Recombinant Saccharomyces Cerevisiae Research Articles

Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway’s overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway’s intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

Dilute sulfuric acid fractionation of Korean food waste for ethanol and lactic acid production by yeast

Fermentation of food waste biomass can be used to produce biochemicals such as lactic acid and ethanol in a cost-effective manner. Korean food waste (KFW) dewatered by a screw press contains 23.1% glucan on a dry basis and is a potential raw material for the production of ethanol and lactic acid through fermentation. This study was conducted to optimize the dilute acid fractionation conditions for KFW fermentation with respect to the H2SO4 concentration (0–0.8% w/v), temperature (130–190 °C), and residence time (1–128 min) using response surface methodology. Dilute sulfuric acid fractionation was carried out using a 30-mL stainless steel reactor under conditions, and then the dilute acid fractionation was scaled-up in 1-L and 7-L stainless steel reactors under the optimal conditions. The hydrolysate was concentrated, liquid-liquid extracted and neutralized for lactic acid and ethanol production. The highest concentration of glucose obtained from the KFW was 26.4 g/L using fractionation with 0.37% w/v H2SO4 at 156 °C for 123.6 min. Using recombinant Saccharomyces cerevisiae containing a codon-optimized lactate dehydrogenase, the yield of lactic acid and ethanol was 77% of the theoretical yield for 17.4 g/L of fermentable sugar at pH 5.5. Additionally, the yield of ethanol produced by Issatchenkia orientalis was 89% of the theoretical yield for 25 g/L of fermentable sugar at pH 3.

An indoleamine 2, 3-dioxygenase siRNA nanoparticle-coated and Trp2-displayed recombinant yeast vaccine inhibits melanoma tumor growth in mice

Therapeutic vaccine is a promising approach in cancer therapy. But tumor-associated antigen peptides have weak immunogenicity and cancer patients are often characterized by immunosuppression and tolerance, leading to less efficiency of immunotherapy. We here successfully developed indoleamine 2, 3-dioxygenase (IDO) siRNA nanoparticle-coated and tyrosinase-related protein 2 (Trp2)-displayed recombinant Saccharomyces cerevisiae (YCP). YCPs had positive charges with a diameter of approximately 5μm, resulting in selective phagocytosis by APC cells. YCP-delivered siRNA and Trp2 successfully escaped from phagosomes, efficiently inhibited IDO expression in DCs, promoted the immune reaction of T cell against Trp2, increased the secretion of proinflammatory cytokines such as IFN-γ,TNF-α, and IL-6, and decreased the generation of regulatory T cells. Moreover, YCPs significantly inhibited melanoma tumor growth by alleviating immune tolerance and promoting Trp2-specific CD8+ T cell immune response. These results suggest that Saccharomyces cerevisiae as a combined immunotherapeutic platform to simultaneously delivery IDO-siRNA and Trp2 epitope peptide is a promising vaccine system for melanoma treatment.

Fermentation of oat and soybean hull hydrolysates into ethanol and xylitol by recombinant industrial strains of Saccharomyces cerevisiae under diverse oxygen environments

In this study, we evaluated the capacity of recombinant industrial Saccharomyces cerevisiae YRH 396 and YRH 400 strains to ferment sugars from oat hull and soybean hull hydrolysates into ethanol and xylitol. The strains were genetically modified by chromosomal integration of Pichia stipitis XYLI/XYL2 genes and the overexpression of S. cerevisiae XKS1 genes, in order to have the ability to metabolize xylose, one of the main sugars in lignocellulosic biomass. The strains YRH 396 and YRH 400 were tested by fermenting acid and enzymatic hydrolysates of oat and soybean hull, with different concentrations of sugars, in orbital shaker under conditions of anaerobiosis and oxygen limitation. The YRH 396 strain showed the best kinetic parameters for the production of ethanol and xylitol, thus its metabolism was further studied in bioreactor cultivations. Under anaerobiosis, the maximum consumption of xylose was approximately 35% when using hydrolysates containing similar concentrations of glucose and xylose, whereas when hydrolysates mainly composed of xylose were used, this strain showed a consumption of 73% of the xylose, reaching yields of ethanol of 0.33 g g−1. In bioreactor cultivations under oxygen limitation, xylose consumption reached approximately 65%, and the main product was xylitol, reaching a final concentration of 8.17 g L−1. These results suggest that, in addition to an adaptive evolution process, molecular modifications are necessary for an industrial application of these type of genetically modified strains.

