Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses.

Archaeal viruses are among the most enigmatic members of the virosphere, and their diverse morphologies raise many questions about their infection mechanisms. The study of molecular mechanisms underlying virus-host interactions hinges upon robust model organisms with a system for gene expression and deletion. Currently, there are only a limited number of archaea that have associated viruses and have a well-developed genetic system. Here, we report the development of a genetic system for the euryarchaeon Haloferax gibbonsii LR2-5. This strain can be infected by multiple viruses and is a model for the study of virus-host interactions. We created a Hfx. gibbonsii LR2-5 ∆pyrE strain, resulting in uracil auxotrophy, which could be used as a selection marker. An expression plasmid carrying a pyrE gene from the well-established Haloferax volcanii system was tested for functionality. Expression of a GFP-MinD fusion under a tryptophan inducible promoter was fully functional and showed similar cellular localization as in Hfx. volcanii. Thus, the plasmids of the Hfx. volcanii system can be used directly for the Hfx. gibbonsii LR2-5 genetic system, facilitating the transfer of tools between the two. Finally, we tested for the functionality of gene deletions by knocking out two genes of the archaeal motility structure, the archaellum. These deletion mutants were as expected non-motile and the phenotype of one deletion could be rescued by the expression of the deleted archaellum gene from a plasmid. Thus, we developed a functional genetic toolbox for the euryarchaeal virus host Hfx. gibbonsii LR2-5, which will propel future studies on archaeal viruses. IMPORTANCE: Species from all domains of life are infected by viruses. In some environments, viruses outnumber their microbial hosts by a factor of 10, and viruses are the most important predators of microorganisms. While much has been discovered about the infection mechanisms of bacterial and eukaryotic viruses, archaeal viruses remain understudied. Good model systems are needed to study their virus-host interactions in detail. The salt-loving archaeon Haloferax gibbonsii LR2-5 has been shown to be infected by a variety of different viruses and, thus, is an excellent model to study archaeal viruses. By establishing a genetic system, we have significantly expanded the toolbox for this model organism, which will fuel our understanding of infection strategies of the underexplored archaeal viruses.

Novel plasmids in multidrug-resistant Shigella flexneri serotypes from Pakistan.

Shigellosis is the main cause of food and waterborne diarrhea and is an emerging threat to human health. The current study characterized the indigenous multidrug-resistant Shigella flexneri serotypes for their plasmid profiles and genetic diversity, to characterize the plasmid evolutionary patterns and distribution. In total, 199 identified S. flexneri isolates belonging to six different serotypes were analyzed for plasmid profiling, followed by an analysis of whole genome sequencing. All isolates of S. flexneri resistant to antibiotics harbored multiple copies of plasmids with sizes ranging from 1.25 kbp to 9.4 kbp. These isolates were clustered into 22 distinct plasmid patterns, labeled as p1-p22. Among these, p1 (24%) and p10 (13%) were the predominant plasmid profiles. All S. flexneri strains were grouped into 12 clades with a 75% similarity level. Also, a significant association was observed among the plasmid patterns, p23 and p17 with the drug-resistant patterns AMC, SXT, C (19.5%) and OFX, AMC, NA, CIP (13.5%), respectively. Moreover, the most widespread plasmid patterns p4, p10, and p1 showed a significant association with the serotypes 1b (29.16%), 2b (36%), and 7a (100%), respectively. After plasmid sequence assembly and annotation analysis, a variety of small plasmids that vary in size from 973 to 6200 bp were discovered. Many of these plasmids displayed high homology and coverage with plasmids from non-S. flexneri. Several novel plasmids of small size were discovered in multidrug-resistant S. flexneri. The data also showed that plasmid profile analysis is more consistent than antibiotic susceptibility pattern analysis for identifying epidemic strains of S. flexneri isolated in Pakistan.

D-xylose accelerated death of pentose metabolizing Saccharomyces cerevisiae.

