Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.Abbreviations NacGlc N-acetyl-D-glucosamine - x total biomass (dry cellular weight) concentration     

Interfertility as basis for the delimitation of Kluyveromyces marxianus

Hybridization studies between strains of Kluyveromyces marxianus and the remaining species of the genus involving the use of auxotrophic mutants, are reported. K. marxianus was found to be interfertile with K. bulgaricus, K. cicerisporus, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis, K. phaseolosporus, K. vanudenii and K. wikenii. Accepting interfertility as criterion for conspecificity, these nine syngamous taxa are relegated to the status of biotypes or physiologic races of a single species K. marxianus.     

Kluyveromyces marxianus: a potential source of diacetyl reductase

Kluyveromyces marxianus had a higher specific activity of diacetyl reductase (EC 1.1.1.5) than all other organisms previously reported. The enzyme was NADH-dependent and irreversibly catalysed the conversion of diacetyl to acetoin with an optimum pH of 7.0. It was stable at 40°C but lost 50% of its activity at 50°C in 30 min. The K _m and V _max values for diacetyl were 1.8 mm and 0.053 mm/min, respectively.The authors are with the Department of Food Science and Technology, Comell University, Geneva, New York 14456, USA     

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.     

A simple and rapid method for lithium acetate-mediated transformation of Kluyveromyces marxianus cells

Efficient plasmid transformation of Kluyveromyces marxianus cells of 1.9 × 10³ transformant μg⁻¹ DNA with an episomal plasmid was achieved by the use of a simple lithium acetate method with the addition of 10 mM DTT and an increased heat shock temperature of 47 °C. This method is shown to be also efficient for replicative plasmids. Therefore, we suggest its use as a routine method to transform K. marxianus cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Utilisation of cheese whey as an alternative growth medium for recombinant strains of Kluyveromyces marxianus

Kluyveromyces marxianus KMDB-1, a plasmid-bearing recombinant, not carrying any particular gene of relevance, derived from auxotrophic strain KMS-2 (ura ^–), grew in cheese whey with a maximum specific growth rate of 0.34 h^–1. This recombinant strain showed the same lactose uptake and extracellular protease production kinetics as the wild type CBS6556 with no evidence of catabolite repression. The plasmid was retained in 50% of cells after 36 h of batch culture. The presence of this vector in Kluyveromyces marxianus, which possesses no natural plasmids, together with the absence of any metabolic loading effect, creates a suitable microbial system for cheese whey processing for potential value-added product formation.     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

Production of High-content Galacto-oligosaccharide by Enzyme Catalysis and Fermentation with Kluyveromyces marxianus

Of three β-galactosidases from Aspergillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto- oligosaccharides (GOS) from lactose, the latter produced the highest yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing β-galactosidase glucose oxidase. The low-content GOS syrups, produced either by β-galactosidase alone or by the mixed enzyme system, were subjected to the fermentation by Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight basis of high-content GOS with the yields of 31% and 32%, respectively. An erratum to this article can be found at     

Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 U_SOD mg_prot ⁻¹) and the highest volumetric yield (8.8 × 10⁵ U_SOD l⁻¹) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu²⁺ and Zn²⁺ supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 × 10⁶ U_SOD l⁻¹. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aroma compounds produced by Kluyveromyces marxianus in solid state fermentation on a packed bed column bioreactor

Studies were carried out for the production of aroma compounds by Kluyveromyces marxianus grown on cassava bagasse in solid state fermentation using packed bed reactors, testing two different aeration rates. Respirometric analysis was used to follow the growth of the culture. Headspace analysis of the culture by gas chromatography showed the production of 11 compounds, out of which nine were identified. Ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Lower aeration rate (0.06l h^–1 g^–1 of initial dry matter) increased total volatile (TV) production and the rate of production was also increased at this aeration rate. Using an aeration rate of 0.06l h^–1 g^–1 maximum TV concentrations were reached at 24 h and at 40 h with 0.12l h^–1 g^–1.     