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.

Comparative metabolomics profiling of engineered Saccharomyces cerevisiae lead to a strategy that improving β-carotene production by acetate supplementation.

A comparative metabolomic analysis was conducted on recombinant Saccharomyces cerevisiae strain producing β-carotene and the parent strain cultivated with glucose as carbon source using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based approach. The results showed that most of the central intermediates associated with amino acids, carbohydrates, glycolysis and TCA cycle intermediates (acetic acid, glycerol, citric acid, pyruvic acid and succinic acid), fatty acids, ergosterol and energy metabolites were produced in a lower amount in recombinant strain, as compared to the parent strain. To increase β-carotene production in recombinant strain, a strategy that exogenous addition of acetate (10 g/l) in exponential phase was developed, which could enhance most intracellular metabolites levels and result in 39.3% and 14.2% improvement of β-carotene concentration and production, respectively, which was accompanied by the enhancement of acetyl-CoA, fatty acids, ergosterol and ATP contents in cells. These results indicated that the amounts of intracellular metabolites in engineered strain are largely consumed by carotenoid formation. Therefore, maintaining intracellular metabolites pool at normal levels is essential for carotenoid biosynthesis. To relieve this limitation, rational supplementation of acetate could be a potential way because it can partially restore the levels of intracellular metabolites and improve the production of carotenoid compounds in recombinant S. cerevisiae.

Enhanced production of xylitol from xylose by expression of Bacillus subtilis arabinose:H+ symporter and Scheffersomyces stipitis xylose reductase in recombinant Saccharomyces cerevisiae.

Inefficient transport of xylose into Saccharomyces cerevisiae is a major hurdle for production of xylitol, a natural sweetener with five carbons. To facilitate the xylose transport and hence increase xylose conversion to xylitol, the araE gene encoding an arabinose:H+ symporter (AraE) from Bacillus subtilis and the XYL1 gene from Scheffersomyces stipitis were expressed in Saccharomyces cerevisiae EBY.VW4000, a hxt null mutant. The resulting strain of EXHA exhibited 4.1 fold increases in xylose consumption rate and xylitol productivity, relative to the control strain without AraE. Also, overexpression of AraE in wild type S. cerevisiae D452-2 having all hexose transporters and the XYL1 gene increased both xylose consumption and xylitol production considerably. In a glucose-limited fed-batch culture with intermittent addition of xylose, the DXXA strain with multiple copies of araE and XYL1 produced 177.8g/L xylitol with 2.47g/L-h productivity, which were 26.9 and 17.6 times higher than those for a batch culture of the DX strain expressing the XYL1 gene only, respectively. It was concluded that B. subtilis AraE might be a potent xylose transporter and conferred much higher xylose-consuming and xylitol-producing abilities to S. cerevisiae.

Life cycle assessment of β-Galactosidase enzyme production

Many consumers are unable to enjoy the benefits of milk due to intolerance or even allergy to lactose. This inability to digest lactose is caused by a deficiency of the enzyme β-Galactosidase in the digestive tract, which performs the hydrolysis of lactose to form glucose and galactose, two sugars which are easily absorbed into the bloodstream. This study reports the Life Cycle Assessment (LCA) outcomes of the β-Galactosidase production in an industrial -scale facility from a cradle-to-gate perspective. Among the different alternatives of the production process of the β-Galactosidase enzyme, the use of recombinant Saccharomyces cerevisiae yeast expressing the lacA gene of Aspergillus niger was considered due to its higher production yield in comparison with wild strains. The primary source of environmental impact is the downstream processing related subsystem, specially attributed to the separation, concentration and purification of the enzyme as well as Cleaning-in-Place activities, required to maintain aseptic and sterile operational conditions. The use of alternative chemicals to the conventional ones in this latter stage could imply environmental improvements of the production process.

