Optimal Extraction and Deproteinization Method for Mannoprotein Purification from Kluyveromyces marxianus.

Background: Mannoproteins, mannose-glycosylated proteins, play an important role in biological processes and have various applications in industries. Several methods have been already used for the extraction of mannoproteins from yeast cell-wall. The aim of this study was to evaluate the extraction and deproteinization of mannan oligosaccharide from the Kluyveromyces (K.) marxianus mannoprotein. Methods: To acquire crude mannan oligosaccharides, K. marxianus mannoproteins were deproteinized by the Sevage, trichloroacetic acid, and hydrochloric acid (HCL) methods. Total nitrogen, crude protein content, fat, carbohydrate and ash content were measured according to the monograph prepared by the meeting of the Joint FAO/WHO Expert Committee and standard. Mannan oligosaccharide loss, percentage of deproteinization, and chemical composition of the product were assessed to check the proficiency of different methods. Results: Highly purified (95.4%) mannan oligosaccharide with the highest deproteinization (97.33 ± 0.4%) and mannan oligosaccharide loss (25.1 ± 0.6%) were obtained following HCl method. Conclusion: HCl, was the most appropriate deproteinization method for the removal of impurities. This preliminary data will support future studies to design scale-up procedures.

Biological Properties of Yeast-based Mannoprotein for Prospective Biomedical Applications.

BACKGROUND: Natural products constitute more than half of all biomolecules lately being used in clinical settings. Mannoprotein derived from the yeast cell wall has found full biotechnological applications. OBJECTIVE: This study was intended to investigate the antioxidant, anticancer, and toxicological properties of Kluyveromyces marxianus mannoprotein (KM). METHODS: The KM extract was obtained through a sequence of operations, including centrifugation for cell isolation, precipitation with potassium citrate/sodium metabisulfite, and recovery and purification. Its antioxidant, growth inhibition, macrophage mitogenic, and toxic activities were evaluated for its future use in the biomedical field. RESULTS: Significant inhibitory effects of KM were obtained on reactive species. It showed antiproliferative activity against HeLa (human cervical adenocarcinoma) and MCF-7 (human breast cancer) cell lines with no toxic effects on HUVECs (human umbilical vein endothelial cells). The in vitro model of CHO-K1 (Chinese hamster ovary) cell lines did not show the cytotoxic and genotoxic of KM. Moreover, it enhanced macrophage activity in terms of nitric oxide (NO) production and viability. No sign of acute toxicity was found in BALB/c mice, and body weight remained unchanged in guinea pigs over three months. CONCLUSION: Comprehensive biological evaluations in this study are expected to expand the potential of KM as a natural material.

The antioxidant function of Sco proteins depends on a critical surface-exposed residue.

BACKGROUND: Besides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity. METHODS: Native and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays. RESULTS: A functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways. CONCLUSION: This study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology. GENERAL SIGNIFICANCE: These findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

BACKGROUND: Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS: Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS: The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of ß-Galactosidase from Kluyveromyces lactis.

Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis ß-galactosidase (ß-Gal). The ß-Gal-catalyzed hydrolysis of o-nitrophenyl-ß-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, ß-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying ß-Gal from K. lactis.

Structural insights into telomere protection and homeostasis regulation by yeast CST complex.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance. Despite extensive studies, only structural information of individual domains of CST is available; the architecture of CST still remains unclear. Here, we report crystal structures of Kluyveromyces lactis Cdc13-telomeric-DNA, Cdc13-Stn1 and Stn1-Ten1 complexes and propose an integrated model depicting how CST assembles and plays its roles at telomeres. Surprisingly, two oligonucleotide/oligosaccharide-binding (OB) folds of Cdc13 (OB2 and OB4), previously believed to mediate Cdc13 homodimerization, actually form a stable intramolecular interaction. This OB2-OB4 module of Cdc13 is required for the Cdc13-Stn1 interaction that assembles CST into an architecture with a central ring-like core and multiple peripheral modules in a 2:2:2 stoichiometry. Functional analyses indicate that this unique CST architecture is essential for both telomere capping and homeostasis regulation. Overall, our results provide fundamentally valuable structural information regarding the CST complex and its roles in telomere biology.

Whole-Cell Biocatalysis in Seawater: New Halotolerant Yeast Strains for the Regio- and Stereoselectivity Reduction of 1-Phenylpropane-1,2-Dione in Saline-Rich Media.

The application of green chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwanniomyces for catalytic processes run in this medium. In the present work, we select other yeasts based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible. The best ones were obtained for Saccharomyces cerevisiae FY86 and Kluyveromyces marxianus, for which acyloins with different stereochemistry were obtained with good to excellent enantiomeric excess.

Structural basis for the evolution of cyclic phosphodiesterase activity in the U6 snRNA exoribonuclease Usb1.

U6 snRNA undergoes post-transcriptional 3' end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3' end of U6 and, in humans, leaves a 2',3' cyclic phosphate that is recognized by the Lsm2-8 complex. Saccharomycescerevisiae Usb1 has additional 2',3' cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3' phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5'-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n - 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.

