A yeast-based in vivo assay system for analyzing efflux of sugars mediated by glucose and xylose transporters.

Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications.

A label-free real-time method for measuring glucose uptake kinetics in yeast.

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.

Glucose-induced internalization of the S. cerevisiae galactose permease Gal2 is dependent on phosphorylation and ubiquitination of its aminoterminal cytoplasmic tail.

The hexose permease Gal2 of Saccharomyces cerevisiae is expressed only in the presence of its physiological substrate galactose. Glucose tightly represses the GAL2 gene and also induces the clearance of the transporter from the plasma membrane by ubiquitination and subsequent degradation in the vacuole. Although many factors involved in this process, especially those responsible for the upstream signaling, have been elucidated, the mechanisms by which Gal2 is specifically targeted by the ubiquitination machinery have remained elusive. Here, we show that ubiquitination occurs within the N-terminal cytoplasmic tail and that the arrestin-like proteins Bul1 and Rod1 are likely acting as adaptors for docking of the ubiquitin E3-ligase Rsp5. We further demonstrate that phosphorylation on multiple residues within the tail is indispensable for the internalization and possibly represents a primary signal that might trigger the recruitment of arrestins to the transporter. In addition to these new fundamental insights, we describe Gal2 mutants with improved stability in the presence of glucose, which should prove valuable for engineering yeast strains utilizing complex carbohydrate mixtures present in hydrolysates of lignocellulosic or pectin-rich biomass.

Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production.

The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6-C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations.

The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae.

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit Ciso (AroY-Ciso), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso, although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-Ciso, suggesting that mitochondrial localization has no major impact on cofactor biosynthesis.IMPORTANCE In Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.

Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae.

Isobutanol is a superior biofuel compared to ethanol, and it is naturally produced by yeasts. Previously, Saccharomyces cerevisiae has been genetically engineered to improve isobutanol production. We found that yeast cells engineered for a cytosolic isobutanol biosynthesis secrete large amounts of the intermediate 2,3-dihydroxyisovalerate (DIV). This indicates that the enzyme dihydroxyacid dehydratase (Ilv3) is limiting the isobutanol pathway and/or yeast exhibit effective transport systems for the secretion of the intermediate, competing with isobutanol synthesis. Moreover, we found that DIV cannot be taken up by the cells again. To identify the responsible transporters, microarray analysis was performed with a DIV producing strain compared to a wild type. Altogether, 19 genes encoding putative transporters were upregulated under DIV-producing conditions. Thirteen of these were deleted together with five homologous genes. A yro2 mrh1 deletion strain showed reduced DIV secretion, while a hxt5 deletion mutant showed increased isobutanol production. However, a strain deleted for all the 18 genes secreted even slightly increased amounts of the intermediates and less isobutanol. The lactate transporter Jen1 turned out to transport the intermediate 2-ketoisovalerate, but not DIV. The results suggest that the transport of DIV is a rather complex process and several unspecific transporters seem to be involved.

Optimisation of trans-cinnamic acid and hydrocinnamyl alcohol production with recombinant Saccharomyces cerevisiae and identification of cinnamyl methyl ketone as a by-product.

Trans-cinnamic acid (tCA) and hydrocinnamyl alcohol (HcinOH) are valuable aromatic compounds with applications in the flavour, fragrance and cosmetic industry. They can be produced with recombinant yeasts from sugars via phenylalanine after expression of a phenylalanine ammonia lyase (PAL) and an aryl carboxylic acid reductase. Here, we show that in Saccharomyces cerevisiae a PAL enzyme from the bacterium Photorhabdus luminescens was superior to a previously used plant PAL enzyme for the production of tCA. Moreover, after expression of a UDP-glucose:cinnamate glucosyltransferase (FaGT2) from Fragaria x ananassa, tCA could be converted to cinnamoyl-D-glucose which is expected to be less toxic to the yeast cells. Production of tCA and HcinOH from glucose could be increased by eliminating feedback-regulated steps of aromatic amino acid biosynthesis and diminishing the decarboxylation step of the competing Ehrlich pathway. Finally, an unknown by-product resulting from further metabolisation of a carboligation product of cinnamaldehyde (cinALD) with activated acetaldehyde, mediated by pyruvate decarboxylases, could be identified as cinnamyl methyl ketone providing a new route for the biosynthesis of precursors, such as (2S,3R) 5-phenylpent-4-ene-2,3-diol, necessary for the chemical synthesis of specific biologically active drugs such as daunomycin.

Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.

All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.

On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

High-Throughput Screening of an Octanoic Acid Producer Strain Library Enables Detection of New Targets for Increasing Titers in Saccharomyces cerevisiae.

Octanoic acid is an industrially relevant compound with applications in antimicrobials or as a precursor for biofuels. Microbial biosynthesis through yeast is a promising alternative to current unsustainable production methods. To increase octanoic acid titers in Saccharomyces cerevisiae, we use a previously developed biosensor that is based on the octanoic acid responsive pPDR12 promotor coupled to GFP. We establish a biosensor strain amenable for high-throughput screening of an octanoic acid producer strain library. Through development, optimization, and execution of a high-throughput screening approach, we were able to detect two new genetic targets, KCS1 and FSH2, which increased octanoic acid titers through combined overexpression by about 55% compared to the parental strain. Neither target has yet been reported to be involved in fatty acid biosynthesis. The presented methodology can be employed to screen any genetic library and thereby more genes involved in improving octanoic acid production can be detected in the future.

Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists.

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids' non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5-1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid-base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a ß-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.

Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.

Identification of a glucose-insensitive variant of Gal2 from Saccharomyces cerevisiae exhibiting a high pentose transport capacity.

As abundant carbohydrates in renewable feedstocks, such as pectin-rich and lignocellulosic hydrolysates, the pentoses arabinose and xylose are regarded as important substrates for production of biofuels and chemicals by engineered microbial hosts. Their efficient transport across the cellular membrane is a prerequisite for economically viable fermentation processes. Thus, there is a need for transporter variants exhibiting a high transport rate of pentoses, especially in the presence of glucose, another major constituent of biomass-based feedstocks. Here, we describe a variant of the galactose permease Gal2 from Saccharomyces cerevisiae (Gal2N376Y/M435I), which is fully insensitive to competitive inhibition by glucose, but, at the same time, exhibits an improved transport capacity for xylose compared to the wildtype protein. Due to this unique property, it significantly reduces the fermentation time of a diploid industrial yeast strain engineered for efficient xylose consumption in mixed glucose/xylose media. When the N376Y/M435I mutations are introduced into a Gal2 variant resistant to glucose-induced degradation, the time necessary for the complete consumption of xylose is reduced by approximately 40%. Moreover, Gal2N376Y/M435I confers improved growth of engineered yeast on arabinose. Therefore, it is a valuable addition to the toolbox necessary for valorization of complex carbohydrate mixtures.