Xylose and some non-sugar carbon sources cause catabolite repression in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Glucose and other sugars, such as galactose or maltose, are able to cause carbon catabolite repression in Saccharomyces cerevisiae. Although glycolytic intermediates have been suggested as signal for repression, no evidence for such a control mechanism is available. The establishment of a correlation between levels of intracellular metabolites and the extent of catabolite repression may facilitate the identification of potential signal molecules in the process. To set a framework for such a study, the repression produced by xylose, glycerol and dihydroxyacetone upon genes belonging to different repressible circuits was tested, using an engineered strain of S. cerevisiae able to metabolize xylose. Xylose decreased the derepression of various enzymes in the presence of ethanol by at least 10-fold; the corresponding mRNAs were not detected in these conditions. Xylose also impaired the derepression of galactokinase and invertase. Glycerol and dihydroxyacetone decreased 2- to 3-fold the derepression observed in ethanol or galactose but did not affect invertase derepression. For yeast cells grown in media with different carbon sources, no correlation was found between repression of fructose-1,6-bisphosphatase and intracellular levels of glucose 6-phosphate or fructose 1,6-bisphosphate.     

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae

Summary The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.The first article of this series is in Mol Gen Genet 191:31–38On a leave absence from Nikka Whisky Co.     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Characterizing the memory of the GAL regulatory network in Saccharomyces cerevisiae

Genetic regulatory networks respond dynamically to perturbations in the intracellular and extracellular environments of an organism. The GAL system in the yeast Saccharomyces cerevisiae has evolved to utilize galactose as an alternative carbon and energy source, in the absence of glucose in the environment. We present a dynamic model for GAL system in Saccharomyces cerevisiae, which includes a novel mechanism for Gal3p activation upon induction with galactose. The modification enables the model to simulate the experimental observation that in absence of galactose, oversynthesis of Gal3p can also induce the GAL system. We then characterize the memory of the GAL system as the domain of attraction of the steady states.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

Perturbation of the interaction between Gal4p and Gal80p of the Saccharomyces cerevisiae GAL switch results in altered responses to galactose and glucose

In S. cerevisiae, following the Whole Genome Duplication (WGD), GAL1‐encoded galactokinase retained its signal transduction function but lost basal expression. On the other hand, its paralogue GAL3, lost kinase activity but retained its signalling function and basal expression, thus making it indispensable for the rapid induction of the S. cerevisiae GAL switch. However, a gal3Δ strain exhibits delayed growth kinetics due to the redundant signalling function of GAL1. The subfunctionalization between the paralogues GAL1 and GAL3 is due to expression divergence and is proposed to be due to the alteration in the Upstream Activating Sequences (UAS_G). We demonstrate that the GAL switch becomes independent of GAL3 by altering the interaction between Gal4p and Gal80p without altering the configuration of UAS_G. In addition to the above, the altered switch of S. cerevisiae loses ultrasensitivity and stringent glucose repression. These changes caused an increase in fitness in the disaccharide melibiose at the expense of a decrease in fitness in galactose. The above altered features of the ScGAL switch are similar to the features of the GAL switch of K. lactis that diverged from S. cerevisiae before the WGD.     

The <Emphasis Type="Italic">GAL10</Emphasis> Gene is Located 40 kbp Away from the <Emphasis Type="Italic">GAL7</Emphasis>-<Emphasis Type="Italic">GAL1</Emphasis> Region in the Yeast <Emphasis Type="Italic">Kazachstania naganishii</Emphasis>

Of the genes involved in galactose metabolism, GAL7, GAL10, and GAL1 are tightly linked in this order on chromosome II in Saccharomyces cerevisiae. While several species of the order Saccharomycetales have similar gene organization, Kazachstania naganishii is unique, in which GAL7 and GAL1 are close to each other whereas GAL10 is substantially apart from them on chromosome XI. In this study, we inserted the recognition sequence of I-SceI homing-endonuclease into GAL10 and also into the intervening segment of GAL7-GAL1. By cleaving chromosome DNA of the gene-manipulated strain with I-SceI, we obtained evidence that chromosome XI (610 kbp) was replaced with three fragments (305, 265, and 40 kbp). Using appropriate probes, we further found that GAL10 was about 40 kbp apart from the GAL7-GAL1 cluster and that orientation of GAL10 was reversed comparing to the S. cerevisiae counter part. We, therefore, contend that comparison of the organization of the GAL cluster among Saccharomycetales is of importance to elucidate evolution of chromosomes and that the experimental scheme developed in this study is useful for this line of investigation.     

The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of simlarity to a number of UDP glucose-D-gatactose-1-phosphate uridylyitransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MFα, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, β-glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7 p::GUS fusion was used to quantify inducibitity of the GAL7 promoter, the level of enzyme activity was at least 500-fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars

Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.     

Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3',5'-monophosphate.

We studied the following two aspects of the glucose effect on galactose operon expression in Escherichia coli K-12: catabolite repression and inducer exclusion. Using both inducible and constitutive strains and measuring the rate of promoter-proximal enzyme synthesis, we found that the galactose operon did not seem to exhibit catabolite repression. The only glucose effect on galactose operon expression which we observed was inducer exclusion, as shown by the existence of diauxic growth in the presence of glucose and galactose. This diauxie was not relieved by cyclic adenosine 3',5'-monophosphate. Cyclic adenosine 3',5'-monophosphate did not seem to be an antagonist of any glucose effect on galactose operon expression; its only effect was to stimulate promoter-distal gene expression.     

Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans

Summary Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.     

A novel promoter,derived from the isocitrate lyase gene of Candida tropicalis,inducible with acetate in Saccharomyces cerevisiae

When the isocitrate lyase gene, containing 5'-upstream and 3'-flanking regions, of an n-alkane-assimilating yeast Candida tropicalis was introduced into Saccharomyces cerevisiae, the enzyme was functionally overexpressed in the cells grown on acetate. The amount of the recombinant isocitrate lyase expressed in S. cerevisiae was as much as 30% of the total soluble proteins in the cells, being comparable to that with GAL7 functional under the control of galactose. The expression was also observed when the cells were grown on glycerol, lactate, ethanol or oleate. These facts indicate that the isocitrate lyase gene upstream region (UPR-ICL) contains a strong promoter functional in S. cerevisiae. UPR-ICL is active as a promoter on cheap carbon sources such as acetate and nonconventional carbon sources such as oleate, whereas many conventional strong promoters demand relatively expensive sugars or sugars derivatives. Therefore, it is promising to construct an economical recombinant protein production system by using UPL-ICL.     

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.