Search

Lactulose is a prebiotic that has found a wide application in medicine and food industry. Commercial lactulose is usually synthesized by isomerization in alkaline media at high temperatures. Enzymatic methods offer a more sustainable alternative and require more moderate processing conditions. This review covers 44 years of scientific publications (1978–2022) on the enzymatic synthesis and purification of lactulose. The materials were retrieved from Scopus, Web of Science, PubMed, and Elibrary databases. The enzymatic approach to lactose-to-lactulose conversion has two methods: isomerization (direct) and transgalactosylation (via hydrolysis). Isomerization exploits cellulose-2-epimerases, but their safety status is still rather vague. As a result, cellulose-2-epimerases are not commercial. Epilactose is a by-product of isomerization. Transgalactosylation involves β-galactosidases with an official international safety status (GRAS). It is available on the market, and its action mechanism is well understood. This article systematizes various data on the conditions for obtaining the maximal yields of lactulose by different enzymes. The Kluyveromyces lactis yeast and the Aspergillus oryzae mold are the main sources of β-galactosidases in lactulose production. The yield can reach 30% if the processing conditions are optimal. Fructose remains the main problem in the production process. No scientific publications revealed a direct relationship between the maximal yields of lactulose and the molar fructose-tolactose ratios. Cellobiose epimerases make it possible to achieve high yields of lactulose (70–80%). However, these enzymes are associated with genetic engineering and mutagenesis, which challenges their safety status. The most promising trends in lactulose biotechnology include secondary dairy raw materials, immobilized enzymes, membrane reactors, complex production processes, lactose-to-lactulose conversion, and purification of final product.

The processing of dairy products currently generates significant amounts of waste, particularly in the form of liquid whey. The disposal of whey poses a challenge to the environment due to its high organic content and biological oxygen demand. Whey contains lactose, soluble proteins, lipids, and minerals. While Saccharomyces cerevisiae can efficiently utilize glucose, they are unable to metabolize lactose. In contrast, Kluyveromyces spp. encode two genes, Lac12 and Lac4 that enable conversion of lactose to other by-products such as ethanol. Here, we selected five Kluyveromyces yeast inoculated into three different types of whey substrates, cheddar sweet whey, cream cheese acid whey, and yogurt acid whey that could be used to convert lactose into ethanol. We demonstrate that differences exist in ethanol production across different whey substrates inoculated with Kluyveromyces yeast. In sweet whey, K. lactis, K. lactis Y-1205 and K. lactis Y-1564 were the highest ethanol producing strains. The highest amount of ethanol produced was 24.85 ± 3.5 g/L achieved by Y-1564 in sweet whey (96.8% efficiency). K. lactis Y-1205 produced 22.39 ± 5.6 g/L ethanol in yogurt acid whey. In cream cheese acid whey, K. lactis strains produced significantly higher ethanol levels compared to S. cerevisiae and K. marxianus (p < 0.05). Outcomes from this study could provide a simple and cheap solution for small-to medium-sized dairy processing facilities to ferment lactose in whey into ethanol using lactose-consuming yeasts. [ABSTRACT FROM AUTHOR]

In Mexico, agave has a significant cultural and economic impact via its use as a raw material for producing fermented drinks. The agave plant grows in arid and nitrogen-deficient soil where it associates with fungi and bacteria to meet nutrient demands, particularly under extreme conditions of fungal growth; this community of microorganisms is called the microbiome. In the microbiome, aromatic volatile synthesis activates the defense response, resistance to abiotic stress, and plant growth. These aromatic volatiles also impact the organoleptic characteristics of fermented beverages and are produced by microorganisms during agave fermentation. This research presents the behavior of K. marxianus cultivated in the presence of extreme and deficient nitrogen concentrations. In addition, a new biological relationship between K. marxianus ITD0090 from mezcal fermentation and Bacillus tequilensis ITD-UANL-01 is reported, confirmed by fluorescence microscopy and intracellular recovery. Our results also reveal that B. tequilensis ITD-UANL-01 is a nitrogen-fixing bacterium that provides assimilable nitrogen to K. marxianus under nitrogen starvation conditions. This phenomenon allows for 144 h of viability without nitrogen. Furthermore, this is the first report of an endosymbiont of K. marxianus ITD0090 in fermentation. [Display omitted] • Kluyveromyces marxianus ITD0090 remains viable under nitrogen starvation stress for 144 h. • Bacillus tequilensis ITD-UANL-01 is the endosymbiont identified in K. marxianus ITD0090. • B. tequilensis ITD-UANL-01 can fix nitrogen and gives to K. marxianus ITD0090 assimilable nitrogen in NH 4 form. [ABSTRACT FROM AUTHOR]

The processing of dairy products currently generates significant amounts of waste, particularly in the form of liquid whey. The disposal of whey poses a challenge to the environment due to its high organic content and biological oxygen demand. Whey contains lactose, soluble proteins, lipids, and minerals. While Saccharomyces cerevisiae can efficiently utilize glucose, they are unable to metabolize lactose. In contrast, Kluyveromyces spp. encode two genes, Lac12 and Lac4 that enable conversion of lactose to other by-products such as ethanol. Here, we selected five Kluyveromyces yeast inoculated into three different types of whey substrates, cheddar sweet whey, cream cheese acid whey, and yogurt acid whey that could be used to convert lactose into ethanol. We demonstrate that differences exist in ethanol production across different whey substrates inoculated with Kluyveromyces yeast. In sweet whey, K. lactis, K. lactis Y-1205 and K. lactis Y-1564 were the highest ethanol producing strains. The highest amount of ethanol produced was 24.85 ± 3.5 g/L achieved by Y-1564 in sweet whey (96.8% efficiency). K. lactis Y-1205 produced 22.39 ± 5.6 g/L ethanol in yogurt acid whey. In cream cheese acid whey, K. lactis strains produced significantly higher ethanol levels compared to S. cerevisiae and K. marxianus (p < 0.05). Outcomes from this study could provide a simple and cheap solution for small-to medium-sized dairy processing facilities to ferment lactose in whey into ethanol using lactose-consuming yeasts.