A design\u2013build\u2013test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models

BackgroundRecent advancements in omics measurement technologies have led to an ever-increasing amount of available experimental data that necessitate systems-oriented methodologies for efficient and systematic integration of data into consistent large-scale kinetic models. These models can help us to uncover new insights into cellular physiology and also to assist in the rational design of bioreactor or fermentation processes. Optimization and Risk Analysis of Complex Living Entities (ORACLE) framework for the construction of large-scale kinetic models can be used as guidance for formulating alternative metabolic engineering strategies.ResultsWe used ORACLE in a metabolic engineering problem: improvement of the xylose uptake rate during mixed glucose–xylose consumption in a recombinant Saccharomyces cerevisiae strain. Using the data from bioreactor fermentations, we characterized network flux and concentration profiles representing possible physiological states of the analyzed strain. We then identified enzymes that could lead to improved flux through xylose transporters (XTR). For some of the identified enzymes, including hexokinase (HXK), we could not deduce if their control over XTR was positive or negative. We thus performed a follow-up experiment, and we found out that HXK2 deletion improves xylose uptake rate. The data from the performed experiments were then used to prune the kinetic models, and the predictions of the pruned population of kinetic models were in agreement with the experimental data collected on the HXK2-deficient S. cerevisiae strain.ConclusionsWe present a design–build–test cycle composed of modeling efforts and experiments with a glucose–xylose co-utilizing recombinant S. cerevisiae and its HXK2-deficient mutant that allowed us to uncover interdependencies between upper glycolysis and xylose uptake pathway. Through this cycle, we also obtained kinetic models with improved prediction capabilities. The present study demonstrates the potential of integrated “modeling and experiments” systems biology approaches that can be applied for diverse applications ranging from biotechnology to drug discovery.

Synergies in coupled hydrolysis and fermentation of cellulose using a Trichoderma reesei enzyme preparation and a recombinant Saccharomyces cerevisiae strain.

We describe a procedure by which filter paper is digested with a cellulolytic enzyme preparation, obtained from Trichoderma reesei cultivated under solid state fermentation conditions and then fermented by a recombinant Saccharomyces cerevisiae strain. The yeast strain produces a β-glucosidase encoded by the BGL1 gene from Saccharomycopsis fibuligera that quantitatively and qualitatively complements the limitations that the Trichoderma enzyme complex shows for this particular activity. The supplemental β-glucosidase activity fuels the progression of cellulose hydrolysis and fermentation by decreasing the inhibitory effects caused by the accumulation of cellobiose and glucose. Fermentation of filter paper by this procedure yields ethanol concentrations above 70g/L.

Enhanced d-lactic acid production by recombinant Saccharomyces cerevisiae following optimization of the global metabolic pathway.

Utilization of renewable feedstocks for the production of bio-based chemicals such as d-lactic acid by engineering metabolic pathways in the yeast Saccharomyces cerevisiae has recently become an attractive option. In this study, to realize efficient d-lactic acid production by S. cerevisiae, the expression of 12 glycolysis-related genes and the Leuconostoc mesenteroides d-LDH gene was optimized using a previously developed global metabolic engineering strategy, and repeated batch fermentation was carried out using the resultant strain YPH499/dPdA3-34/DLDH/1-18. Stable d-lactic acid production through 10 repeated batch fermentations was achieved using YPH499/dPdA3-34/DLDH/1-18. The average d-lactic acid production, productivity, and yield with 10 repeated batch fermentations were 60.3 g/L, 2.80 g/L/h, and 0.646, respectively. The present study is the first report of the application of a global metabolic engineering strategy for bio-based chemical production, and it shows the potential for efficient production of such chemicals by global metabolic engineering of the yeast S. cerevisiae. Biotechnol. Bioeng. 2017;114: 2075-2084. © 2017 Wiley Periodicals, Inc.

Efficient production of 2,3-butanediol by recombinant Saccharomyces cerevisiae through modulation of gene expression by cocktail δ-integration

In this study, the expression of 4 genes encoding α-acetolactate synthase, α-acetolactate decarboxylase, 2,3-butanediol dehydrogenase, and NADH oxidase was modulated using a previously developed cocktail δ-integration strategy. The resultant strain, YPH499/dPdAdG/BD6-10, was used in a fed-batch cultivation for the production of 2,3-butanediol. The concentration, production rate, and yield obtained were 80.0g/L, 4.00g/L/h, and 41.7%, respectively. The production rate and yield of the compound obtained are higher for this strain compared to reports published for Saccharomyces cerevisiae so far. The cocktail δ-integration strategy allows for modulation of multiple gene expression, without the exact knowledge of rate-limiting steps, and therefore, could be used as a promising strategy for the production of bio-based chemicals in recombinant S. cerevisiae.

