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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]

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 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.

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]

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]

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]

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]

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23's improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields. The CYR1 mutation can enhance a strain's thermotolerance and ability to produce high yields of recombinant protein by simultaneously strengthening stress resistance and the energy system through cAMP signalling cascades. [ABSTRACT FROM AUTHOR]

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts. • Reprogrammed ABA receptor, PYR1, enables dynamic gene regulation in K. marxianus. • Orthogonal PYR1*MANDI/HAB1* module enables simultaneous gene activation and repression. • Dynamic control of pyruvate flux enhances ethyl acetate biosynthesis. • Time-dependent induction of gene regulation further increases ethyl acetate biosynthesis. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. Key points: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance [ABSTRACT FROM AUTHOR]

Abstract\nHIGHLIGHTSAzo dyes are one of the biggest class of the colorants and their removal from wastewater is still a problematic issue. Bioremoval presents an inexpensive and efficient alternative to conventional removal techniques and efficacy of the process can be increased significantly with supplementation of certain chemicals, such as polymers or surfactants. In this context, the effect of the surfactant sodium dodecyl sulfate on the bioremoval of Cu (II) by the yeast Kluyveromyces marxianus was investigated and found as 83% from the medium containing 25 mg/L Cu (II) and bioremoval yield was increased to 98% when the medium contained 0.1 mmol/L sodium dodecyl sulfate. The optimum conditions for Cu (II) bioremoval were observed in the presence of 25 mg/L initial Cu (II) and 0.1 mmol/L SDS at pH 6 when the agitation speed was 100 rpm and temperature was 30 °C. This study showed that surfactant aiding provides a remarkable strategy for Cu (II) bioremoval by K. marxianus.Effect of surfactants of Cu (II) bioremoval of K. marxianus was investigated.SDS has promotive effect on bioremoval and CTAB negatively affected the process.Bioremoval yield was 98% when the medium was supplemented with 0.1 mmol/L SDS.The qm was detected as 52.0 mg/g in 100 mg/L Cu (II) when 0.1 mmol/L SDS was used. [ABSTRACT FROM AUTHOR]

In this study, we pursued the heterologous expression of the xylanase gene from Trichoderma atroviride, a native fungus in the province of Misiones, and used it to enhance the textural properties of baked goods through varying enzymatic concentrations. This marks the inaugural exploration into its functionality in the context of bread production. The recombinant xylanase exhibited improved activity, reaching 36,292 U L−1, achieved by supplementing the culture medium with dextrose. Following the optimization of recombinant xylanase concentration, promising results emerged, notably reducing hardness and chewiness parameters of bread significantly. Our findings underscore the potential of this native fungal enzyme for industrial processes, offering a sustainable and efficient means to enhance the quality of baked goods with broad implications for the food industry. No prior research has been documented on the heterologous expression of the xylanase gene derived from T. atroviride, from the Misiones rainforest, expressed in Kluyveromyces lactis. Practical Application: This research, focusing on the isolation and cloning of xylanase enzyme from Trichoderma atroviride, a native fungus in the province of Misiones, offers a valuable tool for improving the texture of bakery products. By optimizing enzyme concentrations, our findings present a practical approach for the food industry, offering a viable solution to improve the overall quality and consumer satisfaction of bakery products. [ABSTRACT FROM AUTHOR]

Adaptive changes in cell characteristics are key to resolving challenges in xylitol fermentation involving biomass hydrolysate. This study aims to improve oil palm frond (OPF) hydrolysate utilization through phenotype adaptation of Kluyveromyces marxianus ATCC 36907. Phenotype adaptation was conducted through 25 successive batch cultures. Prior to batch fermentation, critical parameters including inoculum size, cell recycling and strain stability were assessed. The findings indicate that the highest xylitol production was attained with 8% inoculum size, which also led to a shorter lag phase. The adapted strain demonstrated consistent xylitol production for up to 3 cycles of batch cultures when recycled in OPF hydrolysate. Enzymatic assays showed that the adapted strain displayed increased xylose reductase activity, signifying enhanced conversion of xylose to xylitol. It exhibited improved growth and xylitol production compared to the wild-type strain. This highlights its stable and distinctive phenotype from the parental strain, achieving a 73% conversion from the initial concentration of 80 g/L. In shake flask cultures, the adapted yeast displayed more than 55% increase in xylose utilization and 45% improvement in yield compared to the wild-type. These results affirm the effectiveness of phenotype adaptation as a viable strategy to boost xylitol productivity in OPF hydrolysate. [Display omitted] • Phenotype adaptation modifies cell characteristics to convert hydrolysate into xylitol. • Hydrolysate utilization and fermentation performance was improved after 3 cycles. • Adapted strain exhibited high specific growth rates and shorter lag phases. • Cell demonstrated enhanced xylose reductase and NADPH activity compared to wild-type. • Restricted oxygen led to excess of NADH and shortage of NADPH impeding xylose fermentation. [ABSTRACT FROM AUTHOR]

