Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 4 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH), synthetic hydrolysate with added lignotoxins (SynH_LT), or synthetic hydrolysate containing acid or amide lignotoxins only (SynH_Acids_Amides). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 6 time points, corresponding to early exponential (Exp1), Mid-exponential (Exp2), Late-exponential (Exp3), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. 3 biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 or GLBRCE1 lacking the plasmid-borne PET cassette (GLBRCE1_pBBR) was grown in AFEX corn stover hydrolysate (ACSH) Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH, and cultures were diluted into ACSH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. One biological replicate was grown in each medium. RNA samples were obtained at 3 time points, corresponding to exponential (Exp), transitional (Trans), and stationary (Stat) growth phases.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. Fermentations were carried out in 0.5L bioreactors (Sartorius) containing 0.3L of SynH, SynH lacking osmoprotectants, SynH+LT, or SynH lacking osmoprotectants but containing lignotoxins and cultures were diluted into SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), early stationary (Stat1), and late stationary (Stat2) growth phases.

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Physiological and molecular characterization of yeast cultures pre-adapted for fermentation of lignocellulosic hydrolysate

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