Mitochondrial DNA of the yeast Kluyveromyces: guanine-cytosine rich sequence clusters.

Mitochondrial DNA from the yeast Kluyveromyces marxianus var. lactis (K.lactis) is a circular molecule of 39 kilobase-pairs. A genetic and physical map was constructed. We found that this genome contained a large number of guanine-cytosine (GC)-rich sequence clusters, many of which are characterized by the presence of SacII restriction sites (CCGCGG). The primary sequence of the GC clusters often showed a palindromic structure. These GC clusters were present in several varieties of K.marxianus, but not in others. The presence of these clusters is a major feature that distinguishes K.lactis strains from those of K.marxianus var. marxianus (including K.fragilis).     

Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus presents several interesting features that make this species a promising industrial yeast for the production of several compounds. In order to take full advantage of this yeast and its particular properties, proper tools for gene disruption and metabolic engineering are needed. The Cre-loxP system is a very versatile tool that allows for gene marker rescue, resulting in mutant strains free of exogenous selective markers, which is a very important aspect for industrial application. As the Cre-loxP system works in some non-conventional yeasts, namely Kluyveromyces lactis, we wished to know whether it also works in K. marxianus. Here, we report the validation of this system in K. marxianus CBS 6556, by disrupting two copies of the LAC4 gene, which encodes a beta-galactosidase activity.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part II – Use of Kluyveromyces marxianus IMB3

It is clear that only a small proportion of all micro-organisms have been isolated and identified. The simple technique of seeking a thermotolerant fermentative yeast from a suitable hot environment has yielded a number of strains. These organisms, identified as strains of Kluyveromyces marxianus var. marxianus, have been shown to have a wide range of metabolic capabilities that could be used in industrial applications. Not only have the metabolic capabilities been elucidated but possible bioreactor configurations and process application options have been investigated. It appears that there are a number of specific situations where this thermotolerant yeast could find industrial applications. A full-scale industrial ethanol production trial using this yeast was successfully carried out in India. K. marxianus IMB3's performance in terms of the ethanol concentrations achieved was comparable to that obtained using the distillery's own yeast strain with an added advantage of eliminating cooling.     

Rapid differentiation of the closely related Kluyveromyces lactis var. lactis and K. marxianus strains isolated from dairy products using selective media and PCR/RFLP of the rDNA non transcribed spacer 2

PCR/RFLP of the NTS2 (IGS2) of rDNA was applied to differentiate two closely related yeast species, Kluyveromyces lactis var. lactis (referred to as K. lactis) and K. marxianus. Using specific primers, the NTS2 region was amplified from DNA of both K. lactis and K. marxianus type and collection strains. AluI restriction of amplified fragments generated patterns characteristic for each species. The NTS2 region from K. lactis var. drosophilarum and related species K. aestuarii, K. africanus, K. dobzhanskii, and K. wickerhamii could also be amplified with the same primers, but AluI patterns generated were clearly different. PCR/RFLP of the NTS2 appears thus to be a convenient method for rapid identification of K. lactis and K. marxianus, frequently found in dairy products. This test was validated therefore on K. lactis and K. marxianus from natural habitats. We showed that all yeast strains collected from whey samples and scoring blue on X-gal glucose plates were either K. lactis or K. marxianus. For application purposes, we propose here an approach for quickly screening for K. lactis/marxianus and Saccharomyces cerevisiae in dairy products using X-gal coloured and lysine growth media.     

Stable isotope labeling of protein by Kluyveromyces lactis for NMR study

Stable isotope labeling for proteins of interest is an important technique in structural analyses of proteins by NMR spectroscopy. Escherichia coli is one of the most useful protein expression systems for stable isotope labeling because of its high-level protein expression and low costs for isotope-labeling. However, for the expression of proteins with numerous disulfide-bonds and/or post-translational modifications, E. coli systems are not necessarily appropriate. Instead, eukaryotic cells, such as yeast Pichia pastoris, have great potential for successful production of these proteins. The hemiascomycete yeast Kluyveromyces lactis is superior to the methylotrophic yeast P. pastoris in some respects: simple and rapid transformation, good reproducibility of protein expression induction and easy scale-up of culture. In the present study, we established a protein expression system using K. lactis, which enabled the preparation of labeled proteins using glucose and ammonium chloride as a stable isotope source.     

