Integration of Genome Content, Enzyme Activities, and Expression Profiles in Assessing Changes in End-Product Yields in Clostridium thermocellum

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dc.contributor.authorRydzak, Thomas
dc.contributor.examiningcommitteeOresnik, Ivan (Microbiology) Court, Deborah (Microbiology) Cicek, Nazim (Biosystems Engineering) Wu, David (Chemical Engineering)en_US
dc.contributor.supervisorSparling, Richard (Microbiology)en_US
dc.date.accessioned2013-11-26T15:46:11Z
dc.date.available2013-11-26T15:46:11Z
dc.date.issued2009-03-25en_US
dc.date.issued2011-01-01en_US
dc.date.issued2012-11-21en_US
dc.date.issued2012-12-18en_US
dc.degree.disciplineMicrobiologyen_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US
dc.description.abstractClostridium thermocellum is a fermentative, Gram-positive, thermophile capable of cellulosome-mediated breakdown of hemicellulose and simultaneous biofuels (ethanol and H2) production, and is thus an excellent candidate for consolidated bioprocessing. However, ethanol and/or H2 production yields are below theoretical maxima due to branched product pathways. Biofuel yields may be improved by manipulation of fermentation conditions or implementation of rational metabolic engineering strategies. However, the latter relies on a thorough understanding of gene content, gene product expression, enzyme activity, and intracellular metabolite levels, which can all influence carbon and electron flux. The thesis work represents the first large-scale attempt in combining bioinformatic, enzymatic, proteomic, and culture perturbation approaches to systematically understand these interactions. C. thermocellum was used to investigate how these parameters affect end-product yields. Enzyme activities involved in conversion of pyruvate to end-products were consistent with end-product profiles and draft genome annotation. NADH and NADPH-dependent alcohol dehydrogenase (ADH) activities were comparable, whereas NADPH-dependent hydrogenase activities were higher than NADH and ferredoxin-dependent hydrogenase activities. While product yields changed in response to exogenous end-product additions, most core fermentative enzyme activities did not, suggesting that these changes may be governed by thermodynamics. The lack of major changes (>2-fold) in expression in response to growth and gas sparging was further confirmed by proteomics and RT-qPCR, respectively, although the latter revealed that ADH expression changes in response to gas sparging. Improved genome curation allowed refinement of metabolic pathways. A genomic and end-product meta-analysis of ethanol and/or H2 producing fermentative bacteria revealed that presence/absence of genes encoding hydrogenases and aldehyde dehydrogenases/ADHs had the greatest impacts on biofuel yields. However, genome content alone did not necessarily explain end-product yields. Given that genomic analysis of C. thermocellum revealed the presence of redundant genes encoding enzymes with analogous functions, shotgun and multiple reaction monitoring proteomics was used to refine which proteins are expressed. Absence/low expression of aldehyde dehydrogenase, ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase suggest that these enzymes may not play a significant role in metabolism. An alternative electron flow pathway is proposed to explain end-product synthesis patterns in response to pyruvate addition or presence of protein inhibitors (CO, hypophosphite).en_US
dc.description.noteFebruary 2014en_US
dc.identifier.citationRydzak T, Levin DB, Cicek N, Sparling R. 2009. Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405. J. Biotechnol. 140:169-175.en_US
dc.identifier.citationRydzak T, Levin DB, Cicek N, Sparling R. 2011. End-product induced metabolic shifts in Clostridium thermocellum ATCC 27405. Appl Microbiol Biotechnol 92:199-209.en_US
dc.identifier.citationRydzak T, McQueen PD, Krokhin OV, Spicer V, Ezzati P, Dwivedi RC, Shamshurin D, Levin DB, Wilkins JA, Sparling R. 2012. Proteomic analysis of Clostridium thermocellum core metabolism: Relative protein expression profiles and growth phase-dependent changes in protein expression. BMC. Microbiol. 12:214.en_US
dc.identifier.citationCarere VR, Rydzak T, Verbeke TJ, Cicek N, Levin DB, Sparling R. 2012. Linking genome contene to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria. BMC. Microbio. 12:295en_US
dc.identifier.urihttp://hdl.handle.net/1993/22280
dc.language.isoengen_US
dc.publisherJournal of Biotechnologyen_US
dc.publisherApplied Microbiology and Biotechnologyen_US
dc.publisherBMC Microbiologyen_US
dc.publisherBMC Microbiologyen_US
dc.rightsopen accessen_US
dc.subjectMetabolismen_US
dc.subjectClostridiumen_US
dc.subjectthermocellumen_US
dc.subjectBiofuelsen_US
dc.subjectHydrogenen_US
dc.subjectEthanolen_US
dc.subjectProteomicsen_US
dc.titleIntegration of Genome Content, Enzyme Activities, and Expression Profiles in Assessing Changes in End-Product Yields in Clostridium thermocellumen_US
dc.typedoctoral thesisen_US
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