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Objective 1 is to determine the functions of K. papyrolyticum GH enzymes. Codon-optimized genes will be synthesized, expressed in E. coli, and screened for activity against both defined and complex polymers. Enzyme kinetic parameters of promising GHs will be determined, particularly primary cellulases. The functional properties of individual GHs are foundational for understanding polysaccharide degradation and to establish the potential contributions of K. papyrolyticum enzymes to lignocellulose depolymerization during spaceflights.
Objective 2 is to determine the broader substrate range and degradation products of K. papyrolyticum. K. papyrolyticum will be screened for growth against a broad group of polymers. For a few polysaccharides, metabolic products will be quantified by untargeted gas chromatography/tandem mass spectrometry. Additionally, gas chromatography with flame ionization detection will be used to quantify volatile fatty acids and alcohols through time courses and to assess the effects of terminal electron acceptor availability (i.e., low or no oxygen) and simulated microgravity on production of chemicals of potential utility. Knowledge of the broader substrate range and products of polysaccharide degradation would be critical to assess biodegradation potential and direct production of biofuels or specialty chemicals.
This work directly addresses the R3 research objective defined in Appendix E under Research Project III: Spaceflight-compatible recycling of non-edible biomass. Although a variety of cellulolytic microorganisms have been described, the current cache of organisms and enzymes is insufficient to degrade the diverse and recalcitrant crop wastes necessary for long-term space flight and colonization. K. papyrolyticum is a member of the bacterial phylum Chloroflexi and is therefore unrelated to other well-described cellulolytic bacteria. By focusing on a highly cellulolytic organism from a poorly explored branch on the tree of life, this project has a strong potential to add to existing cellulolytic organisms and enzymes to address NASA’s spaceflight needs and also provide a template to study other cellulolytic Chloroflexi.
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