Carbon Source-responsive Element Research Articles

Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, PIT350 and PIN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of PIN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of PIN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used PIN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

The transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris.

Protein production in Pichia pastoris is often based on the methanol‐inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1. Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P AOX1 strain in terms of titer and process performance.

Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to selected CSREs and provide evidence that KlSip4 counteracts KlCat8-mediated transcription activation by competing for binding to some but not all CSREs. The finding that the hierarchical relationship of these transcription factors differs between K. lactis and S. cerevisiae and that the sets of target genes have diverged contributes to explaining the phenotypic differences in metabolic life-style.

Differential control of isocitrate lyase gene transcription by non-fermentable carbon sources in the milk yeast Kluyveromyces lactis

The KlICL1 gene, encoding isocitrate lyase in Kluyveromyces lactis, is essential for ethanol utilization. Deletion analyses identified two functional promoter elements, CSRE-A and CSRE-B. Transcription is activated on ethanol, but not on glucose, glycerol or lactate. Expression depends on the KlCat8p transcription factor and KlSip4p binds to the promoter elements. Glycerol diminishes KlICL1 expression and a single carbon source responsive element (CSRE) sequence is both necessary and sufficient to mediate this regulation. The glycerol effect is less pronounced in Saccharomyces cerevisiae than in K. lactis. Mutants lacking KlGUT2 (which encodes the glycerol 3-phosphate dehydrogenase) still show reduced expression in glycerol, whereas mutants deficient in glycerol kinase ( Klgut1) do not. We conclude that a metabolite of glycerol is required for this regulation.

Role of Tos3, a Snf1 Protein Kinase Kinase, during Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

In Saccharomyces cerevisiae, Snf1 protein kinase of the Snf1/AMP-activated protein kinase family is required for growth on nonfermentable carbon sources and nonpreferred sugars. Three kinases, Pak1, Elm1, and Tos3, activate Snf1 by phosphorylation of its activation-loop threonine, and the absence of all three causes the Snf(-) phenotype. No phenotype has previously been reported for the tos3Delta single mutation. We show here that, when cells are grown on glycerol-ethanol, tos3Delta reduces growth rate, Snf1 catalytic activity, and activation of the Snf1-dependent carbon source-responsive element (CSRE) in the promoters of gluconeogenic genes. In contrast, tos3Delta did not significantly affect Snf1 catalytic activity or CSRE function during abrupt glucose depletion, indicating that Tos3 has a more substantial role in activating Snf1 protein kinase during growth on a nonfermentable carbon source than during acute carbon stress. We also report that Tos3 is localized in the cytosol during growth in either glucose or glycerol-ethanol. These findings lend support to the idea that the Snf1 protein kinase kinases make different contributions to cellular regulation under different growth conditions.

Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast Saccharomyces cerevisiae.

The structural genes for gluconeogenesis in the yeast Saccharomyces cerevisiae are activated by the carbon source-responsive element (CSRE) found in the respective upstream regions. Regulatory genes CAT8 and SIP4 both encode zinc-cluster proteins which can bind to CSRE motifs and activate target genes under conditions of glucose deprivation. In this work, we describe a functional analysis of sequence variants containing single mutations within the strongly activating CSRE(ICL1) motif. While the sequence CCNNNNNNCCG was required as the minimal UAS for gene activation by both Cat8 and Sip4, the activators responded differently to sequence variations in the central part of the CSRE. Our results allowed us to derive a consensus sequence for efficient gene activation by Cat8 (YCCNYTNRKCCG), while a more specific motif is required for activation by Sip4 (TCCATTSRTCCGR). Although their zinc cluster domains are clearly related, Cat8 and Sip4 are not isofunctional. This conclusion is further supported by the finding that biosynthetic derepression of Cat8 in the presence of a nonfermentable carbon source precedes that of Sip4 by about 90 min.

Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae.

Sip4 is a Zn(2)Cys(6) transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae.

Glucose-repressible alcohol dehydrogenase II, encoded by the ADH2 gene of the yeast Saccharomyces cerevisiae, is transcriptionally controlled by the activator Adr1, binding UAS1 of the control region. However, even in an adr1 null mutant, a substantial level of gene derepression can be detected, arguing for the existence of a further mechanism of activation. Here it is shown that the previously identified UAS2 contains a distantly related variant of the carbon source-responsive element (CSRE) initially found upstream of gluconeogenic genes. In a mutant defective for the CSRE-binding factor Cat8, derepression of an ADH2-lacZ fusion was reduced to about 12% of the wild-type level. Gene expression in a cat8 adr1 double mutant decreased almost to the basal level of the glucose-repressed promoter. CSRE(ADH2) present in a single copy turned out to be a weak UAS element, while a significant synergism of gene activation was found in the presence of at least two copies. Its importance for regulated gene activation was confirmed by site-directed mutagenesis of the CSRE in the natural ADH2 control region. Direct binding of Cat8 to CSRE(ADH2) could be shown by electrophoretic retardation of the corresponding protein/DNA complex in the presence of a specific antibody. In contrast to what was shown previously for CSRE sequence variants, no significant influence of the isofunctional activator Sip4 on CSRE(ADH2) was detected. In conclusion, these results show a derepression of ADH2 by synergistically acting regulators Adr1 (interacting with UAS1) and Cat8, binding to UAS2 (=CSRE(ADH2)).

