Recombinant Yeast Strains Research Articles

Enhanced biosorption of cadmium ions on immobilized surface-engineered yeast using cadmium-binding peptides

A new type of cadmium (Cd) ion cell surface adsorbent was developed by integrating bacteriophage display peptide library technology with cell surface display technology. Cd2+ chelating resin served as the target molecule in screening experiments, leading to the identification of four Cd2+ −binding peptides. These peptides were introduced into Saccharomyces cerevisiae via the pYD1 plasmid using lithium acetate heat shock transformation. Adsorption efficiency tests indicated that the engineered yeasts adsorbed more Cd2+ than the control strain EBY100 when exposed to the same amount of Cd2+. Among these peptides, sequence 3-containing strain was demonstrated to have the highest Cd2+ adsorption efficiency, being 35% higher than the control strain. Additionally, when this recombinant yeast strain was immobilized using sodium alginate, the adsorption efficiency was increased by 55.7% compared to the control strain.

Engineered Microbial Consortium for De Novo Production of Sclareolide.

Sclareolide, a natural product with bioactive and fragrant properties, is not only utilized in the food, healthcare, and cosmetics industries but also serves as a precursor for the production of ambroxide and some bioactive compounds. Currently, there are three primary methods for producing sclareolide: direct extraction from plants, chemical synthesis using sclareol as a precursor, and the biotransformation of sclareol. Here, we established a platform for producing sclareolide through a modular coculture system with Saccharomyces cerevisiae and Cryptococcus albidus ATCC 20918. S. cerevisiae was engineered for de novo sclareol biosynthesis from glucose, while C. albidus enabled the production of sclareolide via sclareol biotransformation. To enhance the supply of sclareol, a recombinant yeast strain was constructed through metabolic engineering to produce 536.2 mg/L of sclareol. Further improvement of the coculture system for sclareolide production was achieved by incorporating Triton X-100 facilitated intermediate permeability, inoculation proportion adjustment, and culture temperature optimization. These refinements culminated in a sclareolide yield of 626.3 mg/L. This study presents a novel streamlined and efficient approach for sclareolide preparation, showcasing the potential of the microbial consortium in sustainable bioproduction.

Coupling thermotolerance and high production of recombinant protein by CYR1N1546K mutation via cAMP signaling cascades

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23’s improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields.

Enhancing the glycerol utilization of engineered yeast increases its bisabolene production

Bisabolene is a compound commonly found in essential oils of various plants. It has a broad application in sectors such as chemical, pharmaceutical, and health-care products. This study focuses on modifying the glycerol metabolism pathway to obtain a high bisabolene-producing strain of Saccharomyces cerevisiae. To achieve this, the glycerol transporter gene PtFPS2 from Pachysolen tannophilus and the glycerol dehydrogenase gene Opgdh from Ogataea parapolymorpha were overexpressed in engineered yeast YS036, which was equipped with a GAL promoters-enhanced mevalonic acid pathway. Additionally, the glucose-inhibiting transcription factor MIG1 was knocked out to reduce glucose inhibition. The results showed that the GAL promoter transcription levels of the recombinant yeast strains increased, and the co-utilization of sucrose and glycerol was further improved in MIG1-knockout strain. Moreover, the maximum yield of bisabolene in shaking flask fermentation increased to 866.7 mg/L, an 82.2% increase compared to that of the original strain. By modifying the metabolic pathway of carbon sources, the yield of bisabolene was considerably improved. This study offers an effective strategy for enhancing the yield of terpene compounds in engineered yeast.

