Galactose Regulon Research Articles

Reshaping the yeast galactose regulon via GPCR signaling cascade

Dynamically regulated systems are preferable to control metabolic pathways for an improved strain performance with better productivity. Here, we harnessed to the G protein-coupled receptor (GPCR) signaling pathway to reshape the yeast galactose regulon. The galactose-regulated (GAL) system was coupled with the GPCR signaling pathway for mating pheromone via a synthetic transcription factor. In this study, we refabricated the dynamic range, sensitivity, and response time of the GAL system to α factor by modulating the key components of the GPCR signaling cascade. A series of engineered yeasts with self-secretion of α factor were constructed to achieve quorum-sensing behaviors. In addition, we also repurposed the GAL system to make it responsive to heat shock. Taken together, our work showcases the great potential of synthetic biology in creating user-defined metabolic controls. We envision that the plasticity of our genetic design would be of significant interest for the future fabrication of novel gene expression systems.

A Layered Genetic Design Enables the Yeast Galactose Regulon to Respond to Cyanamide.

The commonly used expression systems in Saccharomyces cerevisiae typically rely on either constitutive or galactose-regulated promoters. The lack of inducible systems in S.cerevisiae limits the precise temporal regulation of protein function and yeast metabolism. We herein repurposed the galactose-regulated system to make it respond to cyanamide. By using a cyanamide-inducible DDI2 promoter to control Gal4 expression in CEN.PK2-1C with Δgal80, a tight and graded cyanamide-inducible GAL system with an enhanced signal output was constructed. Subsequently, we demonstrated that the cyanamide-inducible GAL system was capable of tightly regulating the pentafunctional Aro1 protein to achieve conditional shikimate pathway activity. Taken together, the cyanamide-inducible GAL system could be implemented for both fundamental research and applied biotechnology.

Towards universal synthetic heterotrophy using a metabolic coordinator

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Targeted gene manipulation of Leloir pathway genes for the constitutive expression of β-galactosidase and its transgalactosylation product galacto-oligosaccharides from Kluyveromyces lactis GG799 and knockout strains

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical industry. At present, production of GOS involves the enzymatic transformation of lactose by transgalactosylation using β-galactosidase. The yeast Kluyveromyces lactis can utilize lactose as its carbon and energy source. In this species lactose is hydrolyzed by an intracellular β-galactosidase (EC 3.2.1.23) which is induced by its substrate and related compounds like galactose. The molecular details of gene regulation in kluyveromyces lactis, we have used multiple knockout approaches to study the constitutive expression by which galactose induces β-galactosidase. The present study involved carrying out to a method of enhancing the constitutive expression of β-galactosidase through galactose induction and its trans-galactosylation reaction for the production of galacto-oligosaccharides (GOS) in Kluyveromyces lactis (K. Lactis) by applying a knockout based approach on Leloir pathway genes based on fusion-overlap extension polymerase chain reaction and transformation into its genome. The k.lactis strain subjected to Leloir pathway genes knockout, resulted in the accumulation of galactose intracellularly and this internal galactose acts as an inducer of galactose regulon for constitutive expression of β-galactosidase at early stationary phase was due to the positive regulatory function of mutant gal1p, gal7p and both. These resulted strains used for trans-galactosylation of lactose by β – galactosidase is characterized for the production of galacto-oligosaccharides. Galactose-induced constitutive expression of β-galactosidase during the early stationary phase of knockout strains was analysed qualitatively & quantitatively. The activity of β-galactosidase of wild type, gal1z, gal7k and gal1z & gal7k strains were 7, 8, 9 and 11 U/ml respectively using high cell density cultivation medium. Based on these expression differences in β-galactosidase, the trans-galactosylation reaction for GOS production and percentage yield of GOS were compared at 25% w/v of lactose. The percentage yield of GOS production of wild type, Δgal1z Lac4+, Δgal7k Lac4++ and Δgal1z Δgal7k Lac4+++mutants strains were 6.3, 13, 17 and 22 U/ml, respectively. Therefore, we propose that the availability of galactose can be used for constitutive over expression of β – galactosidase in Leloir pathway engineering applications and also for GOS production. Further, increased expression of β – galactosidases can be used in dairy industry by-products like whey to produce added value products such as galacto-oligosaccharides.

A Tunable and Expandable Transactivation System in Probiotic Yeast Saccharomyces boulardii.

Precise transcriptional modulation is a key requirement for developing synthetic probiotics with predictably tunable functionalities. In this study, an expandable and tunable transactivation system was constructed and validated in probiotic yeast Saccharomyces boulardii. The use of nuclease-null Cas9 and scaffold RNA (scRNA) directed regulation enabled transactivation under the control of a synthetic promoter in S.boulardii. A synthetic promoter consisting of the scRNA target sequence and the core GAL7 promoter region restricted interference from the native galactose regulon. The system was readily expanded by introducing new target sequences to the promoter and scRNA. Complementarity between the promoter and scRNA, and binding specificity between scRNA and transcriptional activator, served as two layers of orthogonality of the transactivation. In addition, activator expression under the control of an inducible promoter enabled control of the transactivation via chemical inducer. The described system has the potential to enable engineering of probiotic yeast to more precisely perform therapeutic functions.

