1-02: Analysis of the response of Thermoanaerobacterium saccharolyticum to inhibitors and hemicellulose enabled by genome-scale resources

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that was previously engineered to produce ethanol at near-theoretical yield from a wide array of biomass-derived sugars. Here we present a summary of genome data for strain JW/SL-YS485, and resequencing data from 6 mutagenized and selected strains. The genome consists of a 2.7 Mbp chromosome and a 111 Kbp megaplasmid. An in silico reconstruction of metabolism was generated based on the existing reconstruction of Clostridium thermocellum. Microarrays were designed and used to analyze the effects of water extracts of steam-pretreated hardwood on gene expression. Genes involved in phosphate transport and fatty acid biosynthesis were down regulated. Clusters of upregulated genes contained combinations of transporters, regulators, glycosyl hydrolases and carbohydrate-specific catabolic genes. Other upregulated genes included oxidoreductases that may be related to inhibitor detoxification or intracellular redox balancing. Methods for sampling cultures for intracellular metabolite profiling by GC/MS were validated and then used to analyze the effects of the inhibitors HMF and furfural. Glycolytic intermediates and other metabolites were observed to decrease markedly within 15 minutes after addition of HMF/furfural, then increase 8-17 fold over the course of 1 hr.  Microarray results from the same cultures showed activity of an integrated prophage in some replicates, along with concomitant decrease in expression of CRISPR-associated genes.