Oral vaccination through voluntary consumption of the convict grouper Epinephelus septemfasciatus with yeast producing the capsid protein of red-spotted grouper nervous necrosis virus

Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >103 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination.

Enhancing ethanol yields through d-xylose and l-arabinose co-fermentation after construction of a novel high efficient l-arabinose-fermenting Saccharomyces cerevisiae strain.

Lignocellulose contains two pentose sugars, l-arabinose and d-xylose, neither of which is naturally fermented by first generation (1G) ethanol-producing Saccharomyces cerevisiae yeast. Since these sugars are inaccessible to 1G yeast, a significant percentage of the total carbon in bioethanol production from plant residues, which are used in second generation (2G) ethanol production, remains unused. Recombinant Saccharomyces cerevisiae strains capable of fermenting d-xylose are available on the market; however, there are few examples of l-arabinose-fermenting yeasts, and commercially, there are no strains capable of fermenting both d-xylose and l-arabinose because of metabolic incompatibilities when both metabolic pathways are expressed in the same cell. To attempt to solve this problem we have tested d-xylose and l-arabinose co-fermentation. To find efficient alternative l-arabinose utilization pathways to the few existing ones, we have used stringent methodology to screen for new genes (metabolic and transporter functions) to facilitate l-arabinose fermentation in recombinant yeast. We demonstrate the feasibility of this approach in a successfully constructed yeast strain capable of using l-arabinose as the sole carbon source and capable of fully transforming it to ethanol, reaching the maximum theoretical fermentation yield (0.43 g g-1). We demonstrate that efficient co-fermentation of d-xylose and l-arabinose is feasible using two different co-cultured strains, and observed no fermentation delays, yield drops or accumulation of undesired byproducts. In this study we have identified a technically efficient strategy to enhance ethanol yields by 10 % in 2G plants in a process based on C5 sugar co-fermentation.

Yeast-mediated mRNA delivery polarizes immuno-suppressive macrophages towards an immuno-stimulatory phenotype

Macrophages have increasingly gained interest as a therapeutic target since they represent an integral component of the tumor microenvironment. In fact, M2 macrophage accumulation in solid tumors is associated with poor prognosis and therapy failure. Therefore, reprogramming M2 macrophages towards an M1 phenotype with anti-tumor activity by gene therapy represents a promising therapeutic approach. Herein, we describe recombinant Saccharomyces cerevisiae as a novel gene delivery vehicle for primary human macrophages. Opsonized S. cerevisiae was taken up efficiently by M2 macrophages and initiated the expression of pro-inflammatory cytokines. Recombinant yeast delivered functional nucleic acids to macrophages, especially when constitutively biosynthesized mRNA was used as cargo. Interestingly, expression of the protein encoded for by the delivered nucleic acid was higher in M2 cells when compared to M1 macrophages. Finally, the delivery of mRNA coding for the pro-inflammatory regulators MYD88 and TNF to M2 macrophages induced a prolonged upregulation of pro-inflammatory and cytotoxic cytokines in these cells, suggesting their successful re-education towards an anti-tumor M1 phenotype. Our results suggest the use of yeast-based gene delivery as a promising approach for the treatment of pathologic conditions that may benefit from the presence of M1-polarized macrophages, such as cancer.

Direct bioethanol production from wheat straw using xylose/glucose co-fermentation by co-culture of two recombinant yeasts.

To achieve a cost-effective bioconversion of lignocellulosic materials, a novel xylose/glucose co-fermentation process by co-culture of cellulose-utilizing recombinant Saccharomyces cerevisiae (S. cerevisiae) and xylan-utilizing recombinant Pichia pastoris (P. pastoris) was developed, in which ethanol was produced directly from wheat straw without additional hydrolytic enzymes. Recombinant S. cerevisiae coexpressing three types of cellulase and recombinant P. pastoris coexpressing two types of xylanase were constructed, respectively. All cellulases and xylanases were successfully expressed and similar extracellular activity was demonstrated. The maximum ethanol concentration of 32.6gL-1 with the yield 0.42gg-1 was achieved from wheat straw corresponding to 100gL-1 of total sugar after 80h co-fermentation, which corresponds to 82.6% of the theoretical yield. These results demonstrate that the direct and efficient ethanol production from lignocellulosic materials is accomplished by simultaneous saccharification (cellulose and hemicellulose) and co-fermentation (glucose and xylose) with the co-culture of the two recombinant yeasts.

