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.     

Subcellular distribution of accumulated heavy metals in Saccharomyces cerevisiae and Kluyveromyces marxianus

Summary Kluyveromyces spp. have been found to be more efficient than a CUP1^R strain of S. cerevisiae in heavy metal resistance and accumulation. The present study describes the subcellular distribution of the accumulated metals (Ag, Cd, Cu) in S. cerevisiae and K. marxianus. Absorption by insoluble cellular material of the metals appears as the main mechanism of metal accumulation in both organisms.     

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.     

Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae

The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35 °C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6 g l^?1 were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70 % of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation.     

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.     

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.     

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.     

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.     

Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.     

酿酒酵母基因组位置效应对外源基因表达的影响

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程,而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响,以绿色荧光蛋白为报告基因,通过双交换同源重组方法,对酿酒酵母单基因敲除库进行高通量转化,构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析,构建高表达位点库和低表达位点库,共发现促进绿色荧光蛋白表达的位点428个,抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布,从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考,对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。     

Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus

A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus. A plasmid was constructed containing the Escherichia coli beta-glucuronidase (gus) gene cloned downstream of the yeast tet-off promoter, the tetR-VP16 activator protein gene, and the URA3 gene for selection. The tet-off promoter-gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 microg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000-fold. The tet-off promoter was effective at 30, 37, and 42 degrees C, although the overall Gus activity was highest at 37 degrees C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture.     

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.     

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.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999