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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]

β-galactosidase from Kluyveromyces lactis has been immobilized on chitosan-glutaraldehyde particles employing different enzyme loadings (in a range of 0.7–7 mg/g of support). The biocatalysts had been later utilized to produce lactulose by transglycosylation using milk whey supplemented with fructose. The amount of produced lactulose decreased when the enzyme loading increased, from around 18 mg/mL using the biocatalysts with only 0.7 mg/g to around 7 mg/mL using the biocatalyst with a loading of 7 mg/mL. The results showed in this paper shows the great effect of the substrates and product concentration gradients inside the biocatalyst particle, not only lactulose, but also glucose and galactose. That is, the use of a excessive enzyme loading can lead to a significant worsening of the β-galactosidase biocatalyst performance in kinetically controlled synthesis. [Display omitted] • β-galactosidase was immobilized using different biocatalysts enzyme loadings. • The biocatalysts were used to produce lactulose from lactose and fructose. • The higher the enzyme loading, the lower the lactulose production. • The substrate and products internal gradients explain the results. [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]

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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]

Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, and GAL10) for galactose catabolism through the Leloir pathway, and a "GALLAC" cluster containing the transcriptional activator gene LAC9, second copies of GAL1 and GAL10, and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences. IMPORTANCE This study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia. Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products. [ABSTRACT FROM AUTHOR]

The yeast microflora of various dairy products sold in Russia was studied using RFLP analysis of the 5.8S-ITS rDNA fragment and sequencing of the D1/D2 domain of 26S rDNA. Most of the fermented milk products studied were dominated by lactose-utilizing yeasts Kluyveromyces and Debaryomyces and by lactose-negative yeasts Saccharomyces, Monosporozyma, Pichia, Geotrichum, and Yarrowia. The yeast Kluyveromyces marxianus was present in most of the fermented milk products studied, while the related species K. lactis was found only in some samples of ayran, curds, and cheese. The dominance of K. marxianus is apparently associated with their physiological characteristics (thermo- and osmotolerance), which provide these yeasts with better adaptation to industrial fermentation conditions. The dominant species in mixed-fermentation dairy products, Saccharomyces cerevisiae and Monosporozyma unispora, were completely absent in cheeses and lactic acid fermentation products. In general, the species composition of yeasts largely depended on the fermented milk product, the type of milk, and the specific manufacturer. [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]

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]

Kluyveromyces marxianus, a thermotolerant, fast‐growing, Crabtree‐negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology‐directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR‐Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA‐gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12‐kb cassette from three fragments and marker‐free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production. [ABSTRACT FROM AUTHOR]

As the most abundant by-product of the brewing industry, the valorization of proteins from brewers' spent grains (BSG) is critical to reducing processing waste and meeting the growing demand for plant-derived protein globally. However, the applicability of brewers' spent grain proteins in food manufacturing has been constrained by the high extraction cost and undesirable functionalities. In this research, BSG was used as the substrate to ferment with the mixed strains Bacillus subtilis, Saccharomyces cerevisiae, and Kluyveromyces marxianus and then subjected to protein extraction. The solid-state fermentation (SSF) conditions were optimized using single-factor tests together with Box-Behnken Design to 50% initial moisture content, 10% strain inoculum ratio, fermented at pH 5.8 under 32 °C for 6 days, with the highest soluble protein concentration of 0.678 ± 0.0047 µg/µL (equivalent to bovine serum albumin). Furthermore, proteins were extracted from unmalted raw barley grains and BSG control (with a yield of 0.338 ± 0.016 µg/µL and 0.352 ± 0.015 µg/µL, respectively) and then evaluated for their physiochemical characterizations as a comparison of solid-state fermented proteins. The physicochemical attributes of all the intact proteins were similar, except that the fermented samples showed a smaller molecular mass on the SDS-PAGE gel. In conclusion, these results indicated that SSF is a feasible strategy for the bioconversion of BSG into valuable food ingredients as it enhances protein extraction efficiency while reducing the production cost, thereby being beneficial to alleviate the associated environmental pollution and to broaden its possibilities for further integration into the circular economy. [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]

ABSTRACT 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]

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]

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.

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.

The experiment aimed to screen probiotic yeast and provide a basis for subsequent yeast use as feed additives. Use yeast enrichment medium to isolate and screen yeast from Lanzhou fermented grains and sweet embryos sold, and evaluate their probiotic properties and safety. The results showed that two strains of Pichia kudriavzevii, one strain of Clavispora lusitaniae from Portugal, one strain of Saccharomyces cerevisiae, and one strain of Kluyveromyces marxianus were obtained from Lanzhou fermented grains and sweet embryo. Among them, strain JM-4 had a short fermentation completion time, strong environmental adaptability, and strong vitality. It could survive in intestinal and gastric fluids and had good growth and probiotic properties. At the same time, this strain had no obvious toxic effects in mice, displaying good security. The research indicates that the five experimental strains of yeast show good safety for mice, making them suitable for the preparation of probiotic formulations, providing a reference for the use of yeast as feed additives. [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]

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]

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]

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]

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]

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]

