol-P by adjusting protein glycosylation. Comparative analysis of sequences of DK homologs from different species of eukaryotes, archaea and bacteria and AlphaFold3 structural model studies, allowed us to predict that DK is most likely composed of two structural/functional domains. The activity of predicted K. lactis DK C-terminal domain expressed from the single copy in the chromosome was not sufficient to keep protein glycosylation level necessary for survival of K. lactis. However, the glycosylation level was partially restored by additionally provided and overexpressed N- or C-terminal domain. Moreover, co-expression of the individual N-and C-terminal domains restored the glycosylation of vacuolar carboxypeptidase Y in both K. lactis and Saccharomyces cerevisiae. Despite the differences in length and non-homologous sequences of the N-terminal domains the human and nematode Caenorhabditis elegans DKs successfully complemented DK functions in both yeast species. Additionally, the N-terminal domains of K. lactis and C. elegans DK could functionally substitute for one another, creating active chimeric enzymes. Our results suggest that while the C-terminal domain remains crucial for DK activity, the N-terminal domain may serve not only as a structural domain but also as a possible regulator of DK activity. [ABSTRACT FROM AUTHOR]

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. Key points: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins [ABSTRACT FROM AUTHOR]

The use of probiotics has been gaining popularity in terms of inclusion into human diets over recent years. Based on properties exerted by these organisms, several benefits have been elucidated and conferred to the host. Bacteria have been more commonly used in probiotic preparations compared to yeast candidates; however, yeast exhibit several beneficial properties, such as the prevention and treatment of diarrhea, the production of antimicrobial agents, the prevention of pathogen adherence to intestinal sites, the maintenance of microbial balance, the modulation of the immune system, antibiotic resistance, amongst others. Saccharomyces boulardii is by far the most studied strain; however, the potential for the use of other yeast candidates, such as Kluyveromyces lactis and Debaryomyces hansenii, amongst others, have also been evaluated in this review. Furthermore, a special focus has been made regarding the production considerations for yeast-based probiotics and their formulation into different delivery formats. When drafting this review, evidence suggests that the use of yeasts, both wild-type and genetically modified candidates, can extend beyond gut health to support skin, the respiratory system, and overall immune health. Hence, this review explores the potential of yeast probiotics as a safe, effective strategy for preventative health in humans, highlighting their mechanisms of action, clinical applications, and production considerations. [ABSTRACT FROM AUTHOR]

Sourdough fermentation is rarely used for gluten-free flatbread (GFFB), a product that is challenging to produce, especially when using high-fiber ingredients that bring nutritional benefits but lead to physical deterioration. The aim of this study was therefore to evaluate the fermentation performance of carob flour (CSPF), proso millet flour (PMF), and proso millet bran (PMB) individually and in combination with Limosilactobacillus fermentum and Kluyveromyces marxianus (LF + KM) and to compare the performance of LF + KM with a commercial starter (LIVENDO® LV1). A mixture design (n = 13) was used to evaluate the fermentation performance of LF + KM (total titratable acidity (TTA); lactobacilli and yeast growth; acetic and lactic acid, fructose, glucose, and saccharose content) at 35 °C for 16 h. The comparison of LF + KM with LV1 fermentation was based on the acidity rate, fermentation quotient, TTA, and finally by determining the physical properties (texture, shape, color) of a rice–corn GFFB in which 10% of flour was supplemented with the sourdoughs. PMB promoted the growth of lactobacilli and the production of organic acids, especially in combination of CSPF and PMF. The optimum flour ratio was 2.4:1:1.2 (PMB:PMF:CSPF). LF + KM shortened the sourdough fermentation time by 2.5 times compared to LV1. The use of LF + KM sourdough reduced the hardness (32%) and chewiness (28%) of the GFFB, while the volume (35%) was increased compared to LV1 sourdough. This study shows the potential of using local alternative flours in sourdough fermentation for the production of GFFB. [ABSTRACT FROM AUTHOR]

Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A > 95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 μg/mL and 3.33 μg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use. Key points: • MET and its precursor ProMET were successfully hetero-expressed in K. marxianus • > 95% high-purity MET and ProMET were purified at 1.68 μg/mL and 3.33 μg/mL • MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth [ABSTRACT FROM AUTHOR]

