Thermoanaerobacterthermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Abstract

e Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture 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 thermohydrosulfuricus WC1, 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 diverse Thermoanaerobacter spp. 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 of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

“…Hydrogen in the headspace was measured using an Agilent Technologies 6850 Series II gas chromatograph with a Carboxen 1010 plot column (30 m × 0.53 mm) and a thermal conductivity detector set at 190°C. Hydrogen in the liquid fraction was determined as previously described [ 43 ] accounting for the gas solubility constant [ 44 ]. Carbon dioxide measurements were estimated and represent the sum of the determined values for acetate and biomass production.…”

“…Carbon dioxide measurements were estimated and represent the sum of the determined values for acetate and biomass production. Protein/biomass measurements were also conducted as previously described [ 43 ], whereby total cellular protein was determined via a standard Bradford assay and cellular protein in C. bescii is assumed to be 50% of the cell dry weight [ 45 ]. An elemental composition of C 4 H 7 O 2 N was used for biomass determinations in moles of carbon [ 46 ].…”

Characterization of Mutations That Affect the Nonoxidative Pentose Phosphate Pathway in Sinorhizobium meliloti

“…A quality control assessing biological variation between any two comparison groups (cross state) versus system noise among biological replicates (intrareplicative viability) was conducted. Transformation of different populations into final log 2 expression values (Pnet) for differential expression analyses of cross-state samples was conducted as described previously (67). Cutoff scores of Ϯ1.0 were used to represent proteins observed to be upregulated or downregulated by at least 2-fold versus the comparison condition.…”

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass

“…Frequently, LA is not among the most abundant end‐products of these organisms as in the case of C. cellulovorans , Clostridium thermocellum or Thermoanaerobacterium saccharolyticum . Exceptions include Thermoanaerobacter thermohydrosulfuricus WC1, that is, a recently isolated xylan‐metabolizing strain, whose main fermentation product is LA .…”