Enzymatic hydrolysis of single-use bioplastic items by improved recombinant yeast strains

Single-use bioplastic items pose new challenges for a circular plastics economy as they require different processing than petroleum-based plastics items. Microbial and enzymatic recycling approaches could address some of the pitfalls created by the influx of bioplastic waste. In this study, the recombinant expression of a cutinase-like-enzyme (CLE1) was improved in the yeast Saccharomyces cerevisiae to efficiently hydrolyse several commercial single-use bioplastic items constituting blends of poly(lactic acid), poly(1,4-butylene adipate-co-terephthalate), poly(butylene succinate) and mineral fillers. The hydrolysis process was optimised in controlled bioreactor configurations to deliver substantial monomer concentrations and, ultimately, 29 to 78% weight loss. Product inhibition studies and molecular docking provided insights into potential bottlenecks of the enzymatic hydrolysis process, while FT-IR analysis showed the preferential breakdown of specific polymers in blended commercial bioplastic items. This work constitutes a step towards implementing enzymatic hydrolysis as a circular economy approach for the valorisation of end-of-life single-use bioplastic items.

Peptide extract from spent yeast improves resistance of Saccharomyces cerevisiae to oxidative stress

Yeast cells face various stress factors during industrial fermentations, since they are exposed to harsh environmental conditions, which may impair biomolecules productivity and yield. In this work, the use of an antioxidant peptide extract obtained from industrial spent yeast was explored as supplement for Saccharomyces cerevisiae fermentation to prevent a common bottleneck: oxidative stress. For that, a recombinant yeast strain, producer of β-farnesene, was firstly incubated with 0.5 and 0.7 g/L peptide extract, in the presence and absence of hydrogen peroxide (an oxidative stress inducer), for 1–5 h, and then assayed for intracellular reactive oxygen species, and growth ability in agar spot assays. Results showed that under 2 mM H2O2, the peptide extract could improve cells growth and reduce reactive oxygen species production. Therefore, this antioxidant effect was further evaluated in shake-flasks and 2-L bioreactor batch fermentations. Peptide extract (0.7 g/L) was able to increase yeast resistance to the oxidative stress promoted by 2 mM H2O2, by reducing reactive oxygen species levels between 1.2- and 1.7-fold in bioreactor and between 1.2- and 3-fold in shake-flask fermentations. Moreover, improvements on yeast cell density of up to 1.5-fold and 2-fold, and on biomolecule concentration of up to 1.6-fold and 2.8-fold, in bioreactor and shake-flasks, respectively, were obtained. Thus, culture medium supplementation with antioxidant peptide extracted from industrial spent yeast is a promising strategy to improve fermentation performance while valuing biomass waste. This valorization can promote a sustainable and eco-friendly solution for the biotechnology industry by the implementation of a circular economy model.Key points• Peptide extract from spent yeast applied for the first time on yeast fermentation.• Antioxidant peptide extract enhanced S. cerevisiae oxidative stress resistance.• Fermentation performance under stress improved by peptide extract supplementation.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase (LHyal) is a hyperactive hyaluronic acid (HA) hydrolase that belongs to the hyaluronoglucuronidase family. Traditionally, LHyal is extracted from the heads of leeches, but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides. However, at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps. Moreover, the enzyme is deactivated and removed after single use. To solve this problem, we developed a recyclable LHyal biocatalyst using a yeast surface display (YSD) system. After screening and characterization, we found that the cell wall protein Sed1p displayed stronger anchoring to the P. pastoris cell wall than other cell wall proteins. By optimizing the type and length of the linkers between LHyal and Sed1p, we increased the activity of enzymes displayed on the P. pastoris cell wall by 50.34% in flask cultures. LHyal-(GGGS)6-Sed1p activity further increased to 3.58 × 105 U mL−1 in fed-batch cultivation in a 5 L bioreactor. Enzymatic property analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme. Moreover, displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times. YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification process for HA oligosaccharides. Thus, this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Commercial Yeast Strains Expressing Polygalacturonase and Glucanase Unravel the Cell Walls of Chardonnay Grape Pomace.