Rapid and effective consumption of D-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous D-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming D-xylose, growth inhibition occurs at higher D-xylose concentrations, even abolishing growth completely at 8% D-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in D-xylose metabolism is the phosphorylation of D-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high D-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high D-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust D-xylose metabolism.

Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

Combined roles of exporters in acetic acid tolerance in Saccharomyces cerevisiae.

Acetic acid is a growth inhibitor generated during alcoholic fermentation and pretreatment of lignocellulosic biomass, a major feedstock to produce bioethanol. An understanding of the acetic acid tolerance mechanisms is pivotal for the industrial production of bioethanol. One of the mechanisms for acetic acid tolerance is transporter-mediated secretion where individual transporters have been implicated. Here, we deleted the transporters Aqr1, Tpo2, and Tpo3, in various combinations, to investigate their combined role in acetic acid tolerance. Single transporter deletions did not impact the tolerance at mild acetic acid stress (20 mM), but at severe stress (50 mM) growth was decreased or impaired. Tpo2 plays a crucial role in acetic acid tolerance, while the AQR1 deletion has a least effect on growth and acetate efflux. Deletion of both Tpo2 and Tpo3 enhanced the severe growth defects at 20 mM acetic acid concomitantly with a reduced rate of acetate secretion, while TPO2 and/or TPO3 overexpression in ∆tpo2∆tpo3∆ restored the tolerance. In the deletion strains, the acetate derived from sugar metabolism accumulated intracellularly, while gene transcription analysis suggests that under these conditions, ethanol metabolism is activated while acetic acid production is reduced. The data demonstrate that Tpo2 and Tpo3 together fulfill an important role in acetate efflux and the acetic acid response.

Antimicrobial Resistance of Shigella flexneri in Pakistani Pediatric Population Reveals an Increased Trend of Third-Generation Cephalosporin Resistance.

The rapid emergence of resistance to third-generation cephalosporins in Shigella flexneri is crucial in pediatric shigellosis management. Limited studies have been conducted on molecular pattern of antibiotic resistance of S. flexneri in diarrhea endemic areas of Pakistan. The aim of the study was to analyze the antimicrobial resistance of S. flexneri isolated from pediatric diarrheal patients in Peshawar, Pakistan. A total of 199 S. flexneri isolates (clinical, n = 1 55 and non-clinical, n = 44) were investigated for drug resistance and mutational analysis of selected drug resistance genes. All isolates were found to be highly resistant to amoxicillin/clavulanic acid (88%), followed by trimethoprim-sulfamethoxazole (77%), chloramphenicol (43%), and quinolones (41.6%). About 34.5% S. flexneri isolates were found to be resistant to third-generation cephalosporin. None of the isolates was resistant to imipenem, piperacillin-tazobactam, and amikacin. Interestingly high frequency of third-generation cephalosporin resistance was observed in S. flexneri isolated from non-clinical samples (49%) when compared to clinical samples (30.5%). Furthermore, the most prevalent phenotypic-resistant patterns among third-generation cephalosporin-resistant isolates were AMC,CAZ,CPD,CFM,CRO,SXT (13%) followed by OFX,AMC,CAZ,CPD,CFM,CRO,SXT,NA,CIP (10%). The most frequently detected resistance genes were trimethoprim-sulfamethoxazole (sul2 = 84%), beta-lactamase genes (blaOXA = 87%), quinolones (qnrS = 77%), and chloramphenicol (cat = 64%). No mutation was detected in any drug-resistant genes. We are reporting for the first time the sequence of the blaTEM gene in S. flexneri. Furthermore, high third-generation cephalosporin resistance was observed in the patients who practiced self-medication as compared to those who took medication according to physician prescription. This study shows the high emergence of third-generation cephalosporin-resistant S. flexneri isolates, which is a potential threat to the community in the country. This finding will be helpful to develop a suitable antibiotic prescription regime to treat shigellosis.

Prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw foods in Peshawar, Pakistan.