In recent years, demand for low-alcohol and alcohol-free beers has been rising. Of the many methods of producing such beers, many have expensive implementation requirements or drawbacks in terms of beer quality. The exploration of non-Saccharomyces yeast species presents a promising opportunity to overcome these challenges. These yeasts, with their diverse metabolic capabilities and unique flavor profiles, offer the potential to create innovative and flavorful low-alcohol beers. The study investigates the feasibility of using selected non-Saccharomyces yeasts for brewing low-alcohol beers, focusing on fermentation kinetics, physicochemical parameters, and the sensory attributes of the final product. The evaluated yeast species were Kluyveromyces lactis MG971263, Metschnikowia pulcherrima MG971247 and MG971250, Torulaspora delbrueckii MG971248, Wickerhamomyces anomalus MG971261, and W. onychis MG971246. Two strains of Saccharomyces cerevisiae were used as a control. The results of the study show that selected non-Saccharomyces yeast species might be used to produce low-alcohol beers. The non-Saccharomyces yeast allowed the researchers to obtain beers with an alcohol content in the range of 0.5–1.05%, while the control beer brewed with US-05 had an alcohol content of 3.77%. Among the evaluated strains, the strains M. pulcherrima MG971250 and T. delbrueckii MG971248 were found to be rated better in a sensory evaluation than the brewed and low-alcohol strains of S. cerevisiae. [ABSTRACT FROM AUTHOR]

Background: Currently, the synthesis of compounds based on microbial cell factories is rapidly advancing, yet it encounters several challenges. During the production process, engineered strains frequently encounter disturbances in the cultivation environment or the impact of their metabolites, such as high temperature, acid-base imbalances, hypertonicity, organic solvents, toxic byproducts, and mechanical damage. These stress factors can constrain the efficiency of microbial fermentation, resulting in slow cell growth, decreased production, significantly increased energy consumption, and other issues that severely limit the application of microbial cell factories. Results: This study demonstrated that sterol engineering in Kluyveromyces marxianus, achieved by overexpressing or deleting the coding genes for the last five steps of ergosterol synthase (Erg2-Erg6), altered the composition and ratio of sterols in its cell membrane, and affected its multiple tolerance. The results suggest that the knockout of the Erg5 can enhance the thermotolerance of K. marxianus, while the overexpression of the Erg4 can improve its acid tolerance. Additionally, engineering strain overexpressed Erg6 improved its tolerance to elevated temperature, hypertonic, and acid. YZB453, obtained by overexpressing Erg6 in an engineering strain with high efficiency in synthesizing xylitol, produced 101.22 g/L xylitol at 45oC and 75.11 g/L xylitol at 46oC. Using corncob hydrolysate for simultaneous saccharification and fermentation (SSF) at 46oC that xylose released from corncob hydrolysate by saccharification with hemicellulase, YZB453 can produce 45.98 g/L of xylitol, saving 53.72% of the cost of hemicellulase compared to 42oC. Conclusions: This study elucidates the mechanism by which K. marxianus acquires resistance to various antifungal drugs, high temperatures, high osmolarity, acidity, and other stressors, through alterations in the composition and ratio of membrane sterols. By employing sterol engineering, the fermentation temperature of this unconventional thermotolerant K. marxianus was further elevated, ultimately providing an efficient platform for synthesizing high-value-added xylitol from biomass via the SSF process at temperatures exceeding 45 °C. [ABSTRACT FROM AUTHOR]

Introduction: The cheese microbiota is very complex and is made up of technologically-relevant, spoilage, opportunistic and pathogenic microorganisms. Among them lactic acid bacteria and yeasts are the main ones. One of the most interesting dairy yeasts is Kluyveromyces marxianus because of its technological properties including the ability to produce aroma compounds. Methods: This study investigated the contribution of Kluyveromyces marxianus to the gross composition and aroma profile of cow cheeses. Experimental cheeses were prepared by inoculating a co-culture of K. marxianus FM09 and a commercial strain of Lacticaseibacillus casei and compared with cheeses obtained with only L. casei. The gross composition was determined by a FoodScan™ 2 Dairy Analyser, and free amino acids were evaluated at 507 nm after reaction with Cd-ninhydrin. The volatile organic compounds were extracted by head-space solid phase micro-extraction and analyzed by gas chromatography-mass spectrometry coupled with odor activity values. qRT-PCR was applied to determine the expression of genes involved in esters synthesis and degradation. Results: The inoculation of K. marxianus induced an increase of pH and a reduction of protein content of cheeses, in agreement with the stronger proteolysis detected in these cheeses. K. marxianus influenced the content of aroma compounds both quantitatively and qualitatively. In particular, an increase of higher alcohols, esters and organic acids was observed. Moreover, 12 compounds were detected only in cheeses obtained with the co-culture. These differences were in agreement with the odor activity values (OAV). In fact, only 11 compounds showed OAV > 1 in cheeses obtained with the commercial strain, and 24 in those obtained with the co-culture. The qPCR analysis revealed an over expression of ATF1, EAT1, and IAH1 genes. Conclusion: Kluyveromyces marxianus could act as an important auxiliary starter for cheese production through the development and diversification of compounds related to flavor in short-aged cow cheeses. [ABSTRACT FROM AUTHOR]

