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.     

Simultaneous production of inulase and lactase in batch and continuous cultures of Kluyveromyces fragilis

Under different induction conditions, the industrial yeast Kluyveromyces fragilis is an excellent producer of the enzymes inulase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) and lactase (β-d-galactoside galactohydrolase, EC 3.2.1.23), producing 27 and 1.6 U mg^?1dry cell weight, respectively. In order to improve overall enzyme yields, conditions for the simultaneous production of both enzymes in a one-stage fermentation have been examined. Techniques employed include carbon-limited batch and continuous culture, single and mixed carbon substrates, and the use of a mutant semi-constitutive for inulase production. Synthesis of both enzymes suffered strongly from carbon catabolite repression in batch cultures grown on single and mixed inducing substrates. Only glycerol and dl-malate did not repress either enzyme. The non-metabolizable analogues of lactose, isopropyl-β-d-thiogalactoside and methyl-β-d-thiogalactoside induced lactase in glycerol grown batch cultures, but were ineffective in sucrose grown continuous cultures. They also depressed the normally high levels of inulase in such continuous cultures. The highest simultaneous inulase and lactase activities in the wild-type yeast were obtained in continuous culture on an equal mixture of d-fructose and d-galactose; 25 and 0.78 U mg^?1dry cell weight, respectively. In this fermentation the combined yield per unit carbon substrate of the two enzymes was 141%, compared to a reference value of 100% for the highest yield of each enzyme in separate fermentations. On the same mixture of d-fructose and d-galactose, the mutant produced ～60 and 0.70 U mg^?1dry cell weight, respectively. The combined enzyme yield per unit of carbon substrate was 172%.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate

Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK _m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V _max) are dependent on the dilution rate (D). At D=0.12 h^–1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h^–1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.     

Kluyveromyces fragilis SS-437: An associatively-profiled thermotolerant yeast

The lactose-utilizing Kluyveromyces fragilis SS-437 was found to have an associative temperature profile, but a thermotolerant growth yield behaviour. Cardinal growth temperatures were: 3°C minimum for growth; 41.5°C optimum; 44.5°C final maximum (growth and death rates equalize); 46.1°C initial maximum (maximum limit for growth).     

Manganese accumulation in yeast cells

Summary Manganese accumulation was studied by room-temperature electron spin resonance (ESR) spectroscopy inSaccharomyces cerevisiae grown in the presence of increasing amounts of MnSO₄. Mn²⁺ retention was nearly linear in intact cells for fractions related to both low-molecular-mass and macromolecular complexes (free and bound Mn²⁺, respectively). A deviation from linearity was observed in cell extracts between the control value and 0.1 mM Mn²⁺, indicating more efficient accumulation at low Mn²⁺ concentrations. The difference in slopes between the two straight lines describing Mn²⁺ retention at concentrations lower and higher than 0.1 mM, respectively, was quite large for the free Mn²⁺ fraction. Furthermore it was unaffected by subsequent dialyses of the extracts, showing stable retention in the form of low-molecular-mass complexes. In contrast, the slope of the line describing retention of bound Mn²⁺ at concentrations higher than 0.1 mM became less steep after subsequent dialyses of the cell extracts. This result indicates that the macromolecule-bound Mn²⁺ was essentially associated with particulate structures. In contrast to Cu²⁺, Mn²⁺ had no effect on the major enzyme activities involved in oxygen metabolism except for a slight increase of cyanide-resistant Mn-superoxide dismutase activity, due to dialyzable Mn²⁺ complexes.     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

The twoPhysarum polycephalum profiling are not functionally equivalent in yeast cells