This study was designed to investigate the prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw food samples in Peshawar, Pakistan. A total of 1,020 different samples were collected from various areas of Peshawar between January 2016 and May 2017, followed by identification of S. flexneri through biochemical, serological, and 16S rRNA gene sequencing. Potential risk factors associated with the development and spreading of S. flexneri infection were also investigated. Overall, 45 (4.41%) samples were positive for Shigella species. Among these samples, the predominant species was S. flexneri (n = 44) followed by S. boydii (n = 1). Interestingly, S. sonnei and S. dysenteriae isolates were not found in any sample. The isolation rate of S. flexneri in drinking water samples, market raw milk, and fruits/vegetables from Peshawar were 6.47%, 3.5%, and 2.9%, respectively. The phylogenetic reconstruction showed genetic diversity among three clades, as clades I and II have isolates of S. flexneri that were circulating within the drinking water, milk, fruits/vegetables, while clade III isolates were recovered from milk samples. Most of S. flexneri were detected in June to September. Potential risk factors of S. flexneri were water sources contaminated by toilet wastes (p = 0.04), surface water drainage (p = 0.0002), hospital wastes (p = 0.01), unhygienic handling (p < 0.05), and transportation of raw food (p = 0.04). In conclusion, S. flexneri isolates of closely related lineage originating from non-clinical samples might be associated with an increased human risk to shigellosis in Pakistan, as significant numbers of S. flexneri were observed in the drinking water and retail raw food samples. PRACTICAL APPLICATION: This study demonstrated the presence of S. flexneri in drinking water and retail raw food samples which seem to possess a serious threat to public health. Potential sources of food and water contamination should properly be monitored by public health authorities to reduce cases of shigellosis.

Virulence profiling of Shigella flexneri and emergence of serotype 2b as a highly virulent shigellosis causing strain in Pakistan.

Bacillary diarrhea caused by Shigella flexneri is mediated by various virulence factors which make it the leading agent of diarrhea in developing countries. Previously, a high prevalence of S. flexneri, associated with diarrhea has been reported in Pakistan but no data is available on their virulence profile. The present study reports for the first time analysis of various virulence factors among S. flexneri serotypes isolated from clinical (diarrheal stool) and non-clinical (retail raw foods and drinking water) sources. A total of 199 S. flexneri (clinical: 155, raw foods: 22, water: 22) belonging to various serotypes were subjected to virulence genes detection and virulence profiling. The most frequent virulence gene was found to be ipaH (100%), followed by sat (98%), ial (71.3%), set1B (65.8%) and set1A (38.7%). A high level of virulence was detected in serotype 2b as compared to other serotypes as 32.3% of all serotype 2b have the entire set of five virulence genes including ipaH (100%), ial (100%), sat (37.7%), set1A (89.3%), and set1B (100%). Seven different virulence gene profiles (V1 - V7) were detected and the most frequently observed to be V1 (ipaH+, ial+, sat+, set1A+, set1B+) followed by V3 (ipaH+, ial+, sat+, set1B+). The predominant virulence gene pattern in serotype 2b isolated from clinical and non-clinical samples were V1 and V3. Furthermore, about 32% strains belonging to serotype 2b contain the complete set of five virulence genes isolated from patients with high disease severity. In conclusion, the current finding revealed for the first times that serotype 2b was the most virulent strains in both clinical and non-clinical samples in Pakistan. In addition, the virulence of serotype 2b was well correlated with high disease severity.

D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain.

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Association of Serotype With Antimicrobial Resistance Patterns Among Shigella flexneri Isolates From Pakistan: The Importance of Serotype 2b.

