Project description:Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers often utilize denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen-gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well-described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci. However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to highlight microbial drivers for lignocellulose transformation in woodchip bioreactors with the aim to provide an in-depth characterization of the indigenous microorganisms and their active enzymes. Our findings highlight a microbial community enriched for lignocellulose-degrading denitrifiers with key players from Giesbergeria, Cellulomonas, Azonexus, and UBA5070, including polysaccharide utilization loci from Bacteroidetes. A wide substrate specificity is observed among the many expressed carbohydrate active enzymes (CAZymes), evidencing a swift degradation of lignocellulose, including even enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions.
Project description:Transcript profiles of Phanerochaete chrysosporium grown on ball-milled aspen or ball-milled pine were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignocellulose degradation.
Project description:CRISPR interference screening of 129 protein kinases and 161 transcription factors in S. cerevisiae. Repression effects on yeast growth in oxygen-limited conditions were quantified in synthetic complete media (SCM), SCM supplemented with 10% lignocellulose hydrolysate and SCM supplemented with 45% of a mixture of growth-inhibiting lignocellulosic compounds. The aim of this project was to determine the reproducibility of CRISPRi effects across studies and to characterize CRISPRi for screening of phenotypes relevant for industrial biotechnology. We identify gene functions in general growth in oxygen-limited conditions, and specific for cellular fitness in lignocellulose hydrolysate. A further screen with a cocktail of lignocellulosic compounds enables us to explain hydrolysate-specific gene functions with roles in toxicity.
Project description:The genome of the lignocellulose-degrading, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus encodes genes comprising clusters of glycoside hydrolases, ABC transporters and metabolic enzymes that are transcriptionally responsive to carbohydrates. Transcriptomic and biosolubilization analyses were used to determine if C. saccharolyticus could be deployed as a probe to assess the characteristics of plant biomass feedstocks and efficacy of pre-treatment methods, as these both relate to deconstruction strategies for biofuels production. Based on the response of C. saccharolyticus to plant cell wall polysaccharides, genomic loci were identified that reflected the availability of cellulose, glucomannan, pectin and xylan in biomass to microbial degradation. Furthermore, these loci were useful in assessing how various plant biomass feedstocks (genetically and chemically modified Populus sp., unpretreated Populus sp., and chemically modified switchgrass) were amenable C. saccharolyticus solubilization.
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 5‑hydroxymethylfurfural 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.