Improving biological detoxification of furfural and acetate in lignocellulosic hydrolysates using metabolic engineering

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Crigler, Jacob
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Middle Tennessee State University
Cellulosic ethanol biofuel, made from plant waste products or perennial energy crops like switchgrass, offers many advantages over corn starch-derived ethanol, including less land competition and a lower carbon footprint. However, the efficiency of conversion is currently lower and the cost higher due to the recalcitrance of lignocellulosic biomass. A chemical and/or physical pretreatment step is required to overcome recalcitrance, and common pretreatment methods (e.g., acid, steam, and/or depressurization) release microbial inhibitors, including furfural and acetate, into the hydrolysate, lowering the yield of ethanol via fermentation. Furfural is generated via sugar dehydration, and acetate derives from acetylated xylan in hemicellulose. Utilizing a detoxifying strain is one strategy to overcome the inhibitor dilemma. Biological detoxification potentially allows lower process costs compared to chemical or enzymatic detoxification or alternative pretreatment methods aimed at reducing inhibitor generation. One drawback is time. Thus increasing the rate of furfural and acetate detoxification is desirable. While most microbial species can catabolize acetate, most do not possess the furfural catabolic pathway. A novel Pseudomonas putida isolate ALS1267, with a growth rate of 0.25/h in 10 mM minimal furfural medium, was characterized. The genome was sequenced and the furfural pathway cloned into wild-type P. putida KT2400, which cannot metabolize furfural, creating a novel strain with an improved growth rate of 0.34/h in 10 mM minimal furfural medium. Genomic library screening was used to find targets for engineering increased acetate consumption in Escherichia coli. Sixteen plasmid clones were generated, with growth rate increases of 42.6 to 76.9 percent in 10 g/l minimal acetate medium, which is a highly inhibitory concentration for this strain. Clones included an uncharacterized oxidoreductase, transporters and other outer membrane proteins, carbon scavengers, and stress defense mechanisms. Genomic mutants with improved acetate consumption were also generated during selection, with mutations in the gluconeogenesis gene pck promoter, the '5 UTR of the poorly characterized cold-shock gene ynaE, and the global regulator of secondary carbon sources CRP. The second major drawback to biological detoxification is consumption of the sugars to be used to produce ethanol by the detoxifying strain. Elimination of glucose metabolism in E. coli was studied by characterizing fast-growing revertants in strains engineered to be glucose minus. All of the revertants either altered or overproduced the N-acetylglucosamine phosphotransferase system. Deletion of the N-acetylglucosamine transporter stabilized the glucose minus phenotype and prevented the occurrence of fast-growing revertants.