Interactions between stressful environment and gene deletions alleviate the expected average loss of fitness in yeast

The conjecture that the deleterious effects of mutations are amplified by stress or interaction with one another remains unsatisfactorily tested. It is now possible to reapproach this problem systematically by using genomic collections of mutants and applying stress-inducing conditions with a well-recognized impact on metabolism. We measured the maximum growth rate of single- and double-gene deletion strains of yeast in several stress-inducing treatments, including poor nutrients, elevated temperature, high salinity, and the addition of caffeine. The negative impact of deletions on the maximum growth rate was relatively smaller in stressful than in favorable conditions. In both benign and harsh environments, double-deletion strains grew on average slightly faster than expected from a multiplicative model of interaction between single growth effects, indicating positive epistasis for the rate of growth. This translates to even higher positive epistasis for fitness defined as the number of progeny. We conclude that the negative impact of metabolic disturbances, regardless of whether they are of environmental or genetic origin, is absolutely and relatively highest when growth is fastest. The effect of further damages tends to be weaker. This results in an average alleviating effect of interactions between stressful environment and gene deletions and among gene deletions.     

Epistatic interaction maps relative to multiple metabolic phenotypes

An epistatic interaction between two genes occurs when the phenotypic impact of one gene depends on another gene, often exposing a functional association between them. Due to experimental scalability and to evolutionary significance, abundant work has been focused on studying how epistasis affects cellular growth rate, most notably in yeast. However, epistasis likely influences many different phenotypes, affecting our capacity to understand cellular functions, biochemical networks adaptation, and genetic diseases. Despite its broad significance, the extent and nature of epistasis relative to different phenotypes remain fundamentally unexplored. Here we use genome-scale metabolic network modeling to investigate the extent and properties of epistatic interactions relative to multiple phenotypes. Specifically, using an experimentally refined stoichiometric model for Saccharomyces cerevisiae, we computed a three-dimensional matrix of epistatic interactions between any two enzyme gene deletions, with respect to all metabolic flux phenotypes. We found that the total number of epistatic interactions between enzymes increases rapidly as phenotypes are added, plateauing at approximately 80 phenotypes, to an overall connectivity that is roughly 8-fold larger than the one observed relative to growth alone. Looking at interactions across all phenotypes, we found that gene pairs interact incoherently relative to different phenotypes, i.e. antagonistically relative to some phenotypes and synergistically relative to others. Specific deletion-deletion-phenotype triplets can be explained metabolically, suggesting a highly informative role of multi-phenotype epistasis in mapping cellular functions. Finally, we found that genes involved in many interactions across multiple phenotypes are more highly expressed, evolve slower, and tend to be associated with diseases, indicating that the importance of genes is hidden in their total phenotypic impact. Our predictions indicate a pervasiveness of nonlinear effects in how genetic perturbations affect multiple metabolic phenotypes. The approaches and results reported could influence future efforts in understanding metabolic diseases and the role of biochemical regulation in the cell.     

Probing the performance limits of the Escherichia coli metabolic network subject to gene additions or deletions.

An optimization-based procedure for studying the response of metabolic networks after gene knockouts or additions is introduced and applied to a linear flux balance analysis (FBA) Escherichia coli model. Both the gene addition problem of optimally selecting which foreign genes to recombine into E. coli, as well as the gene deletion problem of removing a given number of existing ones, are formulated as mixed-integer optimization problems using binary 0-1 variables. The developed modeling and optimization framework is tested by investigating the effect of gene deletions on biomass production and addressing the maximum theoretical production of the 20 amino acids for aerobic growth on glucose and acetate substrates. In the gene deletion study, the smallest gene set necessary to achieve maximum biomass production in E. coli is determined for aerobic growth on glucose. The subsequent gene knockout analysis indicates that biomass production decreases monotonically, rendering the metabolic network incapable of growth after only 18 gene deletions. In the gene addition study, the E. coli flux balance model is augmented with 3,400 non-E. coli reactions from the KEGG database to form a multispecies model. This model is referred to as the Universal model. This study reveals that the maximum theoretical production of six amino acids could be improved by the addition of only one or two genes to the native amino acid production pathway of E. coli, even though the model could choose from 3,400 foreign reaction candidates. Specifically, manipulation of the arginine production pathway showed the most promise with 8.75% and 9.05% predicted increases with the addition of genes for growth on glucose and acetate, respectively. The mechanism of all suggested enhancements is either by: 1) improving the energy efficiency and/or 2) increasing the carbon conversion efficiency of the production route.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Quantitative comparison of transient growth of Saccharomyces cerevisiae,Saccharomyces kluyveri,and Kluyveromyces lactis

A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess. However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response. The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S. kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model. It was found that, under transient growth conditions, S. kluyveri behaved like K. lactis, while classification using steady-state conditions would position S. kluyveri close to S. cerevisiae. For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S. cerevisiae, and carbon-driven, such as S. kluyveri and K. lactis, strains. Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents. The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively. The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose. The benefit from using small metabolic flux models is the richness of information and the enhanced insight into intrinsic metabolic pathways without a priori knowledge of adaptation kinetics. Used in an online context, this approach serves as an efficient tool for strain characterization and physiological studies.     

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.     

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae

We observed that removing pantothenate (vitamin B₅), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce β-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, β-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a “metabolic switch” for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.     