Besides organic compounds such as lactose and proteins, cheese whey is rich in other nutrients. Damping of these valuable compounds to the environment, first, harms the environment, and second, it wastes valuable resources. Therefore, this review aims to find out the current progress on the valorization of cheese whey for ethanol production. Efficient ethanol-producing yeasts like Saccharomyces cerevisiae have no pathway to utilize lactose and, therefore, they can be co-cultured with microbes that can produce β-galactosidase. In addition, chemical, biological, and physical hydrolysis of lactose can be used to produce ethanol from cheese whey. Ethanol production from unsterilized or unpasteurized whey is very promising and this reduces the production cost significantly. This suggests that the ethanol-producing yeasts are competent against the lactic acid bacteria that are commonly found in cheese whey. Despite the presence of central metabolic genes associated with ethanol production from different sugars in some yeasts, these yeasts can’t ferment the different sugars and this is basically due to a lack of the different sugar transport systems in the yeasts. Therefore, additions of different sugars to whey to increase the sugar content for economical ethanol production are impaired by catabolite repressions. However, catabolite repression can be significantly reduced by metabolic engineering by targeting sugar transporter proteins like the major facilitator superfamily (MFS), particularly LAC, CEL2, HGT, RAG, and KHT. Therefore, this enhances ethanol production from cheese whey supplemented with a variety of sugars. Currently, nanoparticles and metal-organic frameworks coated immobilization of S. cerevisiae produced higher ethanol from lignocellulosic substrates than the classical carries such as alginates; however, studies of such immobilizing materials on Kluveromyces spp for ethanol production are very limited, and open for research. Electro-fermentation, an emerging bioprocess to control microbial fermentative metabolism, boosts ethanol production, enables the production of 14% (v/v) ethanol, and shortens the fermentation time of high sugar-containing whey. Generally, utilizing efficient yeast (possibly by adaptive evolution and genetic engineering) at optimal fermenting conditions enabled to production of economical ethanol from cheese whey that contains higher sugars (greater than 15%) at the large-scale cheese processing industries.

The enzyme β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, although for its effective application it is necessary to establish techniques for purification, concentration, or polishing, such as membrane separation processes, in particular ultrafiltration. The present study aimed to investigate ultrafiltration and diafiltration applied as initial steps for concentration and salt removal, respectively, in the β-galactosidase purification processes. Additionally, the influence levels of the pH (6.5, 7.7, or 7.5), membrane molecular weight cut-off (30, 50, 60, or 100 kDa), operating pressure (1.5, 2.0, or 2.5 kgf/cm2), and ionic strength of the ultrafiltration using NaCL or KCl (0.01–0.1 M) were evaluated considering the enzyme recovery, purification, retention, and concentration factors in relation to the proteins, volume, activity, and protein flux and yield of the processes. The ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm2 with a 50 kDa polyethersulfone membrane resulted in a volume concentration of the β-galactosidase extract up to 7.1-fold greater, a purification factor 1.2-fold greater, and an enzyme recovery rate of 108.9% by eliminating metabolites during the purification process. In addition, the lowest flux variation range (16.0 to 13.1 L/m2·h) was observed under these same conditions, thereby representing a decrease of 18.0%. An increase in the operating pressure and the addition of salts results in reduced enzyme recovery (up to 38% of the process yield (734.1 to 453.7 U/h) and up to 40% of the enzyme recovery rate (108.9 to 60.6%) during the ultrafiltration using NaCl, respectively). The operation in the diafiltration mode allowed salt removal after the purification of β-galactosidase (enzymatic recovery rates above 93.4%) via precipitation and ion-exchange chromatography elution and as part of an aqueous two-phase system using 6 diafiltration cycles, thereby revealing its application potential. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effects of different feeding strategies in fed-batch cultivations on the production of β-galactosidase fused to a cellulose-binding domain (CBD) by Escherichia coli C41(DE3) using a bench bioreactor. The highest enzyme activities (∼17,000 U/L) were obtained in fed-batch cultivations controlled by DO-stat and induced with lactose (5 g/L) at 18 h of cultivation. Under these cultivation conditions, the maximum values of β-galactosidase yields per substrate, β-galactosidase yields per biomass, and Q P were approximately 950 U/g glucose , 1100 U/g cells , and 540 U/L.h, respectively, using Terrific Broth as culture medium and concentrated feed solution (2 ×). The dairy coproducts were efficient in inducing the expression of the recombinant enzyme, having reached the highest values of β-galactosidase-CBD activity after 40 h of cultivation. The maximum enzyme activities obtained when using whey permeate or ricotta whey as inducers were 22,950 or 24,640 U/L, respectively. Using the optimal temperature of E. coli growth before induction and ricotta whey as an inducer increased enzyme productivity in 50%. Plasmid construction showed high stability (>94%) throughout E. coli cultivation. This study demonstrated the feasibility of using the DO-stat feeding strategy to produce β-galactosidase-CBD employing ricotta whey as an inducer of enzyme expression. [Display omitted] • β-Galactosidase was produced at a benchtop bioreactor in fed-batch cultivations. • Lactose was used to induce the recombinant β-galactosidase expression. • DO-stat feeding strategy resulted in the highest kinetic parameters of cultivations. • The higher β-galactosidase activity was reached using ricotta whey as inducer. [ABSTRACT FROM AUTHOR]