The use of microencapsulated probiotic bacteria, by maintaining the survival of probiotic bacteria during the production and storage process, produces high quality and long-lasting products. Lentinula edodes polysaccharide is a prebiotic that causes the growth and better performance of probiotics and helps to preserve the product for a long time. For this purpose, 20 samples were prepared from the wastewater of dairy factories around Tehran and 10 bacterial isolates and 10 yeast isolates were isolated from the samples based on biochemical, macroscopic and microscopic tests. Heat tolerance test, acid tolerance and resistance to bile salts and antibiogram were performed to measure the probiotic properties. The polysaccharides of Lentinula edodes were then extracted using a diethylaminoethyl Sephadex-25A ion exchange chromatography column and the amount of polysaccharide was determined using the phenol-sulphuric acid method. Fourier spectroscopy was used to detect biochemical compounds. Superior probiotic isolates were identified by PCR. The microencapsulation effect of superior isolates was investigated using sodium alginate and lentinula edodes polysaccharides on orange juice, apple juice and kombucha drink. Kluyveromyces lactis strain CBS 683 and Lactobacillus brevis strain 123-20 were identified and encapsulated with relative probiotic properties, growing more than in the free state. The shelf life and sensory characteristics of drinks containing bacteria and yeast encapsulated with sodium alginate and polysaccharide were also improved compared to the control group, and during 60 days of storage at 4°C, the bacteria and yeast survived and maintained their performance. The best protection was provided by encapsulated Lactobacillus brevis strain 123-20 in orange juice and apple juice. The use of native strains encapsulated with biocompatible compounds preserves the sensory characteristics of food, prevents food spoilage during storage by producing postbiotics and improves the function of the digestive system. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. The bacterium Lactobacillus kefiranofaciens OSU-BDGOA1 and yeast Kluyveromyces marxianus bdgo-ym6 were previously isolated from kefir grains and have shown probiotic traits in mono- and coculture. This research evaluates the effect of introducing probiotic kefir microorganisms in monoculture and in coculture alongside yogurt starter cultures on the physicochemical and rheological properties, volatile flavor compounds, survival of the microorganisms during simulated digestion, and sensory attributes of the final fermented products. The incorporation of L. kefiranofaciens OSU-BDGOA1 in monoculture showed promising outcomes, resulting in a final product showing more solid-like characteristics and potentially improving the texture of the product. There was also a significant increase in the concentration of desirable volatile flavor compounds in the yogurt with the monoculture, particularly 2,3-butanedione, displaying a positive correlation with buttery flavor in the sensory analysis. The inclusion of L. kefiranofaciens in monoculture also promoted better sensory attributes and was significantly better than the yogurt with the coculture with the yeast, showing promising results for the incorporation of this probiotic bacterium into functional fermented dairy products. [ABSTRACT FROM AUTHOR]