Simple SummaryGrape skins, usually discarded during wine making, are a valuable source of cellulose (20–50%), hemicelluloses (15–20%), lignin (17–30%) and other compounds, e.g., polyphenols, which can be used as biomaterials in the manufacturing of a variety of new products, such as bioethanol or pharmaceutical products. However, to obtain these biomaterials, the complex polysaccharides of the grape cell walls must be broken down into smaller molecules to allow the extraction of compounds. The degradation process is often performed enzymatically or hydrothermally. Microorganisms that produce the required enzymes while using this waste product as a growth medium can have interesting economic advantages. Here, we created two genetically engineered wine yeast strains that produce grape cell wall degrading enzymes. These yeasts, when grown on grape pomace, induced enzymatic structural changes to the grape cell walls. A collection of antibodies binding to the different cell wall molecules were used to monitor the impact on the cell wall structure of the enzymes, confirming increased extractability of key cell wall polymers when relatively low levels of enzymes are present, illustrating the potential to develop and optimise yeast for grape waste valorisation applications.Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, β-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation.

Identification of bioactive natural products using yeast：Application to monoacylglycerol lipase inhibitor extraction from Corydalis Rhizoma

Identification of bioactive principles from natural products is considered a challenging task in drug discovery. Recently, ligand fishing has been growing in interest as a sustainable strategy. In this study, a yeast-based drug discovery strategy was investigated to screen and fish active ingredients from natural products. Human monoacylglycerol lipase (MAGL) was first displayed on the cell wall of Pichia pastoris. The expression of MAGL on the cell surface was confirmed by immunofluorescence analysis. The activity toward 7-HCA which was consistent with free enzymes in solution. Recombinant yeast strains were used to screen the potential inhibitors from traditional Chinese medicines. Preliminary screening showed that the extracts of 12 herbs showed inhibition on MAGL activity, among which Corydalis Rhizoma had the highest inhibition rate of 60.66 ± 2.44%. Recombinant yeast fishing combined with HPLC-Q-TOF-MS/MS analysis was further used to identify the potential MAGL inhibitors. Finally, dehydrocorydaline (DHC) in Corydalis Rhizoma was determined as a ligand to MAGL with the IC50 value at 154.7 μΜ. Corydalis Rhizoma has been used as a pain reliever in TCM. Intraperitoneal injection of 7 mg kg− 1 DHC in chronic constriction injury model rats significantly attenuated the mechanical allodynia and thermal hyperalgesia. Meanwhile, 2-arachidonoylglycerol, the major MAGL substrate in the brain, was significantly increased both in the hippocampus and striatum. In conclusion, yeast-based ligand fishing combined with HPLC-Q-TOF-MS/MS is a powerful strategy for drug discovery in complex mixtures and DHC from Corydalis Rhizoma was confirmed with high inhibitory activity to MAGL either in vitro or in vivo .

Preparation of recombinant Kluyveromyces lactis agents for simultaneous degradation of two mycotoxins

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are widely distributed in corns, peanuts, and other cereals, causing serious threat to food safety and human health. As shown by our previous studies, the recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-ZPF1) had the ability of degrading AFB1 and ZEN simultaneously. In this work, the agent preparation process was optimized for K. lactis GG799(pKLAC1-ZPF1), and the storage conditions of the prepared yeast agents were investigated, for obtaining the products with high storage activities and potent mycotoxin degradation efficiency. The optimal preparation process was as follows: centrifugation at 6000 rpm for 15 min for collection of the yeast cells, spray drying with the ratio of protective compounds to yeast cells at 3:1 (w/w) and then stored at − 20 °C. Simultaneous degradation tests of AFB1 and ZEN were performed using the supernatants of reactivated yeast agents after three months of storage, and the degradation ratios for AFB1 and ZEN in reaction system 1 (70.0 mmol/L malonic buffer, pH 4.5, with 1.0 mmol/L MnSO4, 0.1 mmol/L H2O2, 5.0 μg/mL AFB1 and ZEN, respectively) were 48.2 ± 3.2% and 34.8 ± 2.8%, while that for ZEN in reaction system 2 (50.0 mmol/L Tris–HCl, pH 7.5, with 5.0 μg/mL AFB1 and ZEN, respectively) was 30.1 ± 2.7%. Besides, the supernatants of reactivated yeast agents degraded more than 80% of AFB1 and 55% of ZEN in contaminated peanuts after twice treatments. Results of this work suggested that the optimized process for K. lactis GG799(pKLAC1-ZPF1) was with high value for industrial applications.

