Directed evolution using genetically diverse libraries is integral to advancing research in industrial microbial production and protein functionality enhancement. This process typically involves a step of sequence diversification and subsequent selection/screening steps for improved variants. While CRISPR-Cas9 systems are known to offer efficient and targeted modification of genes in vivo, concerns arise regarding off-target effects and the emergence of escaper cells evading Cas9 cleavage. This study investigated a strategy to leverage CRISPR-Cas9 counter-selection in Escherichia coli for targeted chromosomal mutagenesis. By designing gRNAs to target a desired region, the spontaneous mutations occurring at the targeted region will potentially disrupt Cas9 binding and thus allow the cell to avoid death caused by Cas9-induced double-stranded DNA breaks. This population of ‘escaper’ cells surviving the counter-selection will have mutations in the gRNA-targeting region at a higher frequency than their non-escaper counterparts. To optimize this counter-selection method, the design for the CRISPR-Cas9 expression system was improved, Cas9 variants with varied fidelities and activities were investigated, and the strategy of using truncated gRNAs for enhanced mutation selectivity was explored. Using the E. coli rpoB gene as a target for editing, the rifampicin-resistant mutation (caused by mutations in rpoB) frequency was increased by more than five orders of magnitude compared to the control E. coli strain without CRISPR targeting. Nanopore DNA sequencing of the mutants’ rpoB region confirmed the promising targeting efficacy of this approach. This study demonstrates a streamlined method for targeted genetic diversification in vivo, facilitating efficient protein engineering in bacterial systems.

As the utilization of tyrosine is needed by both eukaryotes and prokaryotes, this versatile amino acid contributes towards a variety of operations including protein synthesis, pigment production, and host or habitat impacting metabolite creation. While there are numerous pathways which involve the degradation of tyrosine to create different products, the one that is central in this thesis is a pathway with homogentisate as an intermediate. This pathway brings an interest due to its association with metabolic disorders like Tyrosinemia (I, II, or III), and its impact within an agricultural environment. In other words, for humans and plant microbiomes to maintain their optimal metabolic homeostasis, tyrosine is required to participate in numerous demands. This necessity can ultimately create competition between organisms present in microbial communities, as there are a multitude of species that can metabolize tyrosine for the creation of diverse products. In this work, a primary objective is to characterize the breakdown of tyrosine within a competitive environment where there are multiple available pathways. There are many factors that could influence the catabolism of tyrosine like catalytic efficiency of enzymes, availability of breakdown routes, and pathway regulations. Here, the start will be creating a proof of concept developed by studying the competition for tyrosine utilization by environmental microbial enzymes; 4-hydroxyphenylpyruvate dioxygenase from Streptomyces avermitilis, 4-hydroxymandelate synthase from Amycolatopsis orientalis, and tyrosine ammonia lyase from Flavobacterium johnsoniae. Through phenotypic assays and by quantifying secreted metabolites, rerouting of this pathway is observed. This insight towards the ability of diverting the homogentisate pathway was then utilized for the analysis of contest between human enzyme, 4-hydroxyphenylpyruvate dioxygenase, and gut microbial enzyme, tyrosine ammonia lyase from Bacteroides ovatus. Within both aims it is seen that due to successful diversion of the pathway, there is a reduction in tyrosine with the formation of more favorable products. The strategy of redirecting this tyrosine catabolism pathway will provide baseline knowledge for future efforts to contribute towards alternative methods of intervention to alleviate the burdens from tyrosine metabolic dysfunction and disorders.

Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and hydroxyl radicals occur naturally as a byproduct of aerobic respiration. To mitigate damages caused by ROS, Escherichia coli employs defenses including two cytosolic superoxide dismutases (SODs), which convert superoxide to hydrogen peroxide. Deletion of both sodA and sodB, the genes coding for the cytosolic SOD enzymes, results in a strain that is unable to grow on minimal medium without amino acid supplementation. Additionally, deletion of both cytosolic SOD enzymes in a background containing the relA1 allele, an inactive version of the relA gene that contributes to activation of stringent response by amino acid starvation, results in a strain that is unable to grow aerobically, even on rich medium. These observations point to a relationship between the stringent response and oxidative stress. To gain insight into this relationship, suppressors were isolated by growing the ∆sodAB relA1 cells aerobically on rich medium, and seven suppressors were further examined to characterize distinct colony sizes and temperature sensitivity phenotypes. In three of these suppressor-containing strains, the relA1 allele was successfully replaced by the wild type relA allele to allow further study in aerobic conditions. None of those three suppressors were found to increase tolerance to exogenous superoxides produced by paraquat, which shows that these mutations only overcome the superoxide buildup that naturally occurs from deletion of SODs. Because each of these suppressors had unique phenotypes, it is likely that they confer tolerance to SOD-dependent superoxide buildup by different mechanisms. Two of these three suppressors have been sent for whole-genome sequencing to identify the location of the suppressor mutation and determine the mechanism by which they confer superoxide tolerance.

The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.

Polymers have played a pivotal role in building modern society. Polymers can be classified as synthetic and natural polymers. Accumulation of both synthetic and natural polymer waste leads to environmental pollution. This dissertation aims at developing one-pot bioprocesses for a breakdown of natural polymers like cellulose, and hemicellulose and synthetic polymers like polyethylene terephthalate (PET). First, a one-pot process was developed for hemicellulose breakdown. A signal peptide library of native SEC pathway signal peptides was developed for efficient secretion of endoxylanse enzyme. Furthermore, in situ, the process was successfully created for hemicellulose to xylose with the highest reported xylose titer of 7.1 g/L. In addition, E. coli: B. subtilis coculture bioprocess was developed to produce succinate, ethanol, and lactate from hemicellulose in one pot process. Second, a one-pot process was developed for cellulose breakdown. In vitro enzyme assays were used to select SEC pathway signal peptides for endoglucanase and glucosidase secretion. Then, the breakdown of carboxymethyl cellulose (CMC), a cellulose derivative, was conducted in in situ conditions. U-13C fingerprinting study showed carbon enrichment from CMC when cultures were cofed with CMC and [U-13C] glucose. Further, Whatman filter paper sheets showed a change in shape in recombinant cocultures. SEM images showed continuous orientation in the case of two enzymes confirmed by fast Fourier transform (FFT), suggesting higher crystallinity of residues. Similarly, in microcrystalline cellulose breakdown in in situ conditions, a 72% reduction of avicel cellulose was achieved in a one pot bioprocess. SEM images revealed valleys and crevices on residues of coculture compared to smoother surfaces in monoculture residues pressing the importance of the synergistic activity of enzymes. Finally, one pot deconstruction process was developed for synthetic polymer PET. First, the PET hydrolase secretion strain was developed by selecting a signal peptide library. The first bis(2-hydroxyethyl) terephthalate (BHET) consolidated bioprocess was developed, which produced a terephthalic acid titer of 7.4 g/L. PET breakdown was successfully demonstrated in in vitro conditions with a TPA titer of 4 g/L. Furthermore, PET breakdown was successfully demonstrated in in situ conditions. Consolidated bioprocesses can be an invaluable approach to waste utilization and making cost-effective processes.

In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA concentrations, including HPLC-based and GC-based methods or enzyme-based kits, have hindered research advancement due to their laborious and/or expensive nature. The work herein establishes a novel, rapid, fluorescence-based assay for detecting total FFA concentrations secreted by Synechocystis FFA secretion strains. The novel FFA-detection assay demonstrates the efficacy of using Nile Red as a fluorescent reporter for laurate or palmitate at concentrations up to 500 µM in the presence of