Coffee and coffee by-products contain several chemical compounds of great relevance, such as chlorogenic acid (CGA), trigonelline, and caffeine. Furthermore, yeasts have been the target of studies for their use as probiotics because of their interesting biochemical characteristics. The combined administration of probiotic microorganisms with components that provide health benefits mediated by alginate encapsulation is an alternative that ensures the stability of cells and chemical compounds. In this context, the aim of this work was to co-encapsulate the probiotic yeast Kluyveromyces lactis B10 and extracts of green coffee beans, coffee silverskin, and PVA (black, green or immature, and sour coffee beans). The bioactive composition, antioxidant and antimicrobial activities of the extracts, microcapsule morphological characteristics and encapsulation efficiency, ability of the encapsulation to protect the yeast cells subjected to gastrointestinal conditions, and antioxidant activity of the microcapsules were evaluated. All the evaluated extracts showed antioxidant activity, of which PVA showed 75.7% and 77.0%, green coffee bean showed 66.4% and 45.7%, and coffee silverskin showed 67.7% and 37.4% inhibition of DPPH and ABTS•+ radicals, respectively, and antimicrobial activity against the pathogenic bacteria E. coli, Salmonella, and S. aureus, with high activity for the PVA extract. The microcapsules presented diameters of between 1451.46 and 1581.12 μm. The encapsulation efficiencies referring to the yeast retention in the microcapsules were 98.05%, 96.51%, and 96.32% for green coffee bean, coffee silverskin, and PVA, respectively. Scanning electron microscopy (SEM) showed that the microcapsules of the three extracts presented small deformations and irregularities on the surface. The K. lactis cells encapsulated in all treatments with the extracts showed viability higher than 8.59 log CFU/mL, as recommended for probiotic food products. The addition of green coffee bean, coffee silverskin, and PVA extracts did not reduce the encapsulation efficiency of the alginate microcapsules, enabling a safe interaction between the extracts and the K. lactis cells. [ABSTRACT FROM AUTHOR]

Background: Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539 m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. Results: The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. Conclusions: This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol. [ABSTRACT FROM AUTHOR]

Meiotic recombination is essential for the accurate chromosome segregation and the generation of genetic diversity through crossover and gene conversion events. Although this process has been studied extensively in a few selected model species, understanding how its properties vary across species remains limited. For instance, the ancestral ZMM pathway that generates interference-dependent crossovers has undergone multiple losses throughout evolution, suggesting variations in the regulation of crossover formation. In this context, we first characterized the meiotic recombination landscape and properties of the Kluyveromyces lactis budding yeast. We then conducted a comprehensive analysis of 29,151 recombination events (19, 212 COs and 9, 939 NCOs) spanning 577 meioses in the five budding yeast species Saccharomyces cerevisiae, Saccharomyces paradoxus, Lachancea kluyveri, Lachancea waltii and K. lactis. Eventually, we found that the Saccharomyces yeasts displayed higher recombination rates compared to the non-Saccharomyces yeasts. In addition, bona fide crossover interference and associated crossover homeostasis were detected in the Saccharomyces species only, adding L. kluyveri and K. lactis to the list of budding yeast species that lost crossover interference. Finally, recombination hotspots, although highly conserved within the Saccharomyces yeasts are not conserved beyond the Saccharomyces genus. Overall, these results highlight great variability in the recombination landscape and properties through budding yeasts evolution. Author summary: Meiotic recombination ensures proper chromosome segregation and promoting genetic diversity. Studies in Saccharomyces cerevisiae have provided mechanistic insights into the process of meiotic recombination, but the evolutionary diversity of the meiotic recombination pathway itself is not well understood. Here, we examined recombination in the non-model yeast Kluyveromyces lactis and compared it with four closely related budding yeast species—S. paradoxus, L. waltii, L. kluyveri, S. cerevisiae—to explore the evolution of the crossover pathway. We observed significant variation in recombination rates among these species alongside the loss of the crossover interference pathway. While crossover assurance is essential in S. cerevisiae and S. paradoxus, it seems defective in non-Saccharomyces yeasts, suggesting alternative mechanisms ensuring faithful meiosis. Additionally, recombination hotspots are conserved only within Saccharomyces yeasts but not beyond, highlighting considerable variability in the recombination landscape across budding yeast evolution. [ABSTRACT FROM AUTHOR]

The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe2+ to Fe3+ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products. [ABSTRACT FROM AUTHOR]

Comparative metatranscriptomics of the bacterial and yeast communities of two milk kefir beverages (MKAA1 and MKAA2) was carried out. They were obtained by fermentation with two different frozen stocks of the kefir grain CIDCA AGK1, differing in rheological features and production of organic acids. We hypothesised that the differences in their physicochemical and rheological properties might be due to the microbial activity in each product. The dominance of lactic acid bacteria, yeast, and a marginal amount of acetic acid bacteria characterised the microbiome. The bacterial families Lactobacillaceae and Streptococcaceae accounted for almost all of the bacterial gene transcripts, with Lactobacillus helveticus, L. kefiranofaciens, L. gallinarum, and Lactococcus lactis being most frequent in the microbiome of the MKAA1 beverage and L. kefiranofaciens, Lc. Lactis, and Leuconostoc mesenteroides being the most prevalent in MKAA2. Dipodascaceae and Saccharomycetaceae were the leading yeast families, represented by Yarrowia lipolytica, Saccharomyces unisporus, and Kluyveromyces marxianus. MKAA1 and MKAA2 shared >75% KEGG Ortologs (KOs) in their bacteria and yeast libraries. The considerable decreases in total expressed genes (KEGG Ortologs) assigned to Lactobacillus helveticus and L. gallinarum might be related to the variations in the rheological features of the beverages, probably by compromising the interrelations with L. kefiranofaciens, which might explain the variations in the rheological features of the beverages. [ABSTRACT FROM AUTHOR]