Cat8 and Sip4 mediate regulated transcriptional activation of the yeast malate dehydrogenase gene MDH2 by three carbon source-responsive promoter elements.

Malate dehydrogenase isoenzymes are localized in different cellular compartments and fulfil important functions in intermediary metabolism. In the yeast Saccharomyces cerevisiae, three malate dehydrogenase genes, MDH1, MDH2 and MDH3, encoding mitochondrial, cytosolic and peroxisomal variants, have been identified. We demonstrate the importance of transcriptional activators Hap4, Cat8 and Pip2 for the carbon source-dependent regulation of MDH1, MDH2 and MDH3, respectively. The control region of the MDH2 gene required for gluconeogenic growth with C(2) substrates contains three sequence elements similar to the previously identified carbon source-responsive element (CSRE). In a synthetic test system, each of these sequences turned out to be a weak UAS element showing a strong synergism when present in multiple copies. Cumulative mutagenesis of the natural MDH2 promoter confirmed the contribution of all three elements to transcriptional derepression under non-fermentative growth conditions. The DNA-binding domains of zinc cluster proteins Cat8 and Sip4 synthesized in Escherichia coli could interact in vitro with CSRE motifs of MDH2. This result was confirmed by binding assays using protein extracts from yeast. Deregulated variants of Cat8 and Sip4 modified by heterologous transcriptional activation domains were able to alleviate glucose repression of MDH2 substantially. Although Sip4 turned out as the less effective activator, our findings demonstrate the general significance of both proteins for expression of gluconeogenic structural genes.

Differences in regulation of yeast gluconeogenesis revealed by Cat8p-independent activation of PCK1 and FBP1 genes in Kluyveromyces lactis.

The yeast Kluyveromyces lactis is can utilise a wide range of non-fermentable carbon compounds as sole sources of carbon and energy, and differs from Saccharomyces cerevisiae in being able to carry out oxidative and fermentative metabolism simultaneously. In S. cerevisiae, growth on all non-fermentable carbon sources requires Cat8p, a transcriptional activator that controls the expression of gluconeogenic and glyoxylate cycle genes via CSREs (Carbon Source Responsive Elements). The down-regulation of Cat8p by fermentable carbon sources is the primary factor responsible for the tight repression of gluconeogenesis by glucose in S. cerevisiae. To analyse the regulation of gluconeogenesis in K. lactis, we have cloned and characterised the K. lactis homologue of CAT8 (KlCAT8). The gene was isolated by multicopy suppression of a fog2/klsnf1 mutation, indicating a similar epistatic relationship between KlSNF1 and KlCAT8 as in the case of the S. cerevisiae homologues. KlCAT8 encodes a protein of 1445 amino acids that is 40% identical to ScCat8p. The most highly conserved block is the putative Zn(II)2Cys6 DNA-binding domain, but additional conserved regions shared with members of the zinc-cluster family from Aspergillus define a subfamily of Cat8p-related proteins. KlCAT8 complements the growth defect of a Sccat8 mutant on non-fermentable carbon sources. In K. lactis, deletion of KlCAT8 severely impairs growth on ethanol, acetate and lactate, but not on glycerol. Derepression of enzymes of the glyoxylate cycle--malate synthase and particularly isocitrate lyase--was impaired in a Klcat8 mutant, whereas Northern analysis revealed that derepression of KlFBP1 and KlPCK1 does not require KlCat8p. Taken together, our results indicate that in K. lactis gluconeogenesis is not co-regulated with the glyoxylate cycle, and only the latter is controlled by KlCat8p.

Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source-responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source-independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40-fold derepression with wild-type CAT8, an almost constitutive expression was found with a MET25-CAT8-INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source-dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.

Regulation of the expression of the Kluyveromyces lactis PDC1 gene: carbon source-responsive elements and autoregulation.

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KIPDC1). In order to study the regulation of the expression of KIPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 delta strain showed that KIPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag- phenotype of the Klpdc1 delta mutant strain and it could also repress transcription of the KIPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Sip4, a Snf1 kinase-dependent transcriptional activator, binds to the carbon source-responsive element of gluconeogenic genes.

The carbon source-responsive element (CSRE) mediates transcriptional activation of the gluconeogenic genes during growth of the yeast Saccharomyces cerevisiae on non-fermentable carbon sources. Previous studies have suggested that the Cat8 protein activates the expression of CSRE-binding factors. We show here that one of these factors is Sip4, a glucose-regulated C6 zinc cluster activator which was identified by its interaction with the Snf1 protein kinase. We present genetic evidence that Sip4 contributes to transcriptional activation by the CSRE and biochemical evidence that Sip4 binds to the CSRE. Binding was detected in electrophoretic mobility shift assays with both yeast nuclear extracts and a bacterially expressed Sip4 fusion protein. Evidence suggests that Sip4 also activates the expression of other CSRE-binding proteins. Finally, we show that Cat8 regulates SIP4 expression and that overexpression of Sip4 compensates for loss of Cat8. We propose a model for activation by the CSRE in which Sip4 and Cat8 have related functions, but Cat8 is the primary regulator because it controls Sip4 expression.

