scholarly journals Harnessing Genetic Diversity in Saccharomyces cerevisiae for Fermentation of Xylose in Hydrolysates of Alkaline Hydrogen Peroxide-Pretreated Biomass

2013 ◽  
Vol 80 (2) ◽  
pp. 540-554 ◽  
Author(s):  
Trey K. Sato ◽  
Tongjun Liu ◽  
Lucas S. Parreiras ◽  
Daniel L. Williams ◽  
Dana J. Wohlbach ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Rapid Evolution ◽  
Biofuel Production ◽  
Ecological Niches ◽  
Alkaline Hydrogen Peroxide ◽  
Xylose Metabolism ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Lignocellulosic Biofuel

ABSTRACTThe fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeastSaccharomyces cerevisiaeis known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverseS. cerevisiaestrains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na+, acetate, andp-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts ofpCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose andpCA or FA with a wildS. cerevisiaestrain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

scholarly journals Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Minhye Shin ◽  
Heeyoung Park ◽  
Sooah Kim ◽  
Eun Joong Oh ◽  
Deokyeol Jeong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Sugar Metabolism ◽  
Pentose Phosphate ◽  
Biofuel Production ◽  
Rna Seq ◽  
Loss Of Function ◽  
Lignocellulosic Biofuel ◽  
Pentose Sugar ◽  
Transcriptomic Changes

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

scholarly journals Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

2016 ◽  
Vol 82 (7) ◽  
pp. 2156-2166 ◽  
Author(s):  
Yingying Chen ◽  
Lisa Stabryla ◽  
Na Wei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Metabolic Engineering ◽  
Acid Stress ◽  
Acid Resistance ◽  
Biofuel Production ◽  
Gene Target ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

scholarly journals Improvement of Biomethane Production from Organic Fraction of Municipal Solid Waste (OFMSW) through Alkaline Hydrogen Peroxide (AHP) Pretreatment

Fermentation ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 197
Author(s):  
Alessio Siciliano ◽  
Carlo Limonti ◽  
Giulia Maria Curcio
Keyword(s):  
Hydrogen Peroxide ◽  
Anaerobic Digestion ◽  
Municipal Solid Waste ◽  
Solid Waste ◽  
Volatile Fatty Acids ◽  
Biofuel Production ◽  
Organic Load ◽  
Organic Substrates ◽  
Organic Fraction ◽  
Alkaline Hydrogen Peroxide

The organic fraction resulting from the separate collection of municipal solid waste (OFMSW) is an abundant residue exploitable for biofuel production. Anaerobic digestion (AD) is one of the most attractive technologies for the treatment of organic wastes thanks to the generation of biogas with a high methane content. However, because of its complex composition, the direct digestion of OFMSW can be less effective. To overcome these difficulties, many pretreatments are under development. In this work, the efficacy of alkaline hydrogen peroxide (AHP) oxidation was assessed for the first time as a pretreatment of OFMSW to enhance its anaerobic biodegradability. In this regard, many AHP batch tests were executed at pH 9 and by changing the peroxide dosages up to 1 gH2O2/gCOD, under room temperature and pressure conditions. Afterwards, biomethane potential tests (BMP) were conducted to evaluate the performance of anaerobic digestion both on raw and pretreated OFMSW. The pretreatment tests demonstrated that AHP induces only a weak reduction in the organic load, reaching a maximum COD removal of about 28%. On the other hand, notable productions of volatile fatty acids (VFA) were found. In fact, by applying a peroxide dose of just 0.025 gH2O2/gCOD, there was a doubling in VFA concentration, which increased by five times with the highest H2O2 amount. These results indicate that AHP mainly causes the conversion of complex organic substrates into easily degradable compounds. This conversion made it possible to achieve much better performance during the BMP tests conducted with the pretreated waste compared to that carried out on fresh OFMSW. Indeed, a low methane production of just 37.06 mLCH4/gTS was detected on raw OFMSW. The cumulated CH4 production in the pretreated samples increased in response to the increase in H2O2 dosage applied during AHP. Maximum specific productions of about 463.7 mLCH4/gTS and 0.31 LCH4/gCODremoved were calculated on mixtures subjected to AHP. On these samples, the satisfactory evolution of AD was confirmed by the process parameters calculated by modeling the cumulated CH4 curves through a new proposed formulation of the Gompertz equation.