In yeasts, the Leu3 transcriptional factor regulates the expression of genes encoding enzymes of the leucine biosynthetic pathway, in which the first committed step is catalyzed by α-isopropylmalate synthase (α-IPMS). This enzyme is feedback inhibited by leucine, and its product, α-isopropylmalate (α-IPM), constitutes a Leu3 co-activator. In S. cerevisiae, the ScBAT1 and ScBAT2 genes encode branched-chain aminotransferase isozymes. ScBAT1 transcriptional activation is dependent on the α-IPM concentration and independent of chromatin organization, while that of ScBAT2 is α-IPM-independent but dependent on chromatin organization. This study aimed at understanding whether chromatin remodeling determines the transcriptional regulation of orthologous KlBAT1 and LkBAT1 genes in Kluyveromyces lactis and Lachancea kluyveri under conditions in which the branched-chain amino acids are synthesized or degraded. The results indicate that, in K. lactis, KlBAT1 expression is reduced under catabolic conditions, while in L. kluyveri, LkBAT1 displays a constitutive expression profile. The chromatin organization of KlBAT1 and LkBAT1 promoters did not change, maintaining the Leu3-binding sites free of nucleosomes. Comparison of the α-IPMS sensitivities to feedback inhibition suggested that the main determinant of transcriptional activation of the KlBAT1 and LkBAT1 genes might be the availability of the α-IPM co-activator, as reported previously for the ScBAT1 gene of S. cerevisiae. [ABSTRACT FROM AUTHOR]

The influence of selected yeast starters (Kluyveromyces lactis L39, Kazachstania humilis AG5, Nakazawaea molendinolei IG9, Candida diddensiae IG12, and Candida adriatica L30) on the fermentation and final characteristics of natural-style Gordal and Manzanilla green table olives was investigated. In both cultivars, the addition of yeast starters had no significant influence on the evolution of physicochemical parameters or the final main metabolites compared to noninoculated olives. In the Gordal cultivar, K. lactis L39 originated the greatest enrichment of volatile compounds, whereas K. lactis L39 and C. adriatica L30 gave the best volatile profiles in the Manzanilla cultivar. In both cultivars, the β-glucosidase-positive strains N. molendinolei IG9, C. diddensiae IG12, and C. adriatica L30 produced no significant decrease in the total phenolic content at the end of fermentation. Although the yeast starters had a significant effect on the volatile contents of the fermented products, they did not have a significant influence on the main sensory characteristics perceived by a sensory panel. A significant linear relationship (R2 = 0.815, p < 0.001) was found and validated between the perceived bitterness intensity and the content of total phenols in olive pulp, providing a simple and objective method for the evaluation of bitterness in table olives without the need for sensory analysis. [ABSTRACT FROM AUTHOR]

In many countries, whey from the dairy industry is an abundant waste that generates an important environmental impact. Alternative processes to use the whey and minimize the environmental impact are needed. This work considered six formulations with different ammonium sulfate and L-phenylalanine (L-Phe) concentrations to produce bioethanol in sweet whey fermentation by Kluyveromyces marxianus. The results showed a maximum bioethanol concentration equal to 25.13 ± 0.37 g L−1 (p < 0.05) for formulation F6, with 1 g L−1 of L-Phe and 1.350 g L−1 of ammonium sulfate (96 h). For these conditions, the chemical oxygen demand removal percentage (CODR%) was 67%. The maximum CODR% obtained was 97.5% for formulation F3 (1 g L−1 of L-Phe) at 96 h; however, a significant decrease in bioethanol concentration (14.33 ± 2.58 g L−1) was observed. On the other hand, for formulation, F3, at 48 h of fermentation time, a bioethanol concentration of 23.71 ± 1.26 g L−1 was observed, with 76.5% CODR%. Based on these results, we suggest that the best conditions to obtain a significant bioethanol concentration and CODR% value are those used on the configuration F3 at 48 h.

