Studies on metal resistance system inKluyveromyces marxianus

Through preliminary plate tests,Kluyveromyces marxianus was found to be much more resistant to toxic heavy metals compared to aCUP1 ^R strain ofSaccharomyces cerevisiae. Specific growth rate and maximum dry weights affected by increasing metal concentrations were determined to obtain precise patterns of resistance. Metal biosorption was also monitored during the course of growth in synthetic media containing respective metals at 0.5 mM final concentration. Although Zn- and Co-binding was negligible, as much as 90% of silver, 60% of copper, and 65% of cadmium were found to be absorbed by the end of active growth. Analysis of the protein profiles ofS. cerevisiae andK. marxianus on metal exposure suggested constitutive production of metallothionein inK. marxianus. Furthermore, a smaller protein synthesized byK. marxianus on induction by silver or cadmium accounts for the high resistance of the organism to these metals.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The use of the replicating pDblet plasmid as a cloning vector with enhanced stability in Kluyveromyces marxianus

The replicating plasmid, pDblet, transformed the budding yeast Kluyveromyces marxianus to an efficiency of 10⁴ transformant g^–1 DNA. Transformed cells showed 1% of segregation rate without affecting their growth rate of 0.69 h^–1 and glucose consumption. These results were similar or better than the commonly used pE1 plasmid and suggests that pDblet can be used for cloning genes in K. marxianus.     

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.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Effect of culture conditions of Kluyveromyces marxianus on its autolysis, and process optimization

Cultivation of the lactose-metabolizing yeast Kluyveromyces marxianus var. marxianus (formerly K.?fragilis) on supplemented whey permeate resulted in cellular yield little affected by culture conditions in the ranges pH?=?2.3–5 and T?=?30–40?°C. When autolysis was induced only by energy source deficiency and thermal shock, cellular material solubilization depended slightly on autolysis temperature in the range T?=?45–60?°C. On the contrary, the process was under tight control of culture conditions; when autolysis was carried out at 50?°C with an initial dry cellular concentration of 50?g l^?1, a clear optimum was observed for cells cultivated at pH?=?4.5 and T?=?35?°C. So the critical step of the autolytic process consisted in biosynthesis of lytic enzymes (during cell growth) rather than enzymatic progress (during autolysis). These results were compatible with a model previously proposed for Saccharomyces cerevisiae [1].     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.     

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     

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.     

Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Summary Two strains of Kluyveromyces marxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.     

Improved ethanol production from whey with Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65–70 g/l lactose) by Saccharomyces cerevisiae CCY 10–13–14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/l × h), compared to the fermentation in which the lactose was directly fermented by K. marxianus.     

The use of the GDH gene for molecular identification and phylogenetic analysis of the yeast Kluyveromyces marxianus

Summary Our previous work showed that NADP⁺-dependent glutamate dehydrogenase from K. marxianus behaves similarly to its counterpart in S. cerevisiae. It suggested that the ammonia assimilation pathway might be different between K. marxianus and the genetic closed species K. lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K. marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K. marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K. marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S. cerevisiae GDH1 gene family together with KlGDH gene.