Derepression of a baker's yeast strain for maltose utilization is associated with severe deregulation of HXT gene expression

Aims: We undertook to improve an industrial Saccharomyces cerevisiae strain by derepressing it for maltose utilization in the presence of high glucose concentrations. Methods and Results: A mutant was obtained from an industrial S. cerevisiae strain following random UV mutagenesis and selection on maltose/5‐thioglucose medium. The mutant acquired the ability to utilize glucose simultaneously with maltose and possibly also sucrose and galactose. Aerobic sugar metabolism was still largely fermentative, but an enhanced respirative metabolism resulted in a 31% higher biomass yield on glucose. Kinetic characterization of glucose transport in the mutant revealed the predominance of the high‐affinity component. Northern blot analysis showed that the mutant strain expresses only the HXT6/7 gene irrespective of the glucose concentration in the medium, indicating a severe deregulation in the induction/repression pathways modulating HXT gene expression. Interestingly, maltose‐grown cells of the mutant display inverse diauxy in a glucose/maltose mixture, preferring maltose to glucose. Conclusion: In the mutant here reported, the glucose transport step seems to be uncoupled from downstream regulation, because it seems to be unable to sense abundant glucose, via both repression and induction pathways. Significance and Impact of the Study: We report here the isolation of a S. cerevisiae mutant with a novel derepressed phenotype, potentially interesting for the industrial fermentation of mixed sugar substrates.     

Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast

Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.     

Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxyglucose-resistant mutant of Kluyveromyces marxianus

Fourteen lactose-fermenting strains of Kluyveromyces marxianus , including its anamorph, Candida kefyr , were grown in two media containing 20% (w/v) sugar as either beet molasses or cheese whey. Strain NBRC 1963 of K. marxianus converted sucrose and lactose to ethanol in both media most efficiently. However, ethanol was produced from sucrose and not from lactose by strain NBRC 1963 in the medium containing equal amounts of sugar from beet molasses and cheese whey. The spontaneous mutants resistant to 2-deoxyglucose in the minimal medium composed of galactose as the sole carbon source were isolated from strain NBRC 1963. Among them, strain KD-15 vigorously produced ethanol in the media containing beet molasses, cheese whey, or both. The mutant strain KD-15 was insensitive to catabolite repression, as shown by the observation that β-galactosidase was not repressed in the presence of sucrose from beet molasses.     

牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达

凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。     

Intraspecific gene expression variability in the yeast Kluyveromyces lactis revealed by micro-array analysis

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.     

Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.     

Genetics and molecular physiology of the yeast Kluyveromyces lactis

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.