Background: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. Results: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. Conclusion: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts. Highlights: The broad-spectrum promoters enable broad cross-species functionality. Five Kluyveromyces marxianus promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) can all express genes in Yarrowia lipolytica. Five Y. lipolytica promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) can all express genes in K. marxianus. The Kluyveromyces marxianus promoter km.TEF1 can strongly express RFP in bacteria E. coli and C. glutamicum. [ABSTRACT FROM AUTHOR]

Bioethanol, an alcohol produced by microbial fermentation, is traditionally produced from sugar-rich plants such as sugar cane, sugar beet and maize. However, there is growing interest in the use of lignocellulose, an abundant and inexpensive renewable energy source, as a potential substitute for the production of biofuels and biochemicals. Yeast Saccharomyces cerevisiae, which is commonly used for ethanol fermentation, cannot cope with lignocellulose due to a lack of lignocellulolytic enzymes and the inefficient functioning of the pentose phosphate pathway. The aim of this research was to isolate yeasts that can efficiently produce bioethanol and valuable byproducts from both glucose and xylose in a two-stage fermentation process using brewer's spent grains. This approach should maximize sugar utilization and improve the economic viability of bioethanol production while contributing to waste valorization and sustainability. Kluyveromyces marxianus and Candida krusei were identified and tested with different initial concentrations of glucose and xylose. The results showed that both yeasts produced bioethanol from glucose but were inefficient with xylose, yielding valuable compounds, such as 2,3-butanediol and glycerol instead. A two-stage fermentation was then carried out with weak acidic hydrolysate from brewer's spent grain. In the first stage, glucose was fermented by S. cerevisiae to produce bioethanol; in the second stage, xylose was fermented by K. marxianus and C. krusei to obtain other valuable products. [ABSTRACT FROM AUTHOR]

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L−1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 μmol h−1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. To evaluate the effect of NaCl content on microbiological, biochemical, physicochemical, and sensorial characteristics, Munster cheeses were prepared from pasteurized milk seeded with 3 yeasts (Kluyveromyces marxianus, Debaryomyces hansenii , and Geotrichum candidum) and 5 ripening bacteria (Arthrobacter arilaitensis, Brevibacterium aurantiacum, Corynebacterium casei, Hafnia alvei , and Staphylococcus equorum). Experiments were performed in triplicate under 1.0%, 1.7%, and 2.4% NaCl levels in cheese. Ripening (d 2–27) was carried out at 12°C and 96% relative humidity. These kinetics were both reproducible and repeatable at a 99% confidence level. For each microbial, biochemical, and physicochemical parameter, 2 kinetic descriptors (the maximal or minimal rate and its occurrence time) were defined. On d 2, the physicochemical variables (water activity, dry matter, and water content) were strongly dependent on the salting level. From d 2 to d 27, K. lactis was insensitive to salt, whereas D. hansenii was stimulated. Geotrichum candidum growth appeared very sensitive to salt in cheese: at 1.0% NaCl, G. candidum exhibited overgrowth, negatively affecting rind appearance, underrind consistency and thickness, and off-flavor flaws. A salt concentration of 2.4% induced death of G. candidum. A total of 4 bacteria (A. arilaitensis, B. aurantiacum, C. casei , and H. alvei) were moderately sensitive to salt, but S. equorum was insensitive to it. Salt level in cheese had a significant effect on carbon substrate consumption rates. The lactate consumption rate in 1.0% salted cheeses was approximately twice higher than under 2.4% NaCl. Data analysis of microorganism, biochemical, and physicochemical kinetics, as well as sensory analysis, showed that 1.7% NaCl was the best salt level in Munster-type cheeses to achieve an optimum balance between cheese characteristics, sensory qualities, and marketability. [ABSTRACT FROM AUTHOR]

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes. [ABSTRACT FROM AUTHOR]