Food-Grade Expression of Manganese Peroxidases in Recombinant Kluyveromyces lactis and Degradation of Aflatoxin B1 Using Fermentation Supernatants.

Aflatoxins are naturally occurring high-toxic secondary metabolites, which cause worldwide environmental contaminations and wastes of food and feed resources and severely threaten human health. Thus, the highly efficient methods and technologies for detoxification of aflatoxins are urgently needed in a long term. In this work, we report the construction of recombinant Kluyveromyces lactis strains GG799(pKLAC1-Phsmnp), GG799(pKLAC1-Plomnp), GG799(pKLAC1-Phcmnp), and then the food-grade expression of the three manganese peroxidases in these strains, followed by the degradation of aflatoxin B1 (AFB1) using the fermentation supernatants. The expression of the manganese peroxidases was achieved in a food-grade manner since Kluyveromyces lactis is food-safe and suitable for application in food or feed industries. The inducible expression process of the optimal recombinant strain GG799(pKLAC1-Phcmnp) and the aflatoxin B1 degradation process were both optimized in detail. After optimization, the degradation ratio reached 75.71%, which was an increase of 49.86% compared to the unoptimized results. The degradation product was analyzed and determined to be AFB1-8,9-dihydrodiol. The recombinant strain GG799(pKLAC1-Phcmnp) supernatants degraded more than 90% of AFB1 in the peanut samples after twice treatments. The structural computational analysis for further mutagenesis of the enzyme PhcMnp was also conducted in this work. The food-grade recombinant yeast strain and the enzyme PhcMnp have potential to be applied in food or feed industries.

Fluorescence-Activated Cell Sorting as a Tool for Recombinant Strain Screening.

Metabolic engineering of microbial cells is the discipline of optimizing microbial metabolism to enable and improve the production of target molecules ranging from biofuels and chemical building blocks to high-value pharmaceuticals. The advances in genetic engineering have eased the construction of highly engineered microbial strains and the generation of genetic libraries. Intracellular metabolite-responsive biosensors facilitate high-throughput screening of these libraries by connecting the levels of a metabolite of interest to a fluorescence output. Fluorescent-activated cell sorting (FACS) enables the isolation of highly fluorescent single cells and thus genotypes that produce higher levels of the metabolite of interest. Here, we describe a high-throughput screening method for recombinant yeast strain screening based on intracellular biosensors and FACS.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus β-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Fractionation, Chemical Analysis, and In Vitro Testing Identify Bioactive Components in MC252 Crude Oil

Abstract Crude oil is a complex mixture that includes polycyclic aromatic hydrocarbons (PAHs) as one of its major components. The toxicity of some chemically substituted PAHs found in oil, such as the methylated species, are relatively understudied. A combination of chemical fractionation and analysis coupled with a bioassay was used to identify a subset of oil PAHs that activated aryl hydrocarbon receptor (AHR). Silica gel chromatography was used for primary and secondary oil fractionation, and standard and reverse phase high performance liquid chromatography (HPLC) were used for the final fractionation steps. Both gas chromatography (GC-) and HPLC-coupled with mass spectrometry (MS) were used to separate and identify compounds present in the petroleum fractions. Bioactivity of the individual fractions was identified and measured using a recombinant yeast strain that expressed the human aryl hydrocarbon receptor complex (AHRC) transcription factor that is composed of human AHR and the ARNT proteins. AHRC activation by oil components results in expression of β-galactosidase, and readout from this enzymatic activity is proportional to the amount and potency of the compounds that activated the system. Silica gel separations produced 25–29 fractions that were assessed for bioactivity using the AHRC reporter system. Bioactivity peaked with the fractions that contained larger PAHs that included four ring compounds such as the triphenylenes, benzanthracenes, and chrysenes (MW 228 + additional methyl groups). When tested as individual compounds, the triphenylenes and benzanthracenes were less potent than the chrysenes, so the latter constituted more of the AHRC signaling activity in the oil fractions. The chrysenes in bioactive fractions were mixtures of the parent compound along with mono-, di-, tri-, and tetra-methyl derivatives and other PAHs. The six possible mono-methylchrysenes were obtained and tested for AHRC activity and for their concentrations in oil. Chrysene, 1-, 2-, 3-, and 6-methylchrysene were present, but 4- and 5-methylchrysene were not detected in the bioactive fractions of oil that were resolved by HPLC. When tested individually in the AHRC bioassay, 4-methylchrysene was the most potent ligand, and 5-methylchrysene was the least potent. Synthetic mixtures of PAHs were reconstructed based upon the chemical composition of one fraction with the high AHRC activity. Collectively, these data show that: 1) the six methylchrysene isomers are within an order of magnitude of chrysene in their ability to activate the AHRC bioassay; 2) although they are a minor group, the chrysene compounds in oil potently activate AHRC signaling; 3) chrysenes diminish as oil weathers, while triphenylenes of identical molecular weight persist, 4) this methodology can be useful for identification and characterization of the bioactivity of sub-fractions and individual compounds found in oil.

