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Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture.

ABSTRACT: Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

SUBMITTER: Lo SC

PROVIDER: S-EPMC8282799 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth ssing sterol requirements enables anaerobic growth of the thermotolerant yeast Kluyveromyces marxianus

Project description:Analysis of gene expression changes in S. cerevisiae and K. marxianus in response to oxygen-limitation

2021-01-06 | GSE164344 | GEO

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.

Project description:BackgroundIn spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.ResultsThe highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity.ConclusionsTaken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

| S-EPMC2817671 | biostudies-literature

Complete Genome Sequence of Kluyveromyces marxianus NBRC1777, a Nonconventional Thermotolerant Yeast.

Project description:We determined the genome sequence of the thermotolerant yeast Kluyveromyces marxianus strain NBRC1777. The genome of strain NBRC1777 is composed of 4,912 open reading frames (ORFs) on 8 chromosomes, with a total size of 10,895,581 bp, including mitochondrial DNA.

| S-EPMC4408353 | biostudies-literature

Draft Genome Sequence of the Probiotic Yeast Kluyveromyces marxianus fragilis B0399.

Project description:Here, we report the draft genome sequence of Kluyveromyces marxianus fragilis B0399, the first yeast approved as a probiotic for human consumption not belonging to the genus Saccharomyces The genome is composed of 8 chromosomes, with a total size of 11.44 Mb, including mitochondrial DNA.

| S-EPMC5009987 | biostudies-literature

Physiological growth and galactose utilization by dairy yeast Kluyveromyces marxianus in mixed sugars and whey during fermentation.

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Transcriptional expression level during exponential growth phase in Kluyveromyces marxianus

Project description:To understand functional roles of ncRNAs during exponential growth phase, we captured transcriptome changes by conducting strand-specific RNA-seq.

2016-08-20 | GSE70111 | GEO

Transcriptomic analysis of thermotolerant yeast <i>Kluyveromyces marxianus</i> in multiple inhibitors tolerance.

Project description:During pretreatment of lignocellulosic biomass, toxic compounds were released and inhibited the growth and fermentation of microorganisms. Here the global transcriptional response of K. marxianus to multiple inhibitors including acetic acid, phenols, furfural and HMF, at 42 °C, was studied, via RNA-seq technology. Genes involved in the glycolysis pathway, fatty acid metabolism, ergosterol metabolism and vitamin B6 and B1 metabolic process were enriched in the down-regulated gene set, while genes involved in TCA cycle, respiratory chain, ROS detoxification and transporter coding genes were enriched in the up-regulated gene set in response to the multiple inhibitors stress. Further real time-PCR results with three single inhibitor stress conditions showed that different transporters responded quite differently to different inhibitor stress. Coenzyme assay results showed that the level of NAD+ was increased and both NADH/NAD+ and NADPH/NADP+ ratio decreased. Furthermore, genes involved with transcription factors related to carbohydrate metabolism, sulfur amino acids metabolism, lipid metabolism or those directly involved in the transcriptional process were significantly regulated. Though belonging to different GO categories or KEGG pathway, many differentially expressed genes were enriched in maintaining the redox balance, NAD(P)+/NAD(P)H homeostasis or NAD+ synthesis, energy production, and iron transportation or metabolism. These results suggest that engineering these aspects represents a possible strategy to develop more robust strains for industrial fermentation from cellulosic biomass.

| S-EPMC9079866 | biostudies-literature

Transcriptional expression level during exponential growth phase in Kluyveromyces marxianus

Project description:To understand functional roles of ncRNAs during exponential growth phase, we captured transcriptome changes by conducting strand-specific RNA-seq. Directional RNA-seq (dUTP method by TruSeqÂ®) were generated in two biological replicates by using Illumina MiSeq

2016-08-20 | E-GEOD-70111 | biostudies-arrayexpress

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

Project description:BackgroundTargeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences.ResultsMutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield.ConclusionsWe present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

| S-EPMC4347551 | biostudies-literature

Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

Project description:BACKGROUND:High-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis. RESULTS:The complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions. CONCLUSIONS:This study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.

| S-EPMC4381506 | biostudies-literature

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