Textile industry creates serious amount of dye effluents. These hazardous molecules threat environment, animal, and plants. Therefore, there is an urgent need to diminish the negative effects of these substances. The current study describes reactive dye removal capacities of newly isolated halotolerant yeast. Because of textile wastewaters contain significant amount of salts, 14 yeasts isolates from cheese and pickle factory wastes were screened for their ability to remove different reactive dyes in molasses media contained NaCl. One isolate showed high Reactive Black 5 removal performance in the presence of 4% NaCl (w/v) and this isolate was identified as Kluyveromyces marxianus. Effects of some important parameters such as pH, NaCl concentration, incubation period, inoculum amount, and initial dye concentration on bioremoval efficiency were investigated. K. marxianus was able to remove 70% Reactive Black 5 at an initial dye concentration of 100 mg/L, pH 6 in the presence of 4% NaCl (w/v) and 10% (v/v) inoculum amount. The maximum specific dye uptake (qm) was obtained as 14.8 mg/g when initial dye concentration was 100 mg/L and this value increased to 33.8 mg/g in the presence of 390 mg/L initial dye which is equal to 27.5% removal of the dye. Based on these findings, the current study shows that halotolerant yeast strains can be evaluated for the removal of salt-contained textile wastewaters. [ABSTRACT FROM AUTHOR]

Mezcal is a traditional Mexican distilled beverage, known for its marked organoleptic profile, which is influenced by several factors, such as the fermentation process, where a wide variety of microorganisms are present. Kluyveromyces marxianus is one of the main yeasts isolated from mezcal fermentations and has been associated with ester synthesis, contributing to the flavors and aromas of the beverage. In this study, we employed CRISPR interference (CRISPRi) technology, using dCas9 fused to the Mxi1 repressor factor domain, to down-regulate the expression of the IAH1 gene, encoding for an isoamyl acetate-hydrolyzing esterase, in K. marxianus strain DU3. The constructed CRISPRi plasmid successfully targeted the IAH1 gene, allowing for specific gene expression modulation. Through gene expression analysis, we assessed the impact of IAH1 down-regulation on the metabolic profile of volatile compounds. We also measured the expression of other genes involved in volatile compound biosynthesis, including ATF1, EAT1, ADH1, and ZWF1 by RT-qPCR. Results demonstrated successful down-regulation of IAH1 expression in K. marxianus strain DU3 using the CRISPRi system. The modulation of IAH1 gene expression resulted in alterations in the production of volatile compounds, specifically ethyl acetate, which are important contributors to the beverage's aroma. Changes in the expression levels of other genes involved in ester biosynthesis, suggesting that the knockdown of IAH1 may generate intracellular alterations in the balance of these metabolites, triggering a regulatory response. The application of CRISPRi technology in K. marxianus opens the possibility of targeted modulation of gene expression, metabolic engineering strategies, and synthetic biology in this yeast strain. [ABSTRACT FROM AUTHOR]

Obesity poses a significant threat to various health conditions such as heart diseases, diabetes, high blood pressure, and heart attack, with the gut microbiota playing a crucial role in maintaining the body's energy balance. We identified a novel probiotic fungal strain, Kluyveromyces lactis JSA 18 (K. lactis), which was isolated from yak milk and was found to possess anti-obesity properties. Additionally, Lactobacillus plantarum CGMCC 8198 (LP8198) from our previous study was also included to evaluate its anti-obesity properties. The findings indicated that K. lactis caused a notable reduction in weight gain, liver and fat indexes, and hyperlipidemia in mice fed a high-fat diet (HFD). Administering K. lactis and LP8198 to mice on a high-fat diet resulted in a reduction of serum triglyceride levels. Furthermore, the supplements reduced ALT and AST activity, and inhibited the production of inflammatory cytokines such as TNF-α and IL-1β. In addition, lipid metabolism was enhanced by the downregulation of ACC1, PPAR-γ, SREBP-1, and Fasn. Moreover, this study found that K. lactis and LP8198 have little effect on gut bacteria. Additionally, K. lactis partially influenced intestinal fungi, while LP8198 had a minor influence on gut mycobiota. The main goal of this research was to show how effective K. lactis can be as a probiotic in combating obesity. [ABSTRACT FROM AUTHOR]