Dahi and chhurpi are the homemade, mildly acidic and mouthfeel fermented dairy products of Sikkim in India. Since yeasts co-exist among traditional fermented dairy foods, we believe that some species of yeasts may have some probiotic properties. Hence, the present study is aimed at screening some probiotic yeasts from dahi and chhurpi. A total of 3438 yeasts were isolated from 40 samples of dahi (1779 isolates) and 40 chhurpi (1659 isolates) and were preliminarily screened for probiotic properties on the basis of survival in low pH, resistance to bile salts and the percentage of hydrophobicity, out of which only 20 yeasts were selected for in vitro and genetic screening of probiotic properties. Saccharomyces cerevisiae DJT-2 and Debaryomyces prosopidis CPA-55 showed the highest hydrophobicity of 97.54% and 98.33%, respectively. S. cerevisiae DRC-42 and S. cerevisiae CGI-29 showed 93.88% and 91.69% auto-aggregation, respectively. All yeasts showed co-aggregation properties against pathogenic bacteria. Kluyveromyces marxianus DPA-41 and Pichia kudriavzevii CNT-3 showed excellent deconjugation activities. Probiotic genes for acid tolerance, bile tolerance, adhesion and antimicrobial activity were detected in S. cerevisiae DAO-17, K. marxianus DPA-41, S. cerevisiae CKL-10 and P. kudriavzevii CNT-3. Based on the results of in vitro and genetic screening of probiotic yeasts strains, S. cerevisiae DAO-17 (dahi), S. cerevisiae CKL-10 (chhurpi), P. kudriavzevii CNT-3 (chhurpi) and K. marxianus DPA-41(dahi) were selected as the potential probiotic yeasts. [ABSTRACT FROM AUTHOR]

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

The conserved decapping enzyme Dcp2 recognizes and removes the 5' eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K. lactis Dcp1-Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1-Dcp1-Dcp2-Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis.

The central dogma processes of DNA replication, transcription, and translation are responsible for the maintenance and expression of every gene in an organism. An orthogonal central dogma may insulate genetic programs from host regulation and allow expansion in the roles of these processes within the cell.

Fighting food waste caused by spoilage yeasts and developing more natural forms of preservatives are critical points for the food industry. Plant antimicrobial peptides have biological activity against a broad spectrum of microorganisms, which could represent a valid alternative to the current preservative agents. In this work, the Pharabitis nil antimicrobial peptides Pn-AMP1 and Pn-AMP2 were chemically synthesised, and the potency of their antiyeast activity against common spoilage yeasts was measured. Pn-AMP1 showed a better inhibitory effect than Pn-AMP2, therefore it was further characterised. Pn-AMP1 displayed fast inhibitory activity against yeast strains Kluyveromyces lactis and Zygosaccharomyces bailii, and the antiyeast mechanism of membrane permeabilisation was detected. Its safety in terms of human consumption was studied and no adverse effects were found. Lastly, the stability of the peptide in different environments and conditions, such as heat, high salt, and a range of pH were studied, in addition to their antiyeast activity in different food matrices such as a soft drink and dairy product, further supporting Pn-AMP1’s potential for use in food preservation.

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., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