The Neurospora crassa Inducible Q System Enables Simultaneous Optogenetic Amplification and Inversion in Saccharomyces cerevisiae for Bidirectional Control of Gene Expression.

Bidirectional optogenetic control of yeast gene expression has great potential for biotechnological applications. Our group has developed optogenetic inverter circuits that activate transcription using darkness, as well as amplifier circuits that reach high expression levels under limited light. However, because both types of circuits harness Gal4p and Gal80p from the galactose (GAL) regulon they cannot be used simultaneously. Here, we apply the Q System, a transcriptional activator/inhibitor system from Neurospora crassa, to build circuits in Saccharomyces cerevisiae that are inducible using quinic acid, darkness, or blue light. We develop light-repressed OptoQ-INVRT circuits that initiate darkness-triggered transcription within an hour of induction, as well as light-activated OptoQ-AMP circuits that achieve up to 39-fold induction. The Q System does not exhibit crosstalk with the GAL regulon, allowing coutilization of OptoQ-AMP circuits with previously developed OptoINVRT circuits. As a demonstration of practical applications in metabolic engineering, we show how simultaneous use of these circuits can be used to dynamically control both growth and production to improve acetoin production, as well as enable light-tunable co-production of geraniol and linalool, two terpenoids implicated in the hoppy flavor of beer. OptoQ-AMP and OptoQ-INVRT circuits enable simultaneous optogenetic signal amplification and inversion, providing powerful additions to the yeast optogenetic toolkit.

Parallel experimental evolution reveals a novel repressive control of GalP on xylose fermentation in Escherichia coli.

Efficient xylose utilization will facilitate microbial conversion of lignocellulosic sugar mixtures into valuable products. In Escherichia coli, xylose catabolism is controlled by carbon catabolite repression (CCR). However, in E. coli such as the succinate-producing strain KJ122 with disrupted CCR, xylose utilization is still inhibited under fermentative conditions. To probe the underlying genetic mechanisms inhibiting xylose utilization, we evolved KJ122 to enhance its xylose fermentation abilities in parallel and characterized the potential convergent genetic changes shared by multiple independently evolved strains. Whole-genome sequencing revealed that convergent mutations occurred in the galactose regulon during adaptive laboratory evolution potentially decreasing the transcriptional level or the activity of GalP, a galactose permease. We showed that deletion of galP increased xylose utilization in both KJ122 and wild-type E. coli, demonstrating a common repressive role of GalP for xylose fermentation. Concomitantly, induced expression of galP from a plasmid repressed xylose fermentation. Transcriptome analysis using RNA sequencing indicates that galP inactivation increases transcription levels of many catabolic genes for secondary sugars including xylose and arabinose. The repressive role of GalP for fermenting secondary sugars in E. coli suggests that utilization of GalP as a substitute glucose transporter is undesirable for conversion of lignocellulosic sugar mixtures.

A semi-synthetic regulon enables rapid growth of yeast on xylose

Nutrient assimilation is the first step that allows biological systems to proliferate and produce value-added products. Yet, implementation of heterologous catabolic pathways has so far relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. In contrast, natural systems prefer nutrient-responsive gene regulation (called regulons) that control multiple cellular functions necessary for cell survival and growth. Here, in Saccharomyces cerevisiae, by partially- and fully uncoupling galactose (GAL)-responsive regulation and metabolism, we demonstrate the significant growth benefits conferred by the GAL regulon. Next, by adapting the various aspects of the GAL regulon for a non-native nutrient, xylose, we build a semi-synthetic regulon that exhibits higher growth rate, better nutrient consumption, and improved growth fitness compared to the traditional and ubiquitous constitutive expression strategy. This work provides an elegant paradigm to integrate non-native nutrient catabolism with native, global cellular responses to support fast growth.

Exploiting Single-Cell Quantitative Data to Map Genetic Variants Having Probabilistic Effects.

Despite the recent progress in sequencing technologies, genome-wide association studies (GWAS) remain limited by a statistical-power issue: many polymorphisms contribute little to common trait variation and therefore escape detection. The small contribution sometimes corresponds to incomplete penetrance, which may result from probabilistic effects on molecular regulations. In such cases, genetic mapping may benefit from the wealth of data produced by single-cell technologies. We present here the development of a novel genetic mapping method that allows to scan genomes for single-cell Probabilistic Trait Loci that modify the statistical properties of cellular-level quantitative traits. Phenotypic values are acquired on thousands of individual cells, and genetic association is obtained from a multivariate analysis of a matrix of Kantorovich distances. No prior assumption is required on the mode of action of the genetic loci involved and, by exploiting all single-cell values, the method can reveal non-deterministic effects. Using both simulations and yeast experimental datasets, we show that it can detect linkages that are missed by classical genetic mapping. A probabilistic effect of a single SNP on cell shape was detected and validated. The method also detected a novel locus associated with elevated gene expression noise of the yeast galactose regulon. Our results illustrate how single-cell technologies can be exploited to improve the genetic dissection of certain common traits. The method is available as an open source R package called ptlmapper.

Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6

Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 ± 0.38 mM and a maximum velocity (V max) of 0.96 ± 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae.

Regulating the Regulon

In the presence of galactose, the yeast Saccharomyces cerevisiae transports the sugar into its cytoplasm, where the galactose binds the Gal3 protein, which in turn sequesters the Gal80 repressor away from the transcriptional activator Gal4, resulting in the induction of galactose-metabolizing enzymes. The induction is so rapid that it has been suggested that another unknown factor must somehow be involved in this "galactose regulon." Kumar et al. have identified this factor as nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP). In the Gal4:Gal80 complex, NAD is sandwiched between the two proteins. NADP, on the other hand, inhibits the interaction between Gal4 and Gal80. Mutations that have the potential to affect the NAD-binding site in Gal80 result in even faster induction, which suggests that Gal80 is sensing the balance between NAD and NADP and thus the metabolic state of the cell. P. R. Kumar, Y. Yu, R. Sternglanz, S. A. Johnston, L. Joshua-Tor, NADP regulates the yeast GAL induction system. Science 319 , 1090-1092 (2008). [Abstract] [Full Text]

Signal integration in the galactose network of Escherichia coli

The gal regulon of Escherichia coli contains genes involved in galactose transport and metabolism. Transcription of the gal regulon genes is regulated in different ways by two iso-regulatory proteins, Gal repressor (GalR) and Gal isorepressor (GalS), which recognize the same binding sites in the absence of d-galactose. DNA binding by both GalR and GalS is inhibited in the presence of d-galactose. Many of the gal regulon genes are activated in the presence of the adenosine cyclic-3',5'-monophosphate (cAMP)-cAMP receptor protein (CRP) complex. We studied transcriptional regulation of the gal regulon promoters simultaneously in a purified system and attempted to integrate the two small molecule signals, d-galactose and cAMP, that modulate the isoregulators and CRP respectively, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that in some cases the activity of the promoter and the cognate gene can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to construct an integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network, feedback loops are by far the most common motif.

The Cysteine-Peptidase Bleomycin Hydrolase Is A Member of the Galactose Regulon in Yeast

Bleomycin hydrolase is a cysteine peptidase discovered through its ability to detoxify the anti-cancer glycopeptide, bleomycin. Although found in all tissues in mammals and in both eukaryotes and prokaryotes, the normal cellular function of this peptidase is not known. We had previously reported the purification of bleomycin hydrolase from yeast based on its unexpected ability to bind DNA. Recently we collaborated in solving the crystal structure of this protein, revealing a hexameric ring organization. We now report that the molecular characterization of the gene encoding yeast bleomycin hydrolase is also surprising. The transcription of the gene is regulated by galactose. Furthermore, this regulation is conveyed by a binding site for the Gal4 regulatory protein in its promoter, prompting the designation of this gene as GAL6. Gal6p also appears to have a negative effect on the GAL system as a deletion of the gene leads to a 2-5-fold higher expression of the GAL1, GAL2, GAL7, and MEL1 genes. The GAL6 deletion does not affect the expression of another inducible gene, HSP26. Neither the peptidase nor the nucleic acid binding activity of Gal6p as assayed is apparently required to convey this regulation, implying yet another function for this new member of the GAL regulon.

Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression.

Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins.

Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.

A single GAL4 dimer can maximally activate transcription under physiological conditions.

Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner. A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill. In this model, various combinations of protein-protein interactions control the level of gene expression. However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo. Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding. These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon.

Gal80 Proteins of Kluyveromyces lactis and Saccharomyces cerevisiae Are Highly Conserved but Contribute Differently to Glucose Repression of the Galactose Regulon

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

Interaction of positive and negative regulatory proteins in the galactose regulon of yeast

Transcriptional regulation in the galactose regulon of yeast is determined by an interplay between a positive regulatory protein, GAL4, and a negative regulatory protein, GAL80. We show that derivatives of GAL4 missing as few as 28 carboxy-terminal amino acids are not responsive to GAL80 regulation, implying that the carboxyl terminus of GAL4 is required for interaction with GAL80. Furthermore, a lesion in GAL4 that genetically defines the GAL80-interactive region maps to the 3′ end of the gene. Since the carboxyl terminus of GAL4 has also been implicated in interaction with a transcriptional factor, we propose a model for negative regulation in which GAL80 and this factor compete for binding a common region of GAL4, and the on/off state of transcription is determined by the relative affinities of the negative regulator and the transcriptional factor for this region.