Summary Profilin is a ubiquitous actin-monomer-binding protein. The protistPhysarum polycephalum contains two profilins, ProA and ProP, present in amoebae and plasmodia, respectively. We have used mutantSaccharomyces cerevisiae cells in an attempt to observe distinct functions for the two profilins. Profilin-deficient yeast cells (pfy1) have delocalized actin cortical patches, do not contain visible actin cables, have reduced mating efficiency and do not grow at 37 °C or in the presence of caffeine. Deletion of theSRV2 gene (srv2), coding for the adenylyl cyclase-associated protein, also results in an altered actin distribution and an inability to survive on rich medium. We found that the pfy1 and srv2 mutant phenotypes were corrected equally well by the overexpression of Physarum ProA or yeast Pfy1p profilins. The pfy1 cells overexpressing ProP have improved mating efficiency and a normal distribution of actin cortical patches. These cells, however, have barely detectable actin cables, do not grow at 37 °C, and are sensitive to caffeine. Also, the expression of ProP does not correct the growth defect of the srv2 cells. These results suggest that the two Physarum proteins are not functionally equivalent in yeast cells. No difference was detected in the affinity of ProA and ProP for poly-L-proline, while ProA has a slightly greater affinity than ProP for phosphatidylinositol 4,5-biphosphate.Abbreviations FITC tfluorescein isothiocyanate - PIP₂ phosphatidylinositol 4,5-biphosphate - YPD yeast extract peptone dextrose     

Hydrolysis of whey lactose using CTAB-permeabilized yeast cells

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to β-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 °C.     

Solvent treatment for β-d-galactosidase release from yeast cells

A method is presented for the release of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from yeast cells. Enzyme release is attained by mixing yeast cells with concentrated solvents (20 to 95%) and subsequently suspending and agitating the cells in buffer. Many solvents, including isopropanol, ethanol and methanol, were found to be effective. Enzyme release into buffer was relatively slow: 10–20 h was required for maximum yields. The release of protease and β-d-galactosidase was monitored. β-d-Galactosidase solubilization was achieved in high yield: 90% of the intracellular enzyme was released into the buffer. Because this method exhibits resistance to yield loss due to microbial degradation and is not sensitive to small changes in solvent in buffer concentration or treatment time, it is particularly suited to industrial-scale enzyme recoveries.     

Thermotolerant single cell protein production byKluyveromyces marxianus var.marxianus

Summary Amino acid analyses were undertaken on single cell protein (SCP) produced by thermotolerant strains ofKluyveromyces marxianus var.marxianus grown on sugar cane molasses at 40°C. The maximum conversion of available sugars to biomass at 45°C was only 10.8% (g dry wt.·g^–1 total sugars). The amino acid composition of the SCP did not differ markedly from that reported for other yeast species.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Responses to experimental fish manipulations in a shallow,hypereutrophic lake: the relative importance of benthic nutrient recycling and trophic cascade

The genetic polymorphism of twelve enzyme systems in two parthenogenetic tetraploidArtemia populations from N. Greece has been studied, using starch gel electrophoresis. The genetic distance within and among the two Greek populations was calculated. The high degree of genetic identity between the populations indicates that they belong to the same species. Each is composed of electrophoretically identifiable clones; the fitness of these clones under different environmental conditions is discussed.     

Utilization of molasses for the production of fat by an oleaginous yeast,Rhodotorula glutinis IIP-30

Summary Rhodotorula glutinis is known to produce fat when cultivated under nitrogen-limiting conditions. Economically, molasses is an ideal substrate, however, due to the presence of nitrogen in molasses, the lipid yield obtained is much lower than that obtained from glucose or sucrose. Higher yields were obtained using molasses in a fed batch fermentation supplemented with glucose or sucrose during the lipid accumulation phase. The fatty acids profile of the lipids thus produced, using a very simple and economical medium, was similar to that obtained from glucose and sucrose.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

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.     

Prevalence and distribution of introns in non-ribosomal protein genes of yeast

Relatively few genes in the yeast Saccharornyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an effort to quantify this bias we have estimated the prevalence of intron sequences among non-ribosomal protein genes by assessing the number of prp2-sensitive mRNAs in an in vitro translation assay. These results, combined with an updated survey of the GenBank DNA database, support an estimate of 2.5% for intron-containing non-ribosomal protein genes. Furthermore, our observations reveal an intriguing distinction between the distributions of ribosomal protein and non-ribosomal protein intron lengths, suggestive of distinct, gene class-specific evolutionary pressures.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Heterologous protein production in yeast

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeastSaccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application.In the following review a selection from the different yeast systems is described and compared.