Progress in studies on production of chemicals from xylose by Saccharomyces cerevisiae

Effective utilization of xylose is a basis for economic production of biofuels or chemicals from lignocellulose biomass. Over the past 30 years, through metabolic engineering, evolutionary engineering and other strategies, the metabolic capacity of xylose of the traditional ethanol-producing microorganism Saccharomyces cerevisiae has been significantly improved. In recent years, the reported results showed that the transcriptome and metabolome profiles between xylose and glucose metabolism existed significant difference in recombinant yeast strains. Compared with glucose, the overall process of xylose metabolism exhibits Crabtree-negative characteristics, including the limited glycolytic pathway activity, which reduces the metabolic flux of pyruvate to ethanol, and the enhanced cytosolic acetyl-CoA synthesis and respiratory energy metabolism. These traits are helpful to achieve efficient synthesis of downstream products using pyruvate or acetyl-CoA as precursors. This review provides a detailed overview on the modification and optimization of xylose metabolic pathways in S. cerevisiae, the characteristics of xylose metabolism, and the construction of cell factories for production of chemicals using xylose as a carbon source. Meanwhile, the existed difficulties and challenges, and future studies on biosynthesis of bulk chemicals using xylose as an important carbon source are proposed.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Engineering prokaryotic regulator IrrE to enhance stress tolerance in budding yeast

BackgroundStress tolerance is one of the important desired microbial traits for industrial bioprocesses, and global regulatory protein engineering is an efficient approach to improve strain tolerance. In our study, IrrE, a global regulatory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved tolerance to the inhibitors in lignocellulose hydrolysates or high temperatures.ResultsThree IrrE mutations were developed through directed evolution, and the expression of these mutants could improve the yeast fermentation rate by threefold or more in the presence of multiple inhibitors. Subsequently, the tolerance to multiple inhibitors of single-site mutants based on the mutations from the variants were then evaluated, and 11 mutants, including L65P, I103T, E119V, L160F, P162S, M169V, V204A, R244G, Base 824 Deletion, V299A, and A300V were identified to be critical for the improved representative inhibitors, i.e., furfural, acetic acid and phenol (FAP) tolerance. Further studies indicated that IrrE caused genome-wide transcriptional perturbation in yeast, and the mutant I24 led to the rapid growth of Saccharomyces cerevisiae by primarily regulating the transcription level of transcription activators/factors, protecting the intracellular environment and enhancing the antioxidant capacity under inhibitor environments, which reflected IrrE plasticity. Meanwhile, we observed that the expression of the wild-type or mutant IrrE could also protect Saccharomyces cerevisiae from the damage caused by thermal stress. The recombinant yeast strains were able to grow with glucose at 42 ℃.ConclusionsIrrE from Deinococcus radiodurans can be engineered as a tolerance-enhancer for Saccharomyces cerevisiae. Systematic research on the regulatory model and mechanism of a prokaryotic global regulatory factor IrrE to increase yeast tolerance provided valuable insights for the improvements in microbial tolerance to complex industrial stress conditions.

