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Abstract
Researchers around the world are in search of sources for renewable fuels. Ethanol from corn-starch cannot provide enough biofuel to satisfy our nations energy needs. A diverse catalogue of biomasses, fuel types and technologies will be required to meet the production goals set forth by the U.S. government. Lignocellulosic material rich in pectin is a promising biomass due to its low concentrations of lignin, a complex, highly recalcitrant chemical compound. Lignin degradation is expensive and produces chemical inhibitors. Instead of lignin, some plants have high concentrations of pectin, a polysaccharide mostly composed of galacturonic acid. Pectin can be degraded by saccharification enzymes, potentially reducing cost and inhibitor production.This dissertation focuses on increasing the feasibility of producing bioethanol from pectin-rich materials. Ethanologens (Saccharomyces cerevisiae and Escherichia coli) and fermentation processes were tailored to the biomass to alleviate the need for costly commercial enzymes and to increase ethanol concentrations.Commercial enzymes to saccharify pectin-rich peaches using S. cerevisiae XR122N were reduced by over 85%. Commercial pectinase was eliminated with the addition of a single enzyme, pectate lyase B (PelB), from Paenibacillus amyloliticus 27C64. S. cerevisiae 09-448 produced its own pectinase, which supported maximum ethanol production in the absence of commercial pectinase.Use of commercial enzymes in E. coli fermentations was decreased through bioengineering of E. coli KO11 to produce heterologous saccharification proteins. The final strain, JP08C, contained cellobiose phosphotransferase genes (CasAB) from Klebsiella oxytoca, pectate lyase E (PelE), oligogalacturonide lyase (Ogl) and the Out secretion system from Erwinia chrysanthemi. JP08C was able to produce more ethanol from sugar beet pulp (SBP) fermentations at lower enzyme concentrations than KO11.However, large concentrations of lactic, acetic and formic acids were formed. To decrease side-products, the genes for lactic acid dehydrogenase (ldhA) and a putative pyruvate formate lyase (ybiW) were deleted. Pyruvate dehydrogenase (Pdh) was activated under anaerobic conditions through the replacement of the lpd gene with an anaerobically active lpd mutant (E354G). The active Pdh has a low Km for pyruvate and can siphon carbon away from the pyruvate formate lyase (Pfl) pathway and increased ethanol production in 10% w/v model sugar fermentations.