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- Genre: Doctoral Dissertation
Description
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background.
ContributorsLeiser, Owen Paul (Author) / Misra, Rajeev (Thesis advisor) / Jacobs, Bertram (Committee member) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2010