Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

Hector, Ronald E.; Dien, Bruce S.; Cotta, Michael A.; Qureshi, Nasib

doi:10.1007/s10295-010-0896-1

Cited by 77 publications

(63 citation statements)

References 33 publications

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“…Although S. cerevisiae is the most commonly used organism for industrial ethanol production, its inefficient fermentation of pentoses has hampered the production of ethanol from lignocellulosic biomass (21). Recombinant yeast strains expressing transgenes that facilitate integration of xylose into central carbon metabolism have been constructed (22,23). However, when they are grown in mixed glucose/xylose cultures, which would be encountered in lysates of cellulosic biomass, they rapidly ferment all available glucose and then shift into a respiratory metabolic state in which little if any ethanol is produced (23).…”

Section: Significancementioning

confidence: 99%

Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

Michael

Maier

Brown

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The ability to rationally manipulate the transcriptional states of cells would be of great use in medicine and bioengineering. We have developed an algorithm, NetSurgeon, which uses genomewide gene-regulatory networks to identify interventions that force a cell toward a desired expression state. We first validated NetSurgeon extensively on existing datasets. Next, we used NetSurgeon to select transcription factor deletions aimed at improving ethanol production in Saccharomyces cerevisiae cultures that are catabolizing xylose. We reasoned that interventions that move the transcriptional state of cells using xylose toward that of cells producing large amounts of ethanol from glucose might improve xylose fermentation. Some of the interventions selected by NetSurgeon successfully promoted a fermentative transcriptional state in the absence of glucose, resulting in strains with a 2.7-fold increase in xylose import rates, a 4-fold improvement in xylose integration into central carbon metabolism, or a 1.3-fold increase in ethanol production rate. We conclude by presenting an integrated model of transcriptional regulation and metabolic flux that will enable future efforts aimed at improving xylose fermentation to prioritize functional regulators of central carbon metabolism.gene-regulatory networks | regulatory systems biology | transcriptome | engineering | Saccharomyces cerevisiae T he central premise of regulatory systems biology is that a systematic map of a cell's regulatory machinery will enable us to understand, predict, and rationally manipulate the cell's state or behavior. Manipulation of cellular state has many promising applications, including stem cell biology and regenerative medicine, biofuel production, and gene therapy. Progress toward cellular state control has been driven by both the systems biology and the synthetic biology research communities. Systems biology has produced whole-genome regulatory network maps (1), but relatively little research has focused on using these maps for predicting and manipulating cellular behavior (2). Regulatory synthetic biology has focused on creating molecular circuits that can be placed into a cell to control the transcription of a small number of transgenes, but genome-scale engineering of the cell's native regulatory apparatus is still rare, with most systems restricted to a limited set of controlled targets (3). Here, we demonstrate that transcription factor (TF) network mapping, gene expression profiling, and computational modeling can be integrated to rationally engineer transcriptional state. We call this activity, which bridges the gap between systems biology and synthetic biology, "transcriptome engineering

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Section: Significancementioning

confidence: 99%

Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

Michael

Maier

Brown

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Yeast strains employed in industrial applications are subject to continuous selection, often resulting in the development of robust stress-tolerant properties (1). Some of these industrial strains that have been metabolically engineered for xylose utilization outperform laboratory strains in the fermentation of pretreated lignocellulosic hydrolysates (19,51,52). In two specific cases, faster xylose fermentation has been independently obtained by engineered industrial strains evolved on defined media containing a cocktail of inhibitors found in dilute acid-pretreated spruce hydrolysate (53) or selected on SO 2 -impregnated steam explosion-pretreated spruce hydrolysate (54).…”

mentioning

confidence: 99%

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

Sato

Liu

Parreiras

et al. 2014

Appl Environ Microbiol

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hThe fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is 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 diverse S. cerevisiae strains 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, and p-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 of pCA 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 and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

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“…In both cases, eng1 gene expression in yeast, with its native signal sequence for secretion, was under the control of the strong GAP-DH promoter. The HO endonuclease is not expressed in yeast with high ploidy, and is active only in haploid organisms (Hector et al, 2011); deletion of the HO gene does not affect the growth rate and viability of yeast (Baganz et al, 1997;Voth et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Aspergillus niger endo-1,4-β-glucanase ENG1 secreted from Saccharomyces cerevisiae using different expression vectors

et al. 2015

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ABSTRACT. Heterologous expression of Aspergillus niger endo-1,4-β-glucanase (ENG1) in Saccharomyces cerevisiae was tested both with an episomal plasmid vector (YEGAp/eng1) and a yeast vector capable of integration into the HO locus of the S. cerevisiae chromosome (pHO-GAPDH-eng1-KanMX4-HO). In both cases, eng1 gene expression in yeast, with its native signal sequence for secretion, was under the control of the strong glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. We aimed to verify how each expression system affects protein expression, posttranslational modification, and biochemical properties. Expression of eng1 from the episomal plasmid vector YEGAp/eng1 significantly slowed the growth of a yeast cell culture. However, expression of eng1 from the vector integrated into the HO locus of the chromosome did not cause growth suppression, and the enzyme activity in a culture supernatant was maintained throughout the incubation time. ENG1 has optimum catalytic activity at pH 6.0, and (2015) is stable in the pH range 5.0-9.0. The enzyme's optimum temperature for catalytic activity at pH 6.0 is 70°C; importantly, more than 95% of the enzyme's initial activity remained after a 2-h incubation at 60°C. The biochemical characterization of ENG1 confirmed the correct expression of the protein and showed that ENG1 expressed by the pHO-GAPDH-eng1-KanMX4-HO vector, in addition to its N-linked sites, is overglycosylated at its O-glycosylation sites compared with ENG1 expressed by the YEGAp/eng1 vector. It is likely that the O-glycosylated form of the A. niger ENG1 retains more stable activity during continuous cultivation of recombinant yeasts than the form that is only N-glycosylated.

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Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

Cited by 77 publications

References 33 publications

Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast

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

Characterization of Aspergillus niger endo-1,4-β-glucanase ENG1 secreted from Saccharomyces cerevisiae using different expression vectors

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