Mixture designs are employed to systematically change the composition of mixtures and investigate how those changes impact their properties. However, all mixture designs currently available are impractical for analyzing mixtures with relatively large numbers of ingredients. In response, this article presents a novel solution that builds on the construction of a new experimental design called "fractional mixture design". The design involves screening the ingredients in mixtures and enables the subsequent construction of a classical mixture design for optimizing mixtures. The design and its accompanying methodology were developed to analyze native strains found in successful spontaneous fermentations with the goal of constructing a mixed starter culture to transition from spontaneous to directed fermentation in the production of agave distillates. The results showed that a starter culture composed of the native strains Kluyveromyces marxianus, Clavispora lusitaniae, and Kluyveromyces marxianus var. drosophilarum, in respective proportions of 35%, 32%, and 33%, enabled the production of a fermented product with 2.1% alcohol and a broad profile of aromatic compounds. Hence, the results show, for the first time, a tool that addresses the technical challenge that allows studying a relatively large number of ingredients in mixtures and a two-stage sequential methodology to construct optimal mixtures. [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]

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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

Ergosterol, as a precursor for synthesising useful molecules like vitamin D2, possesses significant physiological functions in both fungal and human systems. In fungi, ergosterol plays a crucial role in stress responses. In contrast to Saccharomyces cerevisiae yeast, the changes in specific ergosterol content of Kluyveromyces marxianus under various stress conditions are less known. This study investigated how ergosterol content changes in response to different stress factors. Carbon to nitrogen (C/N) ratio was examined using experimental design. The effects of aeration and shear force beside constant overall volumetric mass transfer coefficient (KLa) were examined. Cell growth and specific ergosterol content were investigated using ethanol stress during a two-stage fermentation. Based on the results, contradictory settings regarding C/N ratio and shear force were found to be favourable for cell growth and specific ergosterol content. However, increased aeration consistently elevated specific ergosterol content and favoured cell growth as well (2.5-fold and 1.5-fold, respectively). In K. marxianus fermentations, higher ergosterol yield can be achieved through a two-stage fermentation (138.9 mg L−1 compared to 52.9 mg L−1), where the first stage provides favourable conditions for cell growth, and the second stage involves stress (beneficial for ergosterol production) conditions. Conclusions drawn from the two-stage fermentation results suggest that early transitioning of cell growth to the second phase will not result higher adaption and specific ergosterol content compared to the transition at the end of exponential growth phase. [ABSTRACT FROM AUTHOR]

Featured Application: In this work, cheese whey, which is a by-product of the cheese industry, was used to obtain lactic acid by biotechnological processes (using strains isolated from the same cheese whey). Lactic acid has several uses in the cosmetic, food, medical, textile, and manufacturing industries. Therefore, while lactic acid can have multiple uses, we want to use it as a raw material for biopolymers for the manufacturing of bioplastics in future research. Cheese whey is a byproduct of the cheese industry that causes high levels of pollution in the environment, but its high lactose content means that it can be used as a source to obtain lactic acid. In this study, two strains, one belonging to a yeast and the other one to a bacteria (Kluyveromyces lactis and Enterococcus faecalis), were isolated from cheese whey and molecularly characterized, and the optimal growth conditions were determined. Then, using proteinized and deproteinized cheese whey, batch fermentation was carried out with the strains arranged in suspension and immobilized. The consumption of lactose and the production of lactic acid were measured through Brix degrees and acidity analysis. Afterwards, the lactic acid was purified, and its yield and physical and chemical characteristics were determined. It was proven that there were differences between each of the strains; arranged in free or encapsulated cells, the proteinized and deproteinized cheese wheys, under the same purification conditions, achieved different yields, colors, and densities of lactic acid. Immobilized Enterococcus faecalis had the highest yield (50.61 ± 34.94 g/L) using the deproteinized cheese whey compared to the immobilized Kluyveromyces lactis (35.70 ± 0.15 g/L) using the proteinized cheese whey. [ABSTRACT FROM AUTHOR]

Whey, a primary byproduct of cheese production, is rich in organic content, largely due to its lactose concentration (45–60 g/L). Cheese production yields three types of residues: sweet whey (SW), acid whey, and ricotta whey (RW). Each has unique physicochemical properties that present challenges for small to medium-sized enterprises because of the expensive treatment required. One solution to this problem is fermenting these substrates to produce value-added products. This study aimed to assess the fermentation of the three RW types using Kluyveromyces marxianus ITD00262 for isoamyl acetate production, a compound that gives a banana-like aroma. Several factors were analyzed: cell growth, pH changes, lactose content reduction, ethanol production, and isoamyl acetate formation. Cell growth was consistent across all whey types. As for pH, there was a drop during the initial 48 h to roughly 4.3, after which it rose to near 7. In acid whey, 50.95 g/L of ethanol was produced by the 120-h mark. The peak isoamyl acetate concentration was observed at 24 h, registering 160 mg/L in YPL medium, while SW peaked at 124 mg/L at 72 h. Lastly, the highest lactose consumption was seen in SW, approximately 76% at 24 h, surpassing that of acid and ricotta whey, which fluctuated between 56 and 58%. [ABSTRACT FROM AUTHOR]

IntroductionThe 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.MethodsThis 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.ResultsThe 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.ConclusionKluyveromyces 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.

Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 22 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 22 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.

Abstract Kluyveromyces marxianus is a food‐safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large‐scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high‐density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.