Current status of Kluyveromyces systematics

A brief outline of the current taxonomic status of the genus Kluyveromyces is presented. Noteworthy are the transfer of several former Kluyveromyces species to other genera, the retention of the name Kluyveromyces for K. lactis, K. marxianus, and four related species, and some recent attempts to clarify the variety status of strains assigned to K. lactis.     

Potential probiotic Kluyveromyces marxianus B0399 modulates the immune response in Caco-2 cells and peripheral blood mononuclear cells and impacts the human gut microbiota in an in vitro colonic model system

Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 10(7) CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.     

Evidence for a lectin in Kluyveromyces sp. that is involved in co-flocculation with Schizosaccharomyces pombe

Co-flocculation is the aggregation of yeasts belonging to different genera or species. Kluyveromyces bulgaricus and Kluyveromyces lactis 5c are self-flocculent, but they can also co-flocculate with the non-flocculent yeast Schizosaccharomyces pombe 972 h(-). This co-flocculation is inhibited by D-galactose and galactose derivatives and involves the binding of a galactose-specific proteinic receptor (or lectin) of Kluyveromyces sp. to the cell wall galactomannans of S. pombe. The proteinic receptor is strongly anchored in the cell wall, it was partially purified by affinity chromatography using immobilized S. pombe galactomannans. This galactose-specific proteinic receptor does not appear to interfere in K. bulgaricus or K. lactis self-flocculation, which is mediated by another galactose-specific lectin weakly linked at the cell wall.     

Modes of lactose uptake in the yeast species Kluyveromyces marxianus

Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis. The strains fell into three groups. Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.     

Phenotypic and genotypic diversity of yeasts isolated from water-buffalo Mozzarella cheese

Water-buffalo Mozzarella (WBM) cheese is one of the several 'pasta filata' or stretched curd cheeses that originated in southern Italy, traditionally manufactured from raw milk employing natural whey starter cultures. Lactose- and galactose-fermenting yeasts isolated from WBM were studied to evaluate their role in the ripening of this cheese. The kinetic parameters of the growth of the yeasts as well as their principal metabolic end-products showed a great variability depending on the species. Moreover, the genetic polymorphism of the yeasts was studied for their differentiation at species level by means of the polymerase chain reaction (PCR) fingerprinting and mitochondrial DNA (mtDNA) restriction analysis. While the differentiation based on metabolic traits was not able to discriminate Kluyveromyces marxianus, Candida kefyr and C. sphaerica, the PCR analysis with primers M13 and RF2 resulted in a reliable and rapid method for differentiating at species level Saccharomyces cerevisiae, K. marxianus, K. lactis and their anamorphic species. Furthermore, mtDNA analysis proved to be more discriminating at strain level.     

Cell surface expression of a GPI-anchored form of mouse acetylcholinesterase in Klpmr1Delta cells of Kluyveromyces lactis

The mouse acetylcholinesterase AChE(H) was expressed in the yeast Kluyveromyces lactis. The AChE(H) activity was detectable in intact cells whereas it was absent in the culture media. Glucanase treatment and immunoelectron microscopy data indicated that AChE(H) is anchored to plasma membrane and that the mouse GPI-signaling is compatible with the K. lactis targeting machinery. The AChE(H) was also expressed in a K. lactis strain carrying an inactivated allele of KlPMR1, the gene coding for a P-type Ca(2+)-ATPase of the Golgi apparatus. This mutant displays changes in protein glycosylation and cell wall structure. The AChE(H) activity detected in Klpmr1Delta cells was more than twofold higher than that observed in wild-type cells. The combination of AChE expression and anchoring with the characteristics of Klpmr1Delta strain of K. lactis resulted in yeast cells displaying high AChE activity. This could be regarded as a novel sensing unit to be employed for detecting AChE inhibitors as pesticides.     