Featured Application: Single-cell protein is a type of protein derived from fungi and it has gained attention as a meat substitute due to its high protein content, low fat content, and specific texture. However, this type of protein can be used in the development of functional foods as well as additive to high-protein products, protein bars, shakes, snacks, dairy alternatives, etc. In recent years, there has been an increasing demand for new sources of protein, both for human and animal nutrition. In addition to alternative sources of protein, such as algae or edible insects, protein obtained from yeast and mold biomass is becoming more and more important. The main fungal protein producers are the yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida utilis, Yarrowia lipolytica, and the molds Fusarium venenatum, Aspergillus oryzae, and Monascus purpureus. The production of fungal protein has many advantages, including the ability to regulate the amino acid composition, high protein content in dry matter, the possibility of production in a continuous process, independence from climatic factors, and the possibility of using waste substrates as ingredients of media. One of the disadvantages is the high content of nucleic acids, which generates the need for additional purification procedures before use in food. However, a number of enzymatic, chemical, and physical methods have been developed to reduce the content of these compounds. The paper presents the current state of knowledge about fungal producers, production and purification methods, the global market, as well as opportunities and challenges for single-cell protein (SCP) production. [ABSTRACT FROM AUTHOR]

Convergent evolution is central in the origins of multicellularity. Identifying the basis for convergent multicellular evolution is challenging because of the diverse evolutionary origins and environments involved. Haploid Kluyveromyces lactis populations evolve multicellularity during selection for increased settling in liquid media. Strong genomic and phenotypic convergence is observed between K. lactis and previously selected S. cerevisiae populations under similar selection, despite their >100-million-year divergence. We find K. lactis multicellularity is conferred by mutations in genes ACE2 or AIM44, with ACE2 being predominant. They are a subset of the six genes involved in the S. cerevisiae multicellularity. Both ACE2 and AIM44 regulate cell division, indicating that the genetic convergence is likely due to conserved cellular replication mechanisms. Complex population dynamics involving multiple ACE2/AIM44 genotypes are found in most K. lactis lineages. The results show common ancestry and natural selection shape convergence while chance and contingency determine the degree of divergence. In this study, the authors retrieve frozen stocks of the K. lactis lineages and reconstruct the evolutionary dynamics of parallel lineages at the genetic level with high throughput sequencing [ABSTRACT FROM AUTHOR]

Yeasts are gaining increasing attention for their potential health benefits as probiotics in recent years. Researchers are actively searching for new yeast strains with probiotic properties (i.e, Debaryomyces hansenii; Kluyveromyces marxianus; Yarrowia lipolytica; Pichia hudriavzevii; and Torulaspora delbrueckii) from various sources, including traditional fermented foods, the human gut, and the environment. This exploration is expanding the pool of potential probiotic yeasts beyond the well-studied Saccharomyces boulardii. Research suggests that specific yeast strains possess properties that could be beneficial for managing conditions like inflammatory bowel disease, irritable bowel syndrome, skin disorders, and allergies. Additionally, probiotic yeasts may compete with pathogenic bacteria for adhesion sites and nutrients, thereby inhibiting their growth and colonization. They might also produce antimicrobial compounds that directly eliminate harmful bacteria. To achieve these goals, the approach that uses probiotics for human health is changing. Next-generation yeast probiotics are emerging as a powerful new approach in the field of live biotherapeutics. By using genetic engineering, scientists are able to equip these tools with specialized capabilities. However, most research on these probiotic yeasts is still in its early stages, and more clinical trials are needed to confirm their efficacy and safety for various health conditions. This review could provide a brief overview of the situation in this field. [ABSTRACT FROM AUTHOR]

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology. Fungi has been widely used as protein and drug production factories, in which efficient heterologous gene expression is fundamental. This paper provides a genome editing tool kit for heterologous gene expression in Kluyveromyces marxianus. [ABSTRACT FROM AUTHOR]

Microorganisms have significant potential to control fungal contamination in various foods. However, the identification of strains that exhibit robust antifungal activity poses challenges due to highly context-dependent responses. Therefore, to fully exploit the potential of isolates as antifungal agents, it is crucial to systematically evaluate them in a variety of biotic and abiotic contexts. Here, we present an adaptable and scalable method using a robotic platform to study the properties of 1022 isolates obtained from maple sap. We tested the antifungal activity of isolates alone or in pairs on M17 + lactose (LM17), plate count agar (PCA), and sucrose–allantoin (SALN) culture media against Kluyveromyces lactis, Candida boidinii, and Saccharomyces cerevisiae. Microorganisms exhibited less often antifungal activity on SALN and PCA than LM17, suggesting that the latter is a better screening medium. We also analyzed the results of ecological interactions between pairs. Isolates that showed consistent competitive behaviors were more likely to show antifungal activity than expected by chance. However, co-culture rarely improved antifungal activity. In fact, an interaction-mediated suppression of activity was more prevalent in our dataset. These findings highlight the importance of incorporating both biotic and abiotic factors into systematic screening designs for the bioprospection of microorganisms with environmentally robust antifungal activity. [ABSTRACT FROM AUTHOR]