Pine needle waste biomass is a troublesome forest biomass from the mid Himalayan region. Every year, it causes number of forest fire incidents causing a significant damage to the Himalayan ecology and environment. However, this forest waste biomass may be utilized to produce various value-added products, including biofuels. Pine needle waste biomass (PNWB) obtained from the Himalayan forests was chopped, ground, and subjected to physico-chemical pre-treatments (1.0% NaOH + Microwave (900 W for 12 min) that resulted in 72.58 ± 0.46% wt/v lignin removal. Saccharification of the resultant lignin-free PNWB with xylanase from Bacillus pumilus XRL5 and cellulase from Bacillus subtilis CPS-66 enzymes (2:4 U/g of pine needles) for 48 h at 45 °C produced 25.64 ± 2.36 mg/ml of fermentable sugars. Subsequent 1.5 l co-fermentation of PNWB fermentable sugars with Schizosaccharomyces sp. EF-3 and Kluyveromyces marxianus MTCC 1498 produced 17.65 g/l ethanol with 86.6% purity and 91.22% fermentation efficiency. Findings of the present study suggest that pine needle waste biomass from the forest could be utilized as an important bioresource for bioethanol production, which will not only help in protecting the Chir-Pine forests from devastating forest fires but will also help in protecting the biodiversity and environment. [ABSTRACT FROM AUTHOR]

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.

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is an indispensable element in many physiological processes, while alterations in its level in the circulating system are signs of pathology-associated diseases. Therefore, its precise and selective detection is critical for clinical applications to monitor the progression of the pathology. In this study, an optical immunoassay biosensor was developed as a model study for detecting recombinant VEGF165. The VEGF165 sample was purified from recombinant Kluyveromyces lactis GG799 yeast cells. Indirect ELISA was used during the detection, wherein iron oxide nanoparticles (FeNPs) were utilized to obtain optical signals. The FeNPs were synthesized in the presence of lactose p-amino benzoic acid (LpAB). VEGF165 antibody was conjugated to the LpAB-FeNPs through EDC/NHS chemistry to convert the iron oxide nanoparticles into VEGF165 specific probes. The specificity of the prepared system was tested in the presence of potential serum-based interferents (i.e., glucose, urea, insulin, C-reactive protein, and serum amyloid A), and validation studies were performed in a simulated serum sample. The proposed immunoassay showed a wide detection range (0.5 to 100 ng/mL) with a detection limit of 0.29 ng/mL. These results show that the developed assay could offer a sensitive, simple, specific, reliable, and high-throughput detection platform that can be used in the clinical diagnostics of VEGF. [ABSTRACT FROM AUTHOR]

Durango State has the denomination of origin for the production of mezcal, which is made from Agave durangensis, mainly in an artisanal way; therefore, differences in the fermentation process affect the quality of the final product. The main objective of the present study was to evaluate the diversity of culturable yeasts involved in the artisanal and semi-technified process of mezcal production in the State of Durango. Three distilleries with different production processes were monitored at different fermentation stages (beginning, mid-fermentation, and end of fermentation) in the spring and summer seasons. A greater diversity was found in the distillery of Nombre de Dios in both the spring and summer production seasons (H' = 1.464 and 1.332, respectively), since it maintains an artisanal production process. In contrast, the distillery of Durango, where a Saccharomyces cerevisiae commercial inoculum is used to start fermentation, presented low diversity indexes (H' = 0.7903 and 0.6442) and only S. cerevisiae, Kluyveromyces marxianus, and, sporadically, Pichia manshurica were found. Results suggest that the yeast microbiota involved in mezcal fermentation during the different seasons is affected by the type of inoculum; changes include the presence of some species that were only identified during a specific season in alcoholic fermentation, such as Torulaspora delbrueckii and Pichia kluyveri. [ABSTRACT FROM AUTHOR]

Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the "beany" off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae (S. cerevisiae) and Kluyveromyces marxianus (K. marxianus) are often used as fermenters in yogurt and alcohol, and have been less studied within meat products. The yeasts were added to sauce meat, and the uninoculated group served as a control in this study to examine and compare the changing patterns of physicochemical and flavor characteristics of S. cerevisiae and K. marxianus on sauce meat during storage. The changes in moisture content, aw, pH, thiobarbituric acid reactive substances (TBARS), and other flavor characteristics were measured in sauce meat during the first, second, fourth, and sixth months after production. The following factors were examined: moisture content, aw, pH, TBARS, peroxide value (POV), acid value (AV), soluble protein (SP), free amino acid (FAA), and volatile flavoring compounds. With VIP > 1 and p < 0.05 as the screening conditions, the partial least squares model (PLS-DA) was used to assess the distinctive flavor components in the sausages. The findings demonstrated that the three groups' changes in sauce meat were comparable during the first two months of storage but differed significantly between the 4th and 6th months. The moisture content, water activity, and pH of the sauce meat decreased gradually with the storage time; TBARS, AV, and FAA increased significantly; SP decreased significantly from 2.61 to 1.72, while POV increased to 0.03 and then decreased to 0.02. The POV and TBARS values of the yeast-infected meat were substantially lower than those of the control group, and the POV and TBARS values of the meat inoculated with S. cerevisiae were particularly decreased (p < 0.05). The POV and TBARS values of SC (S. cerevisiae group) decreased by 49.09% and 40.15%, respectively, compared to CK (the control group) at the time of storage until June. The experimental group (KM: K. marxianus group) significantly increased the SP and FAA values of the sauce meat (p < 0.05) by 32.4% and 29.84% compared to the CK group, respectively. Esters and olefins as well as alcohols and esters were much greater in meat that had been supplemented with S. cerevisiae and K. marxianus than in meat from the control group. In conclusion, inoculating sauce meat with S. cerevisiae can significantly enhance the quality and flavor of sauce meat while it is being stored. [ABSTRACT FROM AUTHOR]

Background: The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins. Results: We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels. Conclusion: The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories. [ABSTRACT FROM AUTHOR]

In this paper, a local isolated yeast strain was studied for bioethanol production using protein-free cheese whey powder (CWP) in batch alcoholic fermentation experiments. Ethanol tolerance tests were performed with different ethanol concentrations (from 0% to 20%). The maximum ethanol tolerance obtained for our strain was 5% (v/v). The influence of initial lactose concentration, temperature and pH on fermentation kinetic was studied. For initial lactose concentration ranging from 57.5 to 260 gL-1, the highest ethanol production 34.8 gL-1 was obtained at 109 gL-1 initial lactose concentration corresponding to 0.413 gg-1 ethanol yield (YP/S); (While it was noticed less ethanol production, namely 0.178 g g-1) at a higher initial whey sugar concentration of 260 gL-1. The effect of the initial pH on the fermentation was studied at three different values: 3, 4 and 5. The ethanol production reached a maximum of 22 g L-1 with a lactose consumption of 77.62% at pH 4. However, an observed decrease occurred in ethanol production and lactose consumption at pH 3, with values of 10.34 gL-1 and 24.46%, respectively. For the fermentation conducted at the three temperatures of 37°C, 40°C and 43°C, the maximum ethanol production and lactose consumption were 34.82 gL-1 and 80.75%, respectively, achieved at 37°C. The experimental data was analyzed using the Gompertz model to obtain the following kinetic parameters: the maximum ethanol concentration (Pm) was found to be 36.97 gL-1, the maximum ethanol production rate (rp,m) was 0.402 gL-1h-1 and the lag phase (tl ) was 10.8 h. The comparison of the isolated Kluyveromyces marxianus TXID4911 strain with the reference strain Kluyveromyces marxianus DSM 5421, showed a relatively close ethanol yield (YP/S) 0.415 and 0.505 gg,-1 respectively [ABSTRACT FROM AUTHOR]

Non-conventional yeasts (NCYs) (i.e., non-Saccharomyces) are used as alternative starters to promote aroma complexity of fermented foods (e.g., bakery products). A total of 66 yeasts isolated from artisanal food matrices (bread and pizza sourdoughs and milk whey) from different geographical areas of the Campania region (Italy) were screened for physiological and technological characteristics such as leavening ability, resistance to NaCl and pH, exopolysaccharide and phytase activity production, and carbohydrate assimilation. Selected and isolated microorganisms were also used to study the leavening kinetics in experimental doughs as mixed inocula of two different strains. Volatile organic compounds (VOCs) of the inoculated doughs were analyzed with solid-phase microextraction/gas chromatography–mass spectrometry (SPME/GC-MS). Most of the strains belonged to non-Saccharomyces species (Pichia kudriavzevii, Kluyveromyces marxianus) and Saccharomyces (S. cerevisiae). Several strains produced exopolysaccharides (EPSs), that are important for dough rheological properties. Moreover, yeasts isolated from whey showed extracellular phytase activity. The mixed starter culture of the S. cerevisiae and NCY strains showed a synergic effect that enhanced the doughs' aroma complexity. The use of non-conventional yeasts mixed with S. cerevisiae strains can be advantageous in the bakery industry because they improve the bread aroma profiles and nutritional properties by bioactive molecule production. [ABSTRACT FROM AUTHOR]