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.     

Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

ABSTRACT: BACKGROUND: In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS- pykA- and PTS- pykF -). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. RESULTS: When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. CONCLUSIONS: Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS- pykA-) and VH35 (PTS- pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.     

Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae.

The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.     

Metabolic modeling for multi-objective optimization of ethanol production in a Synechocystis mutant

Cyanobacteria have potential to produce drop-in bio-fuels such as ethanol via photoautotrophic metabolism. Although model cyanobacterial strains have been engineered to produce such products, systematic metabolic engineering studies to identify optimal strains for the same have not been performed. In this work, we identify optimal ethanol producing mutants corresponding to appropriate gene deletions that result in a suitable redirection in the carbon flux. In particular, we systematically simulate exhaustive single and double gene deletions considering a genome scale metabolic model of a mutant strain of the unicellular cyanobacterium Synechocystis species strain PCC 6803. Various optimization based metabolic modeling techniques, such as flux balance analysis (FBA), method of minimization of metabolic adjustment (MOMA) and regulatory on/off minimization (ROOM) were used for this analysis. For single gene deletion MOMA simulations, the Pareto front with biomass and ethanol fluxes as the two objectives to be maximized was obtained and analyzed. Points on the Pareto front represent maximal utilization of resources constrained by substrate uptake thereby representing an optimal trade-off between the two fluxes. Pareto analysis was also performed for double gene deletion MOMA and single and double gene deletion ROOM simulations. Based on these analyses, two mutants, with combined gene deletions in ethanol and purine metabolism pathways, were identified as promising candidates for ethanol production. The relevant genes were adk, pta and ackA. An ethanol productivity of approximately 0.15 mmol/(gDW h) was predicted for these mutants which appears to be reasonable based on experimentally reported values in literature for other strains.     

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations. In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C. YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene. The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C. YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein. The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space. Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains. Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources.     

Saccharomyces Cerevisiae Null Mutants in Glucose Phosphorylation: Metabolism and Invertase Expression

A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.     

Genome-scale analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture

A dynamic flux balance model based on a genome-scale metabolic network reconstruction is developed for in silico analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture. Metabolic engineering strategies previously identified for their enhanced steady-state biomass and/or ethanol yields are evaluated for fed-batch performance in glucose and glucose/xylose media. Dynamic analysis is shown to provide a single quantitative measure of fed-batch ethanol productivity that explicitly handles the possible tradeoff between the biomass and ethanol yields. Productivity optimization conducted to rank achievable fed-batch performance demonstrates that the genetic manipulation strategy and the fed-batch operating policy should be considered simultaneously. A library of candidate gene insertions is assembled and directly screened for their achievable ethanol productivity in fed-batch culture. A number of novel gene insertions with ethanol productivities identical to the best metabolic engineering strategies reported in previous studies are identified, thereby providing additional targets for experimental evaluation. The top performing gene insertions were substrate dependent, with the highest ranked insertions for glucose media yielding suboptimal performance in glucose/xylose media. The analysis results suggest that enhancements in biomass yield are most beneficial for the enhancement of fed-batch ethanol productivity by recombinant xylose utilizing yeast strains. We conclude that steady-state flux balance analysis is not sufficient to predict fed-batch performance and that the media, genetic manipulations, and fed-batch operating policy should be considered simultaneously to achieve optimal metabolite productivity.     

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

Characterization of the adaptive response and growth upon hyperosmotic shock in Saccharomyces cerevisiae

Molecular and physiological details of osmoadaptation in yeast Saccharomyces cerevisiae are well characterized. It is well known that a cell, upon osmotic shock, delays its growth, produces a compatible solute like glycerol in yeast to maintain the osmotic equilibrium. Many genes are regulated by the hyperosmolarity glycerol (HOG) singling pathway, some of which in turn control the carbon flux in the glycolytic pathway for glycerol synthesis and reduced growth. The whole process of survival of cells under hyperosmotic stress is controlled at multiple levels in signaling and metabolic pathways. To better understand the multi-level regulations in yeast to osmotic shock, a mathematical model is formulated which integrates the growth and the osmoadaptation process. The model included the HOG pathway which consists of Sho1 and Sln1 signaling branches, gene regulation, metabolism and cell growth on glucose and ethanol. Experiments were performed to characterize the effect of various concentrations of salt on the wild-type and mutant strains. The model was able to successfully predict the experimental observations for both the wild-type and mutant strains. Further, the model was used to analyze the effects of various regulatory mechanisms prevalent in the signaling and metabolic pathways which are essential in achieving optimum growth in a saline medium. The analysis demonstrated the relevance of the combined effects of regulation at several points in the signaling and metabolic pathways including activation of GPD1 and GPD2, inhibition of PYK and PDC1, closure of the Fps1 channel, volume effect on the glucose uptake rate, downregulation of ethanol synthesis and upregulation of ALD6 for acetate synthesis. The analysis demonstrated that these combined effects orchestrated the phenomena of adaptation to osmotic stress in yeast.     

Protein production by<Emphasis Type="Italic"> Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> wild-type and <Emphasis Type="Italic">ΔptsG</Emphasis> mutant strains with IPTG induction at the onset

During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate.     

Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux

In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3′ adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (≥1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.