Functional Expression of All Human Sulfotransferases in Fission Yeast, Assay Development, and Structural Models for Isoforms SULT4A1 and SULT6B1.

Cytosolic sulfotransferases (SULTs) catalyze phase II (conjugation) reactions of drugs and endogenous compounds. A complete set of recombinant fission yeast strains each expressing one of the 14 human SULTs was generated, including SULT4A1 and SULT6B1. Sulfation of test substrates by whole-cell biotransformation was successfully demonstrated for all enzymes for which substrates were previously known. The results proved that the intracellular production of the cofactor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) necessary for SULT activity in fission yeast is sufficiently high to support metabolite production. A modified variant of sulfotransferase assay was also developed that employs permeabilized fission yeast cells (enzyme bags). Using this approach, SULT4A1-dependent sulfation of 1-naphthol was observed. Additionally, a new and convenient SULT activity assay is presented. It is based on the sulfation of a proluciferin compound, which was catalyzed by SULT1E1, SULT2A1, SULT4A1, and SULT6B1. For the latter two enzymes this study represents the first demonstration of their enzymatic functionality. Furthermore, the first catalytically competent homology models for SULT4A1 and SULT6B1 in complex with PAPS are reported. Through mechanistic molecular modeling driven by substrate docking, we pinned down the increased activity levels of these two isoforms to optimized substrate binding.

Microbial Cell Factories for Tetrahydroisoquinoline Alkaloid Production.

For decades, plants have represented an inexhaustible source of natural products used in various sectors such as health and industry. However, one recurring problem is the low accumulation of these compounds in planta and, therefore, their production costs and supply. In recent years, unprecedented hope has been brought by the metabolic engineering of microorganisms, which opens up prospects for supply of these molecules at lower cost with high added value. However, many of these productions remained at a laboratory scale. In a recent article published in Nature Communication, Vincent J. J. Martin's team has developed an optimized yeast strain capable of synthesizing not only a huge amount of (S)-reticuline, a major precursor of the plant tetrahydroisoquinoline alkaloid series, but also a whole range of new-to-nature compounds from this prominent family of natural products. This synthesis, reaching industrial scales, thus paves the way to efficient production in microbial cell factories.

Characterization of two sugar transporters responsible for efficient xylose uptake in an oleaginous yeast Candida tropicalis SY005

Microbial conversion of lignocellulosic feedstock to the target bioproduct requires efficient assimilation of its constituent sugars, a large part of which comprises of glucose and xylose. This study aims to identify and characterize sugar transporters capable of xylose uptake in an oleaginous strain of the industrially relevant yeast Candida tropicalis. In silico database mining resulted in two sugar transporter proteins- CtStp1 and CtStp2, containing conserved amino acid residues and motifs that have been previously reported to be involved in xylose transport in other organisms. Several softwares predicted the likelihood of 10–12 transmembrane (TM) helices to be present in both the Stps, while molecular modelling showed 12 TM helices that were organized into a typical structure found in the major facilitator superfamily of transporters. Docking with different sugars also predicted favorable interactions. Heterologous expression in a Saccharomyces cerevisiae strain harboring functional xylose metabolic genes validated the broad substrate specificity of the two Stps. Each transporter supported prominent growth of recombinant S. cerevisiae strains on six sugars including xylose at various concentrations. Expression of CtSTP1 and CtSTP2 along with the xylose metabolic genes in yeast transformants grown in presence of xylose was confirmed by transcript detection. Growth curve and sugar consumption profiles revealed uptake of both glucose and xylose simultaneously by the recombinant yeast strains, though CtStp1 showed relatively less effect of glucose repression in mixed sugars and was a better transporter of xylose than CtStp2. Such glucose-xylose utilizing efficient transporters can be effective tools for developing co-fermenting yeasts through genetic engineering in future, with noteworthy applications in renewable biomass utilization.