BACKGROUND: Shigella flexneri is an emerging threat in low socioeconomic countries including Pakistan. No previous data is available on the association between S. flexneri serotypes and antimicrobial resistance in Pakistan. OBJECTIVES: The objective of the present study was to assess the association between serotypes and antimicrobial resistance patterns among S. flexneri isolated from clinical and nonclinical samples. METHODS: A total of 199 S. flexneri isolates were subjected to molecular serotyping and antibiotic resistance. RESULTS: The most prevalent S. flexneri serotype was 2b (38%) followed by 1b (24%), 7a (20%), 2a (11%), 1d (5%) and Y (2%). The phylogenetic reconstruction showed 12 clades among which the clades II, III, V, VIII, IX and XI have consisted of serotypes that were found both in human population and environment samples. A high level of multidrug resistance (MDR) was observed in serotype 2b (37.68%) followed by 1b (19.5%) and 7a (19.5%), 2a (11.5%), 1d (5%) and Y (2%). All isolates of serotype 2b showed high level of resistance to amoxicillin/clavulanic acid (100%) followed by quinolone (74.6%) and trimethoprim-sulfamethoxazole (54.6%). Interestingly, none of the serotype was resistant to piperacillin-tazobactam, imipenem and amikacin. The most frequently detected resistance genes among serotype 2b were blaOXA (100%) followed by qnrS (88%), cat (81%) and sul2 (63%). CONCLUSION: The most frequent S. flexneri serotype was 2b while 1d and Y was first time reported in Pakistan. High frequency of MDR serotypes of S. flexneri is a serious threat in diarrhea endemic regions and thus require urgent strategies for its continuous monitoring and prevention.

Molecular epidemiology of Shigella flexneri isolated from pediatrics in a diarrhea-endemic area of Khyber Pakhtunkhwa, Pakistan.

Shigella flexneri is considered as an important causative agent of Shigellosis causing diarrhea in the countries with a low socioeconomic status. No study has been carried out on the molecular prevalence of S. flexneri in Khyber Pakhtunkhwa, Pakistan. So this study was designed to evaluate the molecular prevalence of S. flexneri and their associated risk factors. A total of 2014 diarrheal stool samples were collected from January 2016 to May 2017 from pediatrics patients of Khyber Pakhtunkhwa followed by identification of S. flexneri through biochemical, serological, and molecular methods. The overall prevalence of Shigella species was found to be 7.9% (n = 160). The predominant Shigella specie was S. flexneri (n = 155, 96.8%) followed by S. boydii (n = 5, 3.1%). Interestingly, no sample was found positive for S. sonnei and S. dysenteriae. The majority of Shigellosis cases occurred from June to September. Potential risk factors related with Shigellosis were unhygienic latrine usage, bad hand washing, and consumption of unhygienic food and water, and pipe leakage in the sewage system. In this study, we have observed a high number of Shigellosis cases especially those caused by S. flexneri. It is suggested that effective health awareness programs should be organized by the regional health authorities to minimize the magnitude of pediatrics Shigellosis.

Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain.

Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications.

Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process.

Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive l-arabinose uptake.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h-1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.

The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae.

BACKGROUND: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. RESULTS: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. CONCLUSION: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

The amino-terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid.

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d-glucose and 4% d-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d-xylose over d-glucose with high d-xylose transport rates. This mutant supported efficient sugar fermentation of both d-glucose and d-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937-1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae.

BACKGROUND: Engineering of the yeast Saccharomyces cerevisiae for improved utilization of pentose sugars is vital for cost-efficient cellulosic bioethanol production. Although endogenous hexose transporters (Hxt) can be engineered into specific pentose transporters, they remain subjected to glucose-regulated protein degradation. Therefore, in the absence of glucose or when the glucose is exhausted from the medium, some Hxt proteins with high xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely xylose. RESULTS: The low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant), and Hxt5 are subjected to catabolite degradation as evidenced by a loss of GFP fused hexose transporters from the membrane upon glucose depletion. Catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. Therefore, N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination, were replaced for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely xylose concomitantly with markedly stimulated growth on xylose. The mutagenesis also improved the late stages of sugar fermentation when cells are grown on both glucose and xylose. CONCLUSIONS: Substitution of N-terminal lysine residues in the endogenous hexose transporters Hxt1 and Hxt36 that are subjected to catabolite degradation results in improved retention at the cytoplasmic membrane in the absence of glucose and causes improved xylose fermentation upon the depletion of glucose and when cells are grown in d-xylose alone.

An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

BACKGROUND: The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks. RESULTS: Evolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1-7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1-7 and GAL2 genes. CONCLUSIONS: Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.