scholarly journals Leveraging Genetic-Background Effects in Saccharomyces cerevisiae To Improve Lignocellulosic Hydrolysate Tolerance

2016 ◽  
Vol 82 (19) ◽  
pp. 5838-5849 ◽  
Author(s):  
Maria Sardi ◽  
Nikolay Rovinskiy ◽  
Yaoping Zhang ◽  
Audrey P. Gasch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Tolerance ◽  
Genetic Background ◽  
Biofuel Production ◽  
Gene Overexpression ◽  
Lignocellulosic Hydrolysate ◽  
Content Type ◽  
Cellular Targets ◽  
Specific Effects ◽  
New Genes

ABSTRACTA major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because the mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variations in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene overexpression in differentSaccharomyces cerevisiae(yeast) strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six differentS. cerevisiaestrains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to identify common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene overexpression in three different strains, we revealed the breadth of the background-specific effects of gene fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms.IMPORTANCERecent studies on natural variation withinSaccharomyces cerevisiaehave uncovered substantial phenotypic diversity. Here, we took advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated the physiological states of cells when exposed to this stress. We also explored the strain-specific effects of gene overexpression to further identify strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

scholarly journals Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

2013 ◽  
Vol 80 (5) ◽  
pp. 1602-1615 ◽  
Author(s):  
Tobin J. Verbeke ◽  
Vic Spicer ◽  
Oleg V. Krokhin ◽  
Xiangli Zhang ◽  
John J. Schellenberg ◽  
...  
Keyword(s):  
Consolidated Bioprocessing ◽  
Expression Patterns ◽  
Distribution Patterns ◽  
Product Distribution ◽  
Biofuel Production ◽  
Gene Products ◽  
Label Free ◽  
Carbon Utilization ◽  
Content Type ◽  
Lignocellulosic Biofuel

ABSTRACTThermoanaerobacterspp. have long been considered suitableClostridium thermocellumcoculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain,Thermoanaerobacter thermohydrosulfuricusWC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverseThermoanaerobacterspp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding ofT. thermohydrosulfuricusWC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.

scholarly journals Simulating Extracellular Glucose Signals Enhances Xylose Metabolism in Recombinant Saccharomyces cerevisiae

2020 ◽  
Vol 8 (1) ◽  
pp. 100 ◽  
Author(s):  
Meiling Wu ◽  
Hongxing Li ◽  
Shan Wei ◽  
Hongyu Wu ◽  
Xianwei Wu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Networks ◽  
Glucose Sensing ◽  
Biofuel Production ◽  
Xylose Utilization ◽  
High Concentration ◽  
Xylose Metabolism ◽  
Camp Phosphodiesterase ◽  
Xylose Consumption ◽  
Recombinant Saccharomyces Cerevisiae

Efficient utilization of both glucose and xylose from lignocellulosic biomass would be economically beneficial for biofuel production. Recombinant Saccharomyces cerevisiae strains with essential genes and metabolic networks for xylose metabolism can ferment xylose; however, the efficiency of xylose fermentation is much lower than that of glucose, the preferred carbon source of yeast. Implications from our previous work suggest that activation of the glucose sensing system may benefit xylose metabolism. Here, we show that deleting cAMP phosphodiesterase genes PDE1 and PDE2 increased PKA activity of strains, and consequently, increased xylose utilization. Compared to the wild type strain, the specific xylose consumption rate (rxylose) of the pde1Δ pde2Δ mutant strains increased by 50%; the specific ethanol-producing rate (rethanol) of the strain increased by 70%. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. Deletion of either RGT2 or SNF3 reduced expression of HXT1 in strains cultured in 1 g L−1 xylose, which suggests that xylose can bind both Snf3 and Rgt2 and slightly alter their conformations. Deletion of SNF3 significantly weakened the expression of HXT2 in the yeast cultured in 40 g L−1 xylose, while deletion of RGT2 did not weaken expression of HXT2, suggesting that S. cerevisiae mainly depends on Snf3 to sense a high concentration of xylose (40 g L−1). Finally, we show that deletion of Rgt1, increased rxylose by 24% from that of the control. Our findings indicate how S. cerevisiae may respond to xylose and this study provides novel targets for further engineering of xylose-fermenting strains.