Short communication: Ethanol production from cellulose at 45°C using a batch-fed system containing alginate-immobilized Kluyveromyces marxianus IMB3

The thermotolerant yeast, Kluyveromyces marxianus IMB3, was grown in batch culture at 45°C on cellulose-containing media, supplemented with exogenous cellulase activity. At various stages during fermentation, both substrate and enzyme were added in batch mode and fermentation was continued for 220 h. Ethanol production increased to 20 g/l at 200 h, representing 45% of the maximum theoretical yield. In subsequent experiments, the organism was immobilized in calcium alginate beads and these were used in a similar, batch-fed system at 45°C. Again, fermentation was continued for 220 h and ethanol production increased to its maximum, of 28 g/l, within 100 h and this represented in excess of 60% of the maximum theoretical yield.     

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7.The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4–7% of the original dry cell weight. The purified product, at a concentration of 12 g l^–1, formed emulsions that were stable for 3 months when subjected to a range of pH (3–11) and NaCl concentrations (2–50 g l^–1). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4–6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.     

Molecular probes for the detection of Kluyveromyces marxianus chromosomal DNA in electrophoretic karyotypes of intergeneric protoplast fusion products

Random genomic DNA fragments from Kluyveromyces marxianus were cloned in order to identify chromosomal bands in pulsed field electrophoresis patterns of intergeneric hybrid strains which were obtained by protoplast fusion. Molecular hybridization data indicated that the K. marxianus parental strain might be triploid, and it showed strong chromosome length polymorphism. We analyzed the karyotype of two Saccharomyces cerevisiae/K. marxianus hybrid strains (St. 1, St. 46) with our DNA probes and with a Ty1 specific probe. We found indications for recombinational events which lead to the formation of hybrid chromosomal DNA molecules.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

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.     

Isolation of a Gene Greatly Expressed in Kluyveromyces Marxianus at High Temperature

Summary This research provides insight into the expression of thermotolerance-related genes in Kluyveromyces marxianus by differential display polymerase chain reaction (DD-PCR) techniques. Fourteen differential expressed sequence tags (ESTs) were observed and one of them, SHWBY10 was confirmed to be positive by reverse Northern blot analysis. The sequence has the GenBank Accession ID No. CD374838. DNA sequencing showed that it encoded a 279 bp ORF containing 92 amino acids. Analysis of protein sequence indicated that it has significant sequence homology with a peroxisomal protein product (gi 50309315) from Kluyveromyces lactis. This discovery suggests this gene may be related to yeast thermotolerance.     

Heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1) using versatile autonomously replicating vector for a wide range of host

A versatile plasmid shuttle vector system pKDU7 was constructed, which is useful for the heterologous gene expression in a wide range of Kluyveromyces and Saccharomyces strains. This cloning vector was constructed using the 1.6-μm circular plasmid pKD1 of Kluyveromyces drosophilarum, the URA3 gene of K. marxianus as well as the pUC19 sequences. The stability of vector in transformants strongly depends on the integrity of the functionally important elements of pKD1. It was shown by comparison of three recombinant vectors, which possessed the pKD1 sequence inserted in different ways. The efficient transformation and stability maintenance of the vector constructed in various strains of Kluyveromyces and Saccharomyces was shown by the expression of the EPG1 gene of the Kluyveromyces marxianus encoding pectin-degrading endopolygalacturonase.     

A growth kinetic model of<Emphasis Type="Italic"> Kluyveromyces marxianus</Emphasis> cultures on cheese whey as substrate

This work presents a multi-route, non-structured kinetic model for determination of microbial growth and substrate consumption in an experimental batch bioreactor in which -galactosidase is produced by Kluyveromyces marxianus growing on cheese whey. The main metabolic routes for lactose, and oxygen consumption, cell growth, and ethanol production are derived based on experimental data. When these individual rates are combined into a single growth rate, by rewriting the model equations, the model re-interpretation has a complexity similar to that of the usual variations of the Monod kinetic model, available in the literature. Furthermore, the proposed model is in good agreement with the experimental data for different growth temperatures, being acceptable for dynamic simulations, processes optimization, and implementations of model-based control technologies.