Up-concentration of sugars in pretreated-rice straw by an osmotic pressure-driven method

Forward osmosis (FO), driven by high osmotic pressure, was used for the first time for bioethanol production from lignocellulosic biomass. A commercial membrane, TFC-ES (Hydration Technology Innovation), was used and 3.6M triethylamine (TEA) was chosen as the draw solution because the concentration ratio against a model xylose solution was similar to that achieved with 2.5M NaCl. The liquid fraction of hot-water-pretreated rice straw was concentrated by using the FO membrane. The initial sugar concentration of 199mM increased to 825 and 1612mM after 48 and 72h of FO concentration, respectively, and these values were much higher than those obtained from nanofiltration. Simultaneous saccharification and fermentation of the liquid fraction after 48h of FO concentration by xylose-fermenting recombinant Saccharomyces cerevisiae produced 17.7g/L of ethanol after 24h fermentation. Thus, the FO process has tremendous potential to up-concentrate sugars obtained from lignocellulosic biomass.

Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae

To clarify the relationship between NAD(P)+/NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20g/L xylose or 20g/L xylitol at pH 5.0 and 30°C. The TDH3p-GAL2 or gal80Δ strain completely consumed the xylose within 24h and aerobically consumed 92-100% of the xylitol within 96h, but anaerobically consumed only 20% of the xylitol within 96h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD+ generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD+-dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD+ generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25g/L, 5.30g/L, and 4.27g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward.

Cloning and characterization of a serine/threonine protein phosphatase2A-encoding gene IbPP2A1 from Ipomoea batatas (L.) Lam.

Sweet potato is one of the most important food crops with strong environmental adaptability. Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that plays crucial roles in hormone signal transduction and abiotic stress response. Characterization of PP2A can elucidate the mechanisms of stress resistance in this crop. In this study, a total of 15 PP2A transcripts, including nine regulatory subunit-encoding sequences and six catalytic subunit-encoding sequences, were identified from sweet potato and found to be expressed at varying levels. Only one contained a complete open reading frame and encoded a B regulatory subunit (termed IbPP2A1). Ten polymorphic sites were distributed in the coding region of this gene, but most did not result in amino acid change. RNA sequencing-based digital gene expression profiling showed that this PP2A gene was primarily expressed in fibrous roots and developing tuberous roots under natural conditions. After drought, salinity, and alkaline stress treatment, the expression of IbPP2A1 was obviously upregulated in stems and tuberous roots at 2 days, whereas the expression of IbPP2A1 in leaves was only upregulated by drought stress at 2 days. However, heterogeneous expression of IbPP2A1 did not enhance abiotic stress tolerance in recombinant Saccharomyces cerevisiae. The results described here indicate that IbPP2A1 is an abiotic stress-responsive gene, but it could not work alone in vitro. This study provides a preliminary but global insight into PP2A proteins in sweet potato for further investigations on improving stress tolerance in this crop.

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability

Production of ethanol from xylose by recombinant Saccharomyces cerevisiae is suboptimal with slow fermentation rate, compared with that from glucose. In this study, a strain-expressing Scheffersomyces stipitis xylose reductase-xylitol dehydrogenase (XR-XDH) pathway was subjected to adaptive evolution on xylose; this approach generated populations with the significantly improved cell growth and ethanol production rate. Mutants were isolated, and the best one was used for sporulation to generate eight stable mutant strains with improved xylose fermentation ability. They were used in a microarray assay to study the molecular basis of the enhanced phenotype. The enriched transcriptional differences among the eight mutant strains and the native strain revealed novel responses to xylose, which likely contributes to the improved xylose utilization. The upregulated vitamin B1 and B6 biosynthesis indicated that thiamine served as an important cofactor in xylose metabolism and may alleviate the redox stress. The increased expression of genes involved in sulfur amino acid biosynthesis and the decreased expression of genes related to Fe(II) transport may alleviate redox stress as well. Meanwhile, it was remarkable that several glucose-repressible genes, including genes of the galactose metabolism, gluconeogenesis, and ethanol catabolism, had a lower expression level after adaptive evolution. Concomitantly, the expression levels of two regulators of the glucose signaling pathway, Rgs2 and Sip4, decreased, indicating a reshaped signaling pathway to xylose after adaptive evolution. Our findings provide new targets for construction of a superior bioethanol producing strain through inverse metabolic engineering.