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.     

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).     

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.     

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.     

The yeast Kluyveromyces marxianus and its biotechnological potential

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (beta-galactosidase, beta-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71-75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value.     

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase

The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase-p37-that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.     

Construction of thermotolerant yeast expressing thermostable cellulase genes

Kluyveromyces marxianus NBRC1777 was identified as a thermotolerant yeast and was developed as a host for the expression of thermostable cellulase genes. The previously isolated genes for thermostable endo-beta-1,4-glucanase, cellobiohydrolase, and beta-glucosidase were introduced into the chromosome of K. marxianus and successfully expressed under the control of high-expression promoters. The recombinant K. marxianus expressing cellulase genes became able to grow in synthetic medium containing cellobiose or carboxymethyl-cellulose as the single carbon source. Moreover, the recombinant strain produced 43.4 g/L ethanol from 10% cellobiose. These results suggest that K. marxianus may afford a useful host system, which may be applicable to the simultaneous saccharification and fermentation and the foundation of cellulose consolidated bioprocessing.     

Inulase-secreting strain of Saccharomyces cerevisiae produces fructose

The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid.     

Potent hypocholesterolemic activity of the yeast Kluyveromyces marxianus YIT 8292 in rats fed a high cholesterol diet

The hypocholesterolemic activities of 81 yeast strains were examined in rats fed a high cholesterol diet (HCD). Male Wistar rats were fed an HCD or an HCD supplemented with 10% yeast for 7 d. It was found that the hypocholesterolemic activities of the yeasts varied remarkably between strains. Kluyveromyces marxianus YIT 8292 exhibited the most potent hypocholesterolemic activity among the yeasts that were tested. K. marxianus YIT 8292 significantly decreased not only plasma total cholesterol but also liver total cholesterol when administered as a dietary admixture at a concentration of 3%. In contrast, brewer's yeast and baker's yeast, which have been predominantly used for food, did not exhibit hypocholesterolemic activity even when administered at a concentration of 10%. These results suggest that K. marxianus YIT 8292 may be utilized as a novel food material with the ability to contribute to the prevention of hypercholesterolemia.     

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.     

Regulation of bacterial photosynthesis genes by oxygen and light

When grown in the appropriate medium, several yeast species produce pectinases able to degrade pectic substances. It is mainly exocellular endopolygalacturonases that break pectins or pectate down by hydrolysis of alpha-1,4-glycosidic linkages in a random way. Biochemical characterisation of these enzymes has shown that they have an optimal pH in the acidic region and an optimal temperature between 40 and 55 degrees C. Their production by yeasts is a constitutive feature and is repressed by the glucose concentration and aeration. Pectic substances and their hydrolysis products are used as carbon sources by a limited number of yeasts and hence these enzymes must be involved in the colonisation of different parts of plants, including fruits. The first yeast pectic enzyme (encoded by the PSE3 gene) was cloned from Tichosporon penicillatum. Recently, a polygalacturonase-encoding gene from Saccharomyces cerevisiae has been cloned and overexpressed in several strains and the gene for an extracellular endopolygalacturonase from Kluyveromyces marxianus has also been described. Taking all the results together, the idea is now emerging that this type of yeast enzyme could offer an alternative to fungal enzymes for industrial applications.     

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.     

U3 snoRNA genes with and without intron in the Kluyveromyces genus: yeasts can accommodate great variations of the U3 snoRNA 3'-terminal domain.

The U3 snoRNA coding sequences from the genomic DNAs of Kluyveromyces delphensis and four variants of the Kluyveromyces marxianus species were cloned by PCR amplification. Nucleotide sequence analysis of the amplification products revealed a unique U3 snoRNA gene sequence in all the strains studied, except for K. marxianus var. fragilis. The K. marxianus U3 genes were intronless, whereas an intron similar to those of the Saccharomyces cerevisiae U3 genes was found in K. delphensis. Hence, U3 genes with and without intron are found in yeasts of the Saccharomycetoideae subfamily. The secondary structure of the K. delphensis pre-U3 snoRNA and of the K. marxianus mature snoRNAs were studied experimentally. They revealed a strong conservation in yeasts of (1) the architecture of U3 snoRNA introns, (2) the 5'-terminal domain of the mature snoRNA, and (3) the protein-anchoring regions of the U3 snoRNA 3' domain. In contrast, stem-loop structures 2, 3, and 4 of the 3' domain showed great variations in size, sequence, and structure. Using a genetic test, we show that, in spite of these variations, the Kluyveromyces U3 snoRNAs are functional in S. cerevisiae. We also show that S. cerevisiae U3A snoRNAs lacking the stem-loop structure 2 or 4 are functional. Hence, U3 snoRNA function can accommodate great variations of the RNA 3'-terminal domain.     

Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.     

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。     

Utilization of corn silage juice by Klyuveromyces marxianus

Corn silage juice was found to be a favorable substrate for production of fodder yeasts. Kluyveromyces marxianus NRRL Y-610 yielded significantly more cell dry weight than other cultures examined. In shake-flask experiments, the yeast produced over 13 g of cell dry weight per liter of corn silage juice and completely consumed the organic pollutants (lactic acid, acetic acid, and ethanol). The yeast settled rapidly and had a yeast volume index of 21 ml/g. The results indicate that K. marxianus NRRL Y-610 could be used to efficiently remove lactic acid and other organic compounds from corn silage juice with the concomitant production of fodder yeast.     

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.     

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.