Probiotic properties and fatty acid composition of the yeast Kluyveromyces lactis M3. In vivo immunomodulatory activities in gilthead seabream (Sparus aurata).

The aim of this study was to analyze the probiotic potential, fatty acid composition and immunostimulant activities of Kluyveromyces lactis M3 isolated from a hypersaline sediment. For this purpose, K. lactis M3 resistance to different pH, salinities and bile, as well as its antioxidant capability were assayed. Furthermore, total fatty acid composition of the yeast was determined where the dominant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. K. lactis M3 showed no cytotoxic effects on peripheral blood leukocytes. During an in vivo experiment in gilthead seabream (Sparus aurata), dietary K. lactis M3 supplemented at 0.55 or 1.1% of the basal diet enhanced bactericidal activity against Vibrio parahaemolyticus N16, V. harveyi Lg 16/00, and V. anguillarum CECT 43442 compared to fish fed commercial diet (control group). Finally, nitric oxide production, peroxidase activity and skin mucus lectin union levels strongly increased in fish fed K. lactis M3 with respect to the control group. The results suggested that the yeast K. lactis M3 had exhibited high antioxidant capability, and its dietary administration at 0.55 or 1% basal diet had immunostimulant activity for gilthead seabream. For all these reasons, it should be considered an appropriate probiotic candidate for the aquaculture fish industry.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains.

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.

Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses.

Kluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Thermotolerant Yeast Kluyveromyces marxianus Reveals More Tolerance to Heat Shock than the Brewery Yeast Saccharomyces cerevisiae.

The thermotolerant yeast Kluyveromyces marxianus, growing at high temperature (45℃) , showed stronger survival under heat shock at 50℃ than the brewing yeast Saccharomyces cerevisiae, which was unable to grow at 45℃. The survival rate of K. marxianus decreased to 10% during heat shock at 50℃ for 20 min, and to less than 0.01% at 60℃ for 20 min. Cells with damaged cellular membranes were infrequently observed at 50℃ and had decreased significantly from heat shock at 60℃. The metabolic activity of K. marxianus was retained at 50℃, whereas that of S. cerevisiae was not. The trehalose content of K. marxianus was approximately two times that of S. cerevisiae. These results suggest that K. marxianus protects itself from heat shock-induced damage through the use of trehalose (a protective molecule in S. cerevisiae) as well as other different factors.

Crystal Structure of the COMPASS H3K4 Methyltransferase Catalytic Module.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.

Investigation of microorganisms involved in kefir biofilm formation.

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.

Structure of the activated Edc1-Dcp1-Dcp2-Edc3 mRNA decapping complex with substrate analog poised for catalysis.

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.

Production of antioxidant and ACE-inhibitory peptides from Kluyveromyces marxianus protein hydrolysates: Purification and molecular docking.

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC50 = 22.88 µM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC50 = 15.20 µM, 5568 µM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE.

Fatty acid addition and thermotolerance of Kluyveromyces marxianus.

Membrane fatty acid composition has an important role in yeast stress resistance, particularly in temperature tolerance. Most studies investigating temperature and membrane fatty acids use the yeast Saccharomyces cerevisiae without considering other yeasts, such as Kluyveromyces marxianus, which has physiological differences and industrial advantages with respect to S. cerevisiae. One of the primary traits of K. marxianus is its thermotolerance. The effect of fatty acid addition (oleic acid, linoleic acid, linolenic acid and araquidic acid) on the thermotolerance of the K. marxianus strain SLP1 was evaluated. SLP1 yeast exhibited temperature tolerance of up to 50°C; at 55°C, viability was reduced significantly, probably due to an increase in the generation of reactive oxygen chemical species. Externally added fatty acids were incorporated in the yeast membrane, increasing their proportion to approximately 70%, thereby changing membrane fluidity. SLP1 cells supplemented with polyunsaturated fatty acids decreased cell thermotolerance and increased the degree of lipoperoxidation, while arachidic acid addition exhibited a tendency to increase yeast thermotolerance.

Quantitative physiology and elemental composition of Kluyveromyces lactis CBS 2359 during growth on glucose at different specific growth rates.

The yeast Kluyveromyces lactis has received attention both from academia and industry due to some important features, such as its capacity to grow in lactose-based media, its safe status, its suitability for large-scale cultivation and for heterologous protein synthesis. It has also been considered as a model organism for genomics and metabolic regulation. Despite this, very few studies were carried out hitherto under strictly controlled conditions, such as those found in a chemostat. Here we report a set of quantitative physiological data generated during chemostat cultivations with the K. lactis CBS 2359 strain, obtained under glucose-limiting and fully aerobic conditions. This dataset serves [corrected] as a basis for the comparison of K. lactis with the model yeast Saccharomyces cerevisiae in terms of their elemental compositions, as well as for future metabolic flux analysis and metabolic modelling studies with K. lactis.