This study investigated the impact of dietary supplementation with hydrolyzed yeast (Kluyveromyces marxianus) on growth performance, humoral immunity, jejunal morphology, cecal microbiota and metabolic pathways in broilers raised at 45 kg/m 2 . A total of 1,176 mixed sex 1-day-old Ross 308 broilers were distributed into 42 pens and randomly assigned to either the control group, the control + 250 g hydrolyzed yeast (HY)/ton, 250HY group, or the control + 500 g HY/ton, 500HY group for 42 d. HY did not affect growth performance. However, HY reduced (P < 0.05) mortality at 25 to 35 d. Dietary HY lowered the heterophil/lymphocyte ratio and enhanced the villus height/crypt depth ratio and Newcastle disease titer (P < 0.05). Compared with HY250 and the control, HY500 upregulated (P < 0.05) IL-10. HY enhanced the α diversity, inferring the richness and evenness of the ceca microbiota. HY500 had greater β diversity than the control (P < 0.05). Six bacterial phyla, namely, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, and Cyanobacteria, were found. The relative abundance of Firmicutes was greater in the HY500 treatment group than in the HY250 and control groups. HY decreased the abundance of Actinobacteria. HY supplementation altered (P < 0.05) the abundance of 8 higher-level taxa consisting of 2 classes (Bacilli and Clostridia), 1 order (Lactobacillales), 1 family (Streptococcaceae), and five genera (Streptococcus, Lachnospiraceae&#95;uc, Akkermansiaceae, PACO01270&#95;g, and LLKB&#95;g). HY500 improved (P < 0.05) the abundance of Bacilli, Clostridia, Lactobacillales, Streptococcaceae, Streptococcus, PACO01270&#95;g, and Lachnospiraceae&#95;uc, while HY250 enhanced (P < 0.05) the abundance of Akkermansiaceae and LLKB&#95;g. HY improved the abundance of Lactobacillus and Akkermansia spp. Minimal set of pathway analyses revealed that compared with the control, both HY250 and HY500 regulated 20 metabolic pathways. These findings suggest that dietary K. marxianus hydrolysate, especially HY500, improved humoral immunity and jejunal morphology and beneficially altered the composition and metabolic pathways of the cecal microbiota in broilers raised at 45 kg/m 2 ., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Different yeast strains benefit postweaning piglets by promoting intestinal health. The objective of this study was to investigate the effect of a yeast mixture containing Kluyveromyces marxianus fragilis, Pichia guilliermondii, and Saccharomyces cerevisiae (Vetoquinol italia s.r.l., Italy) on gut health parameters and growth performance traits of weaned piglets. Forty-eight postweaning castrated male piglets (27 ± 1.7 days, 7.19 ± 0.54 kg) were randomly allocated to two homogeneous experimental groups and involved in a 28-day trial. Both the groups received a basal diet with (yeast mixture, YM) or without (control, CTR) the inclusion of 0.8% yeast mixture during weeks 1 and 2, and 0.6% during weeks 3 and 4. Individual BW and box feed intake were determined on days 0, 14, and 28, and average daily gain and Gain:Feed ratio were subsequently calculated for each administration period (0-14, 14-28). Individual faecal samples were collected for microbiota analysis on days 4, 14, 21, and 28, and faecal score was evaluated on the same days. At the end of the trial, 12 piglets for each group were sacrificed, and ileal tissue was sampled for morphological analysis and the evaluation of mucins profile, using Alcian-Blue/Periodic Acid-Shiff (PAS) staining. On ileum samples, dividing and differentiated epithelial cells were also identified using proliferating cell nuclear antigen and alkaline phosphatase expression, respectively. Differences in the means between the experimental groups were determined by ANOVA, while the metataxonomics analyses were performed by sequencing for V3 and V4 hypervariable regions of the 16S rRNA gene. Growth performance traits were not different among the two experimental groups when considering the whole trial period, while treated animals showed increased faecal consistency on weeks 1 and 4 (P = 0.036 and 0.021, respectively). Yeast mixture administration increased the abundance of Bifidobacterium (P = 0.006) and Coprococcus 2 (P = 0.015), and decreased Clostridium Sensu Stricto 1 (P = 0.019) at all the considered timepoints. Ileum villous height, villous width, and crypt depth were significantly increased by yeast mixture supplementation (P = 0.019; P = 0.013; P = 0.036, respectively), while no differences were observed for the villous:crypt ratio among the groups. The mucin profile showed no differences among experimental groups for acid and neutral glycoconjugates. However, a higher presence of PAS-positive mucins was highlighted in the villi of YM piglets (P < 0.001) compared to CTR. Overall, the administration of a yeast mixture to postweaning piglets showed positive effects on gut health when compared to piglets not receiving the tested product, improving beneficial genera and intestinal morphology., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Scaffold protein Ste5 and associated kinases, including Ste11, Ste7, and Fus3, are core components of the mating pheromone pathway, which is required to induce a mating response. Orthologs of these proteins are widely present in fungi, but to which extent one protein can be replaced by its ortholog is less well understood. Here, interspecies complementation was carried out to evaluate the functional homology of Ste5 and associated kinases in Kluyveromyces lactis , K. marxianus , and Saccharomyces cerevisiae. These three species occupy important positions in the evolution of hemiascomycetes. Results indicated that Ste5 and associated kinases in K. lactis and K. marxianus could be functionally replaced by their orthologs to different extents. However, the extent of sequence identity, either between full-length proteins or between domains, did not necessarily indicate the extent of functional replaceability. For example, Ste5, the most unconserved protein in sequence, achieved the highest average functional replaceability. Notably, swapping Ste5 between K. lactis and K. marxianus significantly promoted mating in both species and the weakened interaction between the Ste5 and Ste7 might contribute to this phenotype. Consistently, chimeric Ste5 displaying a higher affinity for Ste7 decreased the mating efficiency, while chimeric Ste5 displaying a lower affinity for Ste7 improved the mating efficiency. Furthermore, the length of a negatively charged segment in the Ste7-binding domain of Ste5 was negatively correlated with the mating efficiency in K. lactis and K. marxianus. Extending the length of the segment in KlSte5 improved its interaction with Ste7 and that might contribute to the reduced mating efficiency. Our study suggested a novel role of Ste5-Ste7 interaction in the negative regulation of the pheromone pathway. Meanwhile, Ste5 mutants displaying improved mating efficiency facilitated the breeding and selection of Kluyveromyces strains for industrial applications. [ABSTRACT FROM AUTHOR]

Concerns about fossil fuel depletion and the environmental effects of greenhouse gas emissions have led to widespread fermentation-based production of bioethanol from corn starch or sugarcane. However, competition for arable land with food production has led to the extensive investigation of lignocellulosic sources and waste products of the food industry as alternative sources of fermentable sugars. In particular, whey, a lactose-rich, inexpensive byproduct of dairy production, is available in stable, high quantities worldwide. This review summarizes strategies and specific factors essential for efficient lactose/whey fermentation to ethanol. In particular, we cover the most commonly used strains and approaches for developing high-performance strains that tolerate fermentation conditions. The relevant genes and regulatory systems controlling lactose utilization and sources of new genes are also discussed in detail. Moreover, this review covers the optimal conditions, various feedstocks that can be coupled with whey substrates, and enzyme supplements for increasing efficiency and yield. In addition to the historical advances in bioethanol production from whey, this review explores the future of yeast-based fermentation of lactose or whey products for beverage or fuel ethanol as a fertile research area for advanced, environmentally friendly uses of industrial waste products. [ABSTRACT FROM AUTHOR]

Scaffold protein Ste5 and associated kinases, including Ste11, Ste7, and Fus3, are core components of the mating pheromone pathway, which is required to induce a mating response. Orthologs of these proteins are widely present in fungi, but to which extent one protein can be replaced by its ortholog is less well understood. Here, interspecies complementation was carried out to evaluate the functional homology of Ste5 and associated kinases in Kluyveromyces lactis, K. marxianus, and Saccharomyces cerevisiae. These three species occupy important positions in the evolution of hemiascomycetes. Results indicated that Ste5 and associated kinases in K. lactis and K. marxianus could be functionally replaced by their orthologs to different extents. However, the extent of sequence identity, either between full-length proteins or between domains, did not necessarily indicate the extent of functional replaceability. For example, Ste5, the most unconserved protein in sequence, achieved the highest average functional replaceability. Notably, swapping Ste5 between K. lactis and K. marxianus significantly promoted mating in both species and the weakened interaction between the Ste5 and Ste7 might contribute to this phenotype. Consistently, chimeric Ste5 displaying a higher affinity for Ste7 decreased the mating efficiency, while chimeric Ste5 displaying a lower affinity for Ste7 improved the mating efficiency. Furthermore, the length of a negatively charged segment in the Ste7-binding domain of Ste5 was negatively correlated with the mating efficiency in K. lactis and K. marxianus. Extending the length of the segment in KlSte5 improved its interaction with Ste7 and that might contribute to the reduced mating efficiency. Our study suggested a novel role of Ste5-Ste7 interaction in the negative regulation of the pheromone pathway. Meanwhile, Ste5 mutants displaying improved mating efficiency facilitated the breeding and selection of Kluyveromyces strains for industrial applications.

Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes. [ABSTRACT FROM AUTHOR]

The enzyme β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, although for its effective application it is necessary to establish techniques for purification, concentration, or polishing, such as membrane separation processes, in particular ultrafiltration. The present study aimed to investigate ultrafiltration and diafiltration applied as initial steps for concentration and salt removal, respectively, in the β-galactosidase purification processes. Additionally, the influence levels of the pH (6.5, 7.7, or 7.5), membrane molecular weight cut-off (30, 50, 60, or 100 kDa), operating pressure (1.5, 2.0, or 2.5 kgf/cm2), and ionic strength of the ultrafiltration using NaCL or KCl (0.01–0.1 M) were evaluated considering the enzyme recovery, purification, retention, and concentration factors in relation to the proteins, volume, activity, and protein flux and yield of the processes. The ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm2 with a 50 kDa polyethersulfone membrane resulted in a volume concentration of the β-galactosidase extract up to 7.1-fold greater, a purification factor 1.2-fold greater, and an enzyme recovery rate of 108.9% by eliminating metabolites during the purification process. In addition, the lowest flux variation range (16.0 to 13.1 L/m2·h) was observed under these same conditions, thereby representing a decrease of 18.0%. An increase in the operating pressure and the addition of salts results in reduced enzyme recovery (up to 38% of the process yield (734.1 to 453.7 U/h) and up to 40% of the enzyme recovery rate (108.9 to 60.6%) during the ultrafiltration using NaCl, respectively). The operation in the diafiltration mode allowed salt removal after the purification of β-galactosidase (enzymatic recovery rates above 93.4%) via precipitation and ion-exchange chromatography elution and as part of an aqueous two-phase system using 6 diafiltration cycles, thereby revealing its application potential.

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.

Although only Saccharomyces boulardii has been studied for ulcerative colitis (UC), probiotic yeasts have immense therapeutic potential. Herein, we evaluated the kefir yeast Kluyveromyces marxianus A4 (Km A4) and its anti-inflammatory effect with sulfasalazine in BALB/c mice with dextran sulfate sodium (DSS)-induced colitis. Oral administration continued for 7 days after the mice were randomly divided into seven groups: control (CON, normal mice administered with saline), DSS-induced colitis mice administered saline (DSS), and DSS-induced colitis mice administered sulfasalazine only (S), Km A4 only (A4), Km A4 plus sulfasalazine (A4 + S), S . boulardii ATCC MYA-796 (Sb MYA-796) only (Sb), and Sb MYA-796 plus sulfasalazine (Sb + S). The β-glucan content of Km A4 was significantly higher than that of Sb MYA-796 ( P < 0.05). Body weight gain (BWG) significantly correlated with colon length, cyclooxygenase-2 ( Cox-2 ) levels, and Bacteroides abundance ( P < 0.05). In colitis-induced mice, the A4 + S group had the lowest histological score (6.00) compared to the DSS group (12.67), indicating the anti-inflammatory effects of this combination. The A4 + S group showed significantly downregulated expression of interleukin ( Il )- 6 , tumor necrosis factor-α ( Tnf-α ), and Cox-2 and upregulated expression of Il-10 and occludin ( Ocln ) compared to the DSS group. Mice treated with A4 + S had enhanced Bacteroides abundance in their gut microbiota compared with the DSS group ( P < 0.05). Bacteroides were significantly correlated with all colitis biomarkers (BWG, colon length, Il-6 , Tnf-α , Il-10 , Cox-2 , and Ocln ; P < 0.05). The anti-inflammatory effects of Km A4 could be attributed to high β-glucan content and gut microbiota modulation. Thus, treatment with Km A4 and sulfasalazine could alleviate UC.

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KL-L holds stock options in and is a consultant for Peptone Ltd. T.A.W. is on the scientific advisory board at Metaphore Biotechnologies. The remaining authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The fermented milk matsoni is a traditional, national food product of both Georgia and Armenia. Little is known about the effects of biogeography and milk type on the microbial biodiversity of matsoni or the fungal composition of matsoni fermentations. High-throughput marker-gene sequencing was used to survey the bacterial and fungal communities of matsoni from different milk types and regions throughout Armenia and Georgia. Results demonstrate that both production region and milk type influence matsoni microbiota, suggesting that the traditional production methods preserve the transfer of unique regional microbiota from batch to batch. Bacterial profiles were dominated by Lactobacillus and Streptococcus species. Yeast profiles varied dramatically, with Kluyveromyces marxianus, Candida famata, Saccharomyces cerevisiae, Lodderomyces elongisporus, and Kluyveromyces lactis being the most important species distinguishing production regions and milk types. This survey will enable more detailed capture and characterization of specific microbiota detected within these fermentations.