scholarly journals Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels

2014 ◽  
Author(s):  
Xin Li ◽  
Vivian Y Yu ◽  
Yuping Lin ◽  
Kulika Chomvong ◽  
Raíssa Estrela ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plant Cell ◽  
First Generation ◽  
Plant Cell Wall ◽  
Biofuel Production ◽  
Xylose Metabolism ◽  
Renewable Biomass ◽  
Generation Biofuel ◽  
Utilization Pathway

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungusNeurospora crassaas a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Successful reconstitution of this xylodextrin utilization pathway inSaccharomyces cerevisiaerevealed that fungal xylose reductases act as xylodextrin reductases, and together with two hydrolases, generate intracellular xylose and xylitol. Xylodextrin consumption using xylodextrin reductases and tandem intracellular hydrolases greatly expands the capacity of yeasts to use plant cell wall-derived sugars, and should be adaptable to increase the efficiency of both first-generation and next-generation biofuel production.

scholarly journals Deletion ofPHO13, Encoding Haloacid Dehalogenase Type IIA Phosphatase, Results in Upregulation of the Pentose Phosphate Pathway in Saccharomyces cerevisiae

2014 ◽  
Vol 81 (5) ◽  
pp. 1601-1609 ◽  
Author(s):  
Soo Rin Kim ◽  
Haiqing Xu ◽  
Anastashia Lesmana ◽  
Uros Kuzmanovic ◽  
Matthew Au ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Pentose Phosphate ◽  
Sequencing Analysis ◽  
Xylose Metabolism ◽  
Fermentation Inhibitors ◽  
Haloacid Dehalogenase ◽  
Content Type ◽  
Had Superfamily ◽  
Transcriptomic Level

ABSTRACTThe haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes inSaccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identifiedPHO13to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion ofPHO13(pho13Δ), we investigated the response ofS. cerevisiaetopho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed thatpho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced bypho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus,pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation bypho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function ofS. cerevisiaePho13 and the HAD superfamily enzymes and for developingS. cerevisiaestrains with industrially attractive phenotypes.

scholarly journals Genome-wide association across Saccharomyces cerevisiae strains reveals substantial variation in underlying gene requirements for toxin tolerance

2017 ◽  
Author(s):  
Maria Sardi ◽  
Vaishnavi Paithane ◽  
Michael Place ◽  
De Elegant Robinson ◽  
James Hose ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Variants ◽  
Genetic Architecture ◽  
Plant Biomass ◽  
Biofuel Production ◽  
Genome Wide Association ◽  
Ecological Niches ◽  
Substantial Variation ◽  
Genome Wide ◽  
Strain Background

AbstractCellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement.Author summaryUnderstanding the genetic architecture of complex traits is important for elucidating the genotype-phenotype relationship. Many studies have sought genetic variants that underlie phenotypic variation across individuals, both to implicate causal variants and to inform on architecture. Here we used genome-wide association analysis to identify genes and processes involved in tolerance of toxins found in plant-biomass hydrolysate, an important substrate for sustainable biofuel production. We found substantial variation in whether or not individual genes were important for tolerance across genetic backgrounds. Whether or not a gene was important in a given strain background explained more variation than the alleleic differences in the gene. These results suggest substantial variation in gene contributions, and perhaps underlying mechanisms, of toxin tolerance.

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