Transcriptional control of the yeast acetyl-CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and the pleiotropic repressor UME6.

The ACS1 gene, encoding one out of two acetyl-CoA synthetase isoenzymes of Saccharomyces cerevisiae, is strictly regulated at the transcriptional level by the carbon source of the medium. While ACS1 is poorly expressed in the presence of a high glucose concentration, a several hundred-fold derepression occurs with ethanol as the sole carbon source or under conditions of sugar limitation. The molecular mechanism responsible for the carbon source control of ACS1 turned out to be highly complex. A carbon source-responsive element (CSRE), previously identified upstream of gluconeogenic structural genes, and a binding site of the alcohol dehydrogenase regulator, Adr1p, together mediate about 80% of the derepressed gene activity. Binding of Adr1p synthesized by Escherichia coli to the ACS1 control region was shown by an electrophoretic mobility shift assay. In addition to these activating elements, two URS1 motifs confer negative control on the ACS1 promoter. The URS1 element was found to be a constitutive repression site, which is most effective from a downstream position with respect to an upstream activation site (UAS). In a mutant lacking the URS1-binding factor, Ume6p, ACS1 expression was partially glucose insensitive. Ume6p must counteract transcription factors that are constitutively active. Site-directed mutagenesis of Abf1p binding sites in the ACS1 promoter significantly reduced gene expression in the ume6 mutant, grown under repressing conditions. Thus, a functional balance of the pleiotropic positive factor Abf1p and the negative factor Ume6p is in part responsible for glucose repression of ACS1. The combined influence of the regulated UAS elements, CSRE and Adr1p binding site, mediates a strong increase in ACS1 expression under derepressing conditions.

Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae.

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

Constitutive and carbon source-responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1.

The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium. A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation. No evidence for MLS1 induction by oleic acid was found. By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene. Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1. Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants. This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG). Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE (ICL1) sequence. No competition was observed with a mutated CSRE variant. Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level. The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription. The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements. The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation. Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation. In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation.

Derepression of gene expression mediated by the 5' upstream region of the isocitrate lyase gene of Candida tropicalis is controlled by two distinct regulatory pathways in Saccharomyces cerevisiae.

The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-A1-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8.

The CSRE (carbon source-responsive element) is a sequence motif responsible for the transcriptional activation of gluconeogenic structural genes in Saccharomyces cerevisiae. We have isolated a regulatory gene, DIL1 (derepression of isocitrate lyase, = CAT8), which is specifically required for derepression of CSRE-dependent genes. Expression of CAT8 is carbon source regulated and requires a functional Cat1p (Snf1p) protein kinase. The derepression defect of CAT8 in a cat1 mutant could be suppressed by a mutant Mig1p repressor protein. Derepression of CAT8 also requires a functional HAP2 gene, suggesting a regulatory connection between respiratory and gluconeogenic genes. Carbon source-dependent protein-CSRE complexes detected in a gel retardation analysis with wild-type extracts were absent in cat8 mutant extracts. However, similar experiments with an epitope-tagged CAT8 gene product in the presence of tag-specific antibodies gave evidence against a direct binding of Cat8p to the CSRE. A constitutively expressed GAL4-CAT8 fusion gene revealed a carbon source-dependent transcriptional activation of a UAS(GAL)-containing reporter gene. Activation mediated by Cat8p was no longer detectable in a cat1 mutant. Thus, biosynthetic control of CAT8 as well as transcriptional activation by Cat8p requires a functional Cat1p protein kinase. A model proposing CAT8 as a specific activator of a transcription factor(s) binding to the CSRE is discussed.

Carbon source-dependent regulation of the acetyl-coenzyme A synthetase-encoding gene ACSI from saccharomyces cerevisiae

The yeast ACS1 gene, encoding acetyl-coenzyme A synthetase (ACS), was cloned using colony hybridization and a facA probe from Aspergillus nidulans. The complete sequence of 1.5 kb of the ACSI upstream region was determined. Northern hybridization revealed a strong derepression of ACSI transcripts in a strain grown on the nonfermentable carbon sources, acetate or ethanol. In contrast to a previous report, δacsl null mutants did not exhibit a growth defect on acetate medium. Indeed, enzyme assays showed the presence of an additional constitutively expressed ACS activity in Aacsl mutants. The carbon source-dependent expression was further investigated by the use of an ACSI::lacZ fusion gene, showing complete repression on easily fermentable sugars such as glucose, maltose, sucrose or galactose. Binding sites for the yeast general regulatory factors, Abflp and Reblp, together with a sequence reminiscent of the recently identified carbon source-responsive element ( CSRE), could be detected in the ACSI upstream region, presumably mediating the observed regulatory phenotype of this ACS isoenzyme.