BACKGROUND: A key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase). This pathway is also prevalent in fruit, producing short and medium chain volatile esters during ripening. In this work, a series of six AATases from Saccharomyces and non-Saccharomyces yeasts as well as tomato fruit were evaluated with respect to their activity, intracellular localization, and expression in Saccharomyces cerevisiae and Escherichia coli cell hosts. The series of AATases includes Atf1 and Atf2 from S. cerevisiae, as well as AATases from S. pastorianus, Kluyveromyces lactis, Pichia anomala, and Solanum lycopersicum (tomato). RESULTS: When expressed in S. cerevisiae, Atf1, Atf2, and an AATase from S. pastorianus localized to lipid droplets, while AATases from non-Saccharomyces yeasts and tomato fruit did not localize to intracellular membranes and were localized to the cytoplasm. All AATases studied here formed intracellular aggregates when expressed in E. coli, and western blot analysis revealed that expression levels in E. coli were upwards of 100-fold higher than in S. cerevisiae. Fermentation and whole cell lysate activity assays of the two most active AATases, Atf1 from S. cerevisiae and an AATase from tomato fruit, demonstrated that the aggregates were enzymatically active, but with highly reduced specific activity in comparison to activity in S. cerevisiae. Activity was partially recovered at lower expression levels, coinciding with smaller intracellular aggregates. In vivo and in vitro activity assays from heterologously expressed Atf1 from S. cerevisiae, which localizes to lipid droplets under homologous expression, demonstrates that its activity is not membrane dependent. CONCLUSIONS: The results of these studies provide important information on the biochemistry of AATases under homologous and heterologous expression with two common microbial hosts for biochemical processes, S. cerevisiae and E. coli. All studied AATases formed aggregates with low enzymatic activity when expressed in E. coli and any membrane localization observed in S. cerevisiae was lost in E. coli. In addition, AATases that were found to localize to lipid droplet membranes in S. cerevisiae were found to not be membrane dependent with respect to activity.

Tequila and mezcal are both traditional Mexican liquors that are produced from cooked Agave spp. must fermentation and usually rely on spontaneous or pure Saccharomyces cerevisiae strain inoculation. In order to contribute to the rational selection of yeast starters for tequila and mezcal productions, we tested a collection of 25 yeasts originally isolated from mezcal musts, spanning 10 different yeast species. These strains were first characterized in a semi synthetic medium (labeled as M2, having 90 g/L fructose and 10 g/L glucose of initial hexoses) at 48 h of culture, observing a differential pattern in the consumption of sugars and productivity. Selected Saccharomyces strains left around 10 g/L of fructose and showed higher fermentation performance. However, some non-Saccharomyces strains, specifically from Torulospora (Td), Kluyveromyces (Km), and Zygosaccharomyces (Zb) genera, consumed almost all the sugar (i.e., Km1Y9 with

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.

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

Many flavoring agents on the market are extracted from natural sources or synthesized chemically. Due to the disadvantages of both methods, biotechnology is becoming a promising alternative. In this study, short chain ethyl esters with fruity notes were biosynthesized in UHT whole milk via coupling ethanolic fermentation with lipase (Palatase®) transesterification. Kluyveromyces marxianus, Lactobacillus fermentum and Lb. paracasei were used for fermentation. Milk fat was esterified with in situ produced ethanol by adding lipase at 0, 8 and 24 h of fermentation. Viable cell counts and pH were monitored during 48 h fermentation period. Flavor active ethyl esters, ethanol and free fatty acids were analyzed using headspace SPME-GC. Free fatty acid levels were lower in K. marxianus samples than lactobacilli. K. marxianus produced higher amounts of ethanol and esters than lactic acid bacteria. Viable cell counts decreased after lipase application at 0 and 8 h, possibly due to fatty acid production. Addition of lipase at 24 h resulted in improved cell counts as well as ethanol and ester production in the case of K. marxianus. This study demonstrated that fermenting milk with alcohol producing cultures in conjunction with lipase application can be an alternative to artificial flavorings in fermented milks. [ABSTRACT FROM AUTHOR]

Yeast whole cells have been widely used in modern biotechnology as biocatalysts to generate numerous compounds of industrial, chemical, and pharmaceutical importance. Since many of the biocatalysis-utilizing manufactures have become more concerned about environmental issues, seawater is now considered a sustainable alternative to freshwater for biocatalytic processes. This approach plausibly commenced new research initiatives into exploration of salt-tolerant yeast strains. Recently, there has also been a growing interest in possible applications of microbial biofilms in the field of biocatalysis. In these complex communities, cells demonstrate higher resistance to adverse environmental conditions due to their embedment in an extracellular matrix, in which physical, chemical, and physiological gradients exist. Considering these two topics, seawater and biofilms, in this work, we characterized biofilm formation in seawater-based growth media by several salt-tolerant yeast strains with previously demonstrated biocatalytic capacities. The tested strains formed both air-liquid-like biofilms and biofilms on silicone surfaces, with Debaryomyces fabryi, Schwanniomyces etchellsii, Schwanniomyces polymorphus, and Kluyveromyces marxianus showing the highest biofilm formation. The extracted biofilm extracellular matrices mostly consisted of carbohydrates and proteins. The latter group was primarily represented by enzymes involved in metabolic processes, particularly the biosynthetic ones, and in the response to stimuli. Specific features were also found in the carbohydrate composition of the extracellular matrix, which were dependent both on the yeast isolate and the nature of formed biofilms. Overall, our findings presented herein provide a unique data resource for further development and optimization of biocatalytic processes and applications employing seawater and halotolerant yeast biofilms. Key points • Ability for biofilm formation of some yeast-halotolerant strains in seawater medium • ECM composition dependent on strain and biofilm-forming surface • Metabolic enzymes in the ECM with potential applications for biocatalysis [ABSTRACT FROM AUTHOR]