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The aim of the study was to evaluate the impact of probiotics obtained from an agroindustrial waste substrate fermented with lactic acid bacteria and/or yeasts on the health and changes in the microbiota of the digestive tract of guinea pigs. Eighty male guinea pigs, Kuri breed, 30 days old and 250 g live weight, were randomly selected and divided into four groups of 20 animals each: T0, control; T1, Lactobacillus acidophilus and L. bulgaricus; T2, Saccharomyces cerevisiae and Kluyveromyces fragilis; and T3. L. acidophilus, L. bulgariccus, S. cerevisiae and K. fragilis. T1, T2 and T3 contained molasses-vinasse substrate in their base, the dose administered was 1.00 mL/animal orally every 3 days. The indicators evaluated were weight gain, occurrence of diarrhea and mortality, macroscopic lesions in the digestive tract organs and changes in the microbiota of the stomach, caecum, small and large intestine. Treatments T1, T2 and T3 improved weight gain (p < 0.05) and reduced the presence of guinea pigs with diarrhea (p < 0.05) and there was no mortality; animals in the control group presented a greater amount (p < 0.05) of macroscopic lesions in the digestive tract organs; in the T1, T2 and T3 groups there was an improvement in the natural microbiota. It is concluded that the inclusion of a microbial additive in young guinea pigs improves intestinal health and consequently improves weight gain, reduces diarrhea and deaths and normalizes the natural microbiota of the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, PIT350 and PIN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of PIN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of PIN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used PIN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

Spirulina is widely known as the “Super Nutritious Food of the Future” due to its exceptional nutritional properties. In this article, we investigated the effects of fermentation using a combination of Lactobacillus acidophilus KDB-03 and Kluyveromyces marxianus GDMCC 2.119 on Spirulina. Fermentation significantly increased the total flavonoid and phenol levels in Spirulina, and enhanced the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity and ferric-reducing antioxidant power. Untargeted ultra-performance liquid chromatography-electrospray tandem mass spectrometry-based metabolomics and gas chromatography-ion mobility spectrometry analysis revealed alterations in the metabolites and flavor components of fermented Spirulina (FS). Furthermore, a mice experiment demonstrated that FS could effectively alleviate metabolism-associated fatty liver disease (MAFLD) by regulating liver metabolism, intestinal flora, and facilitating communication between the liver and gut. These findings suggest that fermentation could be a innovative approach to enhance the diversity of Spirulina products, and potentially be used as a strategy for the prevention and treatment of MAFLD.

Compared with the rice-acid soup inoculated with single starter, the synergistically intensified rice-acid soup inoculated with Lactobacillus paracasei H4-11 (L. paracasei H4-11) and Kluyveromyces marxianus L1-1 (K. marxianus L1-1) contained more flavor compounds. Organic acids mainly included L-lactic acid and the main volatile flavor component was ethyl acetate. Moreover, the signal intensity of astringency and bitterness and the total concentration of volatile sulfur compounds were reduced. The combined analysis results of RNA sequencing (RNA-seq) technology and 4D label-free quantitative (4D LFQ) proteomics explained the flavor formation pathways in rice-acid soup inoculated with L. paracasei H4-11 and K. marxianus L1-1. In L. paracasei H4-11, L-lactate dehydrogenase, phosphoglucomutase, acetate kinase, alcohol dehydrogenase and acetyl-CoA were up-regulated and D-lactate dehydrogenase and N-Acetyltransferase were down-regulated. In K. marxianus L1-1, Acetyl-CoA, acetaldehyde dehydrogenase, acyl-coenzyme A, N-acetyltransferase, and L-lactate dehydrogenase were up-regulated and hexokinase, alcohol dehydrogenase, and alcohol O-acetyltransferase were down-regulated. The above up-regulation and down-regulation synergistically promoted the formation of characteristic flavor compounds (mainly L-lactic acid and ethyl acetate). Enzyme-linked immunoassay (ELISA) and parallel reaction monitoring (PRM) quantitative analysis respectively verified that 5 key metabolic enzymes and 27 proteins in L. paracasei H4-11 and K. marxianus L1-1 were associated with the characteristic flavor of rice-acid soup, as confirmed by the quantitative results of 4D LFQ.

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.

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.