Xiaoqu starter serves as the saccharifying and fermenting agent in the production of Cantonese soybean-flavor (Chi-flavor) Baijiu, and the complex microbial communities determine the flavor and quality of the product. Round-Koji-mechanical starter (produced by using an automated starter-making disk machine) is advantageous as it decreases operator influence, labor costs, and fermentation time, but the product quality is lower compared to traditional starter. Thus, two types of starters (traditional and Round-Koji-mechanical starter) from a Cantonese Baijiu factory were compared in a metagenomic analysis to investigate the differences in microbial community composition and core microbes. The results showed that several core microbes related to carbohydrate metabolism, amino acid metabolism and lipid metabolism, were differentially enriched in the traditional starter. Mucor lusitanicus and Rhizopus delemar were significantly positively correlated with the three key metabolic pathways. Saccharomyces cerevisiae, Cyberlindnera fabianii, Kluyveromyces marxianus, Lactobacillus fermentum, Mucor ambiguous, Rhizopus microspores, Rhizopus azygosporus, Mucor circinelloides, and Ascoidea rubescens were significantly positively correlated with two of the three key metabolic pathways. The results of this study provide a basis for understanding the differential core microbes in traditional and Round-Koji-mechanical starters of Chi-flavor Baijiu, and they also provide guidance for improving Round-Koji-mechanical starter. [ABSTRACT FROM AUTHOR]

In a bid to explore the on-site biorefinery approach for conversion of forestry residues, lignocellulosic biomass into value-added products was studied. The bark white pine wood was subjected to the microwave technique of fast and slow hydrolysis under varying acid and biomass concentrations to produce levulinic acid (LA). The HCl (2% v/v) and plant biomass (1% w/v) were identified as the optimum conditions for fast wood hydrolysis (270 ºC for 12 sec), which led to maximum LA yield of 446.68 g/kg PB. The proposed sustainable approach is mild, quick, and utilized a very low concentration of the HCl for the production of LA. The hydrolysate was used as a medium for Kluyveromyces marxianus growth to produce 2-phenylethanol (2-PE). K. marxianus used 74–95% of furfural from hydrolysate as a co-substrate to grow. The proposed model of the integrated biorefinery is an affordable on-site approach of using forest waste into localized solutions to produce LA and 2-PE. [Display omitted] • Levulinic acid was produced using microwave-based hydrolysis of pine wood. • 2% HCl resulted in maximum levulinic acid production of 446.48 g/kg. • Kluyveromyces marxianus was able to grow on pine wood hydrolysate and produce 2-phenylethanol. • K. marxianus consumed 74–90% of furfural as co-substrate. • Pine wood can be an alternative option for Levulinic acid and 2-phenylethanol production. [ABSTRACT FROM AUTHOR]

The present work examined the production of single-cell protein (SCP) by a newly isolated strain of Kluyveromyces marxianus EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors' knowledge, this is the first report examining the ability of Kluyveromyces marxianus species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain's growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of K. marxianus strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0–100.0 g/L) led to increased ethanol production (Ethmax = 35.5 ± 0.2 g/L), suggesting that K. marxianus strain EXF-5288 is "Crabtree-positive". Batch-bioreactor trials shifted the strain's metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly, K. marxianus strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile [ABSTRACT FROM AUTHOR]

Summary: The aim of this study was to develop and evaluate the bioactive properties and storage stability of microcapsules (MCs) obtained by co‐encapsulation of the potential probiotic yeast Kluyveromyces marxianus VM004 and peanut skin polyphenolic extract (PSE) by spray drying, using whey protein concentrate (WPC) and water‐soluble chitosan (WSCh) as wall materials. The results showed that the selected wall materials provided protection to yeast during spray drying, storage, and simulated gastrointestinal conditions, obtaining better results for WPC and higher concentrations of PSE. Moreover, all formulations demonstrated cytoprotective effects against menadione‐induced oxidative stress in normal rat ileum epithelial cells (IEC‐18) at all concentrations and storage temperatures evaluated. These results suggest that the obtained MCs could be potential functional food ingredients, considering their antioxidant, cytoprotective, and potential probiotic properties. [ABSTRACT FROM AUTHOR]