Concerns about fossil fuel depletion and the environmental effects of greenhouse gas emissions have led to widespread fermentation-based production of bioethanol from corn starch or sugarcane. However, competition for arable land with food production has led to the extensive investigation of lignocellulosic sources and waste products of the food industry as alternative sources of fermentable sugars. In particular, whey, a lactose-rich, inexpensive byproduct of dairy production, is available in stable, high quantities worldwide. This review summarizes strategies and specific factors essential for efficient lactose/whey fermentation to ethanol. In particular, we cover the most commonly used strains and approaches for developing high-performance strains that tolerate fermentation conditions. The relevant genes and regulatory systems controlling lactose utilization and sources of new genes are also discussed in detail. Moreover, this review covers the optimal conditions, various feedstocks that can be coupled with whey substrates, and enzyme supplements for increasing efficiency and yield. In addition to the historical advances in bioethanol production from whey, this review explores the future of yeast-based fermentation of lactose or whey products for beverage or fuel ethanol as a fertile research area for advanced, environmentally friendly uses of industrial waste products.

Most models of gene duplication assume that the ancestral functions of the preduplication gene are independent and can therefore be neatly partitioned between descendant paralogs. However, many gene products, such as transcriptional regulators, are components within cooperative assemblies; here, we show that a natural consequence of duplication and divergence of such proteins can be competitive interference between the paralogs. Our example is based on the duplication of the essential MADS-box transcriptional regulator Mcm1, which is found in all fungi and regulates a large set of genes. We show that a set of historical amino acid sequence substitutions minimized paralog interference in contemporary species and, in doing so, increased the molecular complexity of this gene regulatory network. We propose that paralog interference is a common constraint on gene duplicate evolution, and its resolution, which can generate additional regulatory complexity, is needed to stabilize duplicated genes in the genome.

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

To reduce proteases in a commercial lactase preparation from K. lactis, enzyme preparations were diluted by a buffer with KCl and the dilutions were flushed through a weak anion-exchange membrane. For KCl concentration in the range of 0.20 to 0.25 M, more than 90 % of lactase was recovered and protease level was decreased to less than 20 %. Purified and pre-purified lactase solutions added in excess to milk and incubated for 3 months at 30 °C showed degraded milk protein bands on SDS-PAGE by treatment with the pre-purified lactase but not purified lactase. A one-step purification method for protease removal from the K. lactis lactase preparation was performed only by flushing the solution through an anion-exchange membrane. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus. The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Traditional Wagashi cheese and fermented cow milk are among the most popular dairy products appreciated by people from Benin, Niger, and the neighboring region. These products are the main source of protein in the diet of the low-income population in the region. The fermented milk is prepared by spontaneous fermentation without back-slopping. Whereas, the leaf extract of Calotropis procera is used for curdling the milk to prepare the soft Wagashi cheese. The present study aims to provide in-depth analysis of yeast communities associated with these traditional milk products by high-throughput Illumina MiSeq amplicon sequencing of internal transcribed spacer (ITS) region of fungal rRNA genes. A total of 60 samples, 20 samples of fermented milk each from Benin and Niger, and 20 samples of Wagashi cheese from Benin were used for analysis. The metagenomic investigation revealed that Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida parapsilosis, and Sagenomella keratitidis were the predominant yeast species present in the traditional milk products. Furthermore, we noticed a high presence of K. marxianus (61.1% relative abundance) in the Wagashi cheese and S. cerevisiae (28.4% relative abundance) in the fermented milk of Niger. The presence of potential pathogenic yeast C. parapsilosis and S. keratitidis in these African milk products calls for further investigation to assess their safety. The predominant yeast K. marxianus and S. cerevisiae, recognized with generally regarded as safe (GRAS) status, could be further selected as starter culture along with lactic acid bacteria for developing controlled fermentation processes with enhanced product quality and safety.

Objective: Non-albicans Candida species, such as Candida kefyr, are emerging pathogens. Chromogenic media are highly useful for the diagnosis of urinary tract infections (UTIs). The aim was to describe the behavior of this specie on a non-specific chromogenic medium., Methods: A retrospective study of cases of candiduria detected in the Microbiology laboratory of the Virgen de las Nieves Hospital in Granada (Spain) between 2016 and 2021 (N=2,130). Urine samples were quantitatively seeded on non-selective UriSelect™4 chromogenic agar., Results: Between 2016 and 2021, C. kefyr was the seventh most frequent Candida species responsible for candiduria in our setting (n=15). The macroscopic appearance of C. kefyr colonies, punctiform and bluish, allowed the direct identification of these microorganisms., Conclusions: This study provides the first description of the specific behavior of C. kefyr on UriSelect™4 agar, which differentiates it from other Candida species based on its enzymatic characteristics., (©The Author 2023. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Importance: The food industry has always used many strains of microorganisms including fungi in their production processes. These strains have been widely characterized for their biotechnological value, but we still know very little about their interaction capacities with the host at a time when the intestinal microbiota is at the center of many pathologies. In this study, we characterized five yeast strains from food production which allowed us to identify two new strains with high probiotic potential and beneficial effects in a model of intestinal inflammation., Competing Interests: The authors declare no conflict of interest.

Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (de p value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t 1/2 ) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure-function relationship of AKRs, providing a certain guidance for the modification of other AKRs., (© 2023 Wiley Periodicals LLC.)

Telomeres are the protein-nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen "monster" cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.