Nisin production by a mixed-culture system consisting of Lactococcus lactis and Kluyveromyces marxianus.

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.     

Comparison of the gene expression patterns of alcohol dehydrogenase isozymes in the thermotolerant yeast Kluyveromyces marxianus and their physiological functions

Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomyces cerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.     

Sequential gene integration for the engineering of Kluyveromyces marxianus

The attributes of the yeast Kluyveromyces marxianus (rapid growth rate at high temperature, utilization of a wide range of inexpensive carbon sources) make it a promising industrial host for the synthesis of protein and non-protein products. However, no stable multicopy plasmids are currently available for long-term culture of K. marxianus. To allow the stable genetic/metabolic engineering of K. marxianus, a method for integrating precise numbers of the same or different genes was developed for this yeast. A K. marxianus URA3 deletion mutant was constructed and the URA3 blaster (UB) reusable selection cassette from Saccharomyces cerevisiae was used to select sequential, untargeted chromosomal insertions of the Bacillus megaterium lactate dehydrogenase (LDH) gene. Following excision of the UB cassette from the chromosomes, the integrating vector was retransformed into the strain and a second copy of LDH was inserted, demonstrating the success of this method for sequential gene integrations in K. marxianus. LDH activity and lactic acid concentration increased with each gene insertion, further illustrating the success of this method.     

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

The GAL regulatory system is highly conserved in yeast species of Saccharomyces cerevisiae and Kluyveromyces lactis. While the GAL system is a well studied system in S. cerevisiae, the dynamic behavior of the KlGAL system in K. lactis has not been characterized. Here, we have characterized the GAL system in yeast K. lactis by developing a dynamic model and comparing its performance to its not-so-distant cousin S. cerevisiae. The present analysis demonstrates the significance of the autoregulatory feedbacks due to KlGal4p, KlGal80p, KlGal1p and Lac12p on the dynamic performance of the KlGAL switch. The model predicts the experimentally observed absence of bistability in the wild type strain of K. lactis, unlike the short term memory of preculturing conditions observed in S. cerevisiae. The performance of the GAL switch is distinct for the two yeast species although they share similarities in the molecular components. The analysis suggests that the whole genome duplication of S. cerevisiae, which resulted in a dedicated inducer protein, Gal3p, may be responsible for the high sensitivity of the system to galactose concentrations. On the other hand, K. lactis uses a bifunctional protein as an inducer in addition to its galactokinase activity, which restricts its regulatory role and hence higher galactose levels in the medium are needed to trigger the GAL system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9082-7) contains supplementary material, which is available to authorized users.     

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

新疆地区酸马奶中酵母菌的鉴定及其生物多样性分析

从新疆少数民族牧民家庭采集的28份传统工艺酿造酸马奶样品中分离出87株酵母菌,并对其进行了生理生化鉴定、分子生物学鉴定和生物多样性分析。生化试验结果表明,新疆地区酸马奶中的酵母菌为Saccharomyces unisporus(占总分离株的48.3%),Kluyveromyces marxianus(27.6%),Pichia membranaefaciens(15.0%)和Saccharomyces cerevisiae(9.2%)。选取其中的6株酵母菌和1株参考菌株,进行大亚基(26S)rDNA D1/D2区域(600bp左右)碱基序列分析,并通过GenBank进行同源序列搜索以确定各菌株的归属,进一步验证生理生化方法的正确性。从得到的结果中可以看出,S.unisporus和K.marxianus为新疆地区酸马奶中的优势菌。     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Genome Sequence of the Thermotolerant Yeast Kluyveromyces marxianus var. marxianus KCTC 17555

Kluyveromyces marxianus is a thermotolerant yeast that has been explored for potential use in biotechnological applications, such as production of biofuels, single-cell proteins, enzymes, and other heterologous proteins. Here, we present the high-quality draft of the 10.9-Mb genome of K. marxianus var. marxianus KCTC 17555 (= CBS 6556 = ATCC 26548).