Background: Thermotolerance is a highly desirable trait of microbial cell factories and has been the focus of extensive research. Yeast usually tolerate only a narrow temperature range and just two species, Kluyveromyces marxianus and Ogataea polymorpha have been described to grow at reasonable rates above 40 °C. However, the complex mechanisms of thermotolerance in yeast impede its full comprehension and the rare physiological data at elevated temperatures has so far not been matched with corresponding metabolic analyses. Results: To elaborate on the metabolic network response to increased fermentation temperatures of up to 49 °C, comprehensive physiological datasets of several Kluyveromyces and Ogataea strains were generated and used for 13C-metabolic flux analyses. While the maximum growth temperature was very similar in all investigated strains, the metabolic network response to elevated temperatures was not conserved among the different species. In fact, metabolic flux distributions were remarkably irresponsive to increasing temperatures in O. polymorpha, while the K. marxianus strains exhibited extensive flux rerouting at elevated temperatures. Conclusions: While a clear mechanism of thermotolerance is not deducible from the fluxome level alone, the generated data can be valued as a knowledge repository for using temperature to modulate the metabolic activity towards engineering goals. [ABSTRACT FROM AUTHOR]

Traditional Wagashi cheese and fermented cow milk are among the most popular dairy products appreciated by people from Benin, Niger, and the neighboring region. These products are the main source of protein in the diet of the low-income population in the region. The fermented milk is prepared by spontaneous fermentation without back-slopping. Whereas, the leaf extract of Calotropis procera is used for curdling the milk to prepare the soft Wagashi cheese. The present study aims to provide in-depth analysis of yeast communities associated with these traditional milk products by high-throughput Illumina MiSeq amplicon sequencing of internal transcribed spacer (ITS) region of fungal rRNA genes. A total of 60 samples, 20 samples of fermented milk each from Benin and Niger, and 20 samples of Wagashi cheese from Benin were used for analysis. The metagenomic investigation revealed that Kluyveromyces marxianus , Saccharomyces cerevisiae , Candida parapsilosis , and Sagenomella keratitidis were the predominant yeast species present in the traditional milk products. Furthermore, we noticed a high presence of K. marxianus (61.1% relative abundance) in the Wagashi cheese and S. cerevisiae (28.4% relative abundance) in the fermented milk of Niger. The presence of potential pathogenic yeast C. parapsilosis and S. keratitidis in these African milk products calls for further investigation to assess their safety. The predominant yeast K. marxianus and S. cerevisiae , recognized with generally regarded as safe (GRAS) status, could be further selected as starter culture along with lactic acid bacteria for developing controlled fermentation processes with enhanced product quality and safety. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of dietary supplementation of a mixed yeast culture (MYC; Saccharomyces cerevisiae YJM1592 and Kluyveromyces maxianus TB7258 in a 1:1 ratio) on growth performance, nutrient digestibility, meat quality, blood parameters, and gut health of broiler chickens. In total, 576 one-day-old male broilers (Ross 308) with an average initial bodyweight (BW) of 37±0.51 g were used in a 35-dayexperiment with a completely randomized design. The broilers were randomly assigned to 3 dietary treatments: CON, basal diet; TRT1, CON + 0.1% MYC; and TRT 2, CON + 0.2% MYC. From days 8 to 21, the feed conversion rate (FCR) was significantlydecreased in broilers fed MYC-supplemented diets. From days 22 to 35, BW gain (BWG) significantly increased with increasing MYC concentration. Throughout the experiment, BWG increased (linear effect, P=0.002) and FCR decreased with increasing MYC in the diet. MYC supplementation increased the digestibilityof drymatter (DM) in broilers in a dose-dependent manner. Relative organ weight of the bursa of Fabricius linearlyincreased in broilers fed MYC-supplemented diets. The white blood cell count showed linear and quadratic increases in broilers fed increasing concentrations of MYC. The population of Lactobacillus in the excreta linearlyincreased P=0.033, whereas that of Escherichia coli tended to linearlydecrease (P=0.064) in the MYC groups. This studyprovides a basis for future research on MYC as a growth promoter in broilers. [ABSTRACT FROM AUTHOR]

Koumiss is notable for its nutritional functions, and microorganisms in koumiss determine its versatility. In this study, the bacterial and fungal community structures in traditional koumiss from Inner Mongolia, China, were investigated. Our results demonstrated that 6 bacterial phyla represented by 126 genera and 49 species and 3 fungal phyla represented by 59 genera and 57 species were detected in 11 samples of artisanal koumiss. Among them, Lactobacillus was the predominant genus of bacterium, and Kluyveromyces and Saccharomyces dominated at the fungal genus level. In addition, there were no differences in the bacterial and fungal richness and diversity of koumiss from 3 neighboring administrative divisions in Inner Mongolia, and the bacterial and fungal community structures (the varieties and relative abundance of bacterial and fungal genera and species) were clearly distinct in individual samples. This study provides a comprehensive understanding of the bacterial and fungal population profiles and the predominant genus and species, which would be beneficial for screening, isolation, and culture of potential probiotics to simulate traditional fermentation of koumiss for industrial and standardized production in the future. [ABSTRACT FROM AUTHOR]

This study aimed to produce ethanol by lactose conversion of dairy coproducts (cheese whey, and whey permeate) using the yeast Kluyveromyces marxianus URM 7404, isolated from cheese whey obtained from the production of Gorgonzola cheese. The fermentation occurred at 30 °C for 20 h under aerobic, microaerobic, and anaerobic conditions for both substrates. The results indicated that ethanol production was greater when using the whey permeate under anaerobic conditions (8.90 g L−1, 18 h of fermentation) in comparison to cheese whey (8.08 g L−1, 10 h) at the same conditions. However, regarding the ethanol yield (η), the cheese whey substrate was more effective, given that it achieved 76% of yield (η) and a volumetric productivity (QP) of 0.81 g L−1 h−1 in 10 h under fermentation anaerobic conditions. Biomass production and productivity (QX) were more satisfactory in the whey permeate under aerobic conditions (11.48 g L−1 and 0.75 g L−1 h−1, respectively), in 14 h, with a specific growth rate of 0.253 h−1. It has been obtained 86% of COD reduction at the end of the aerobic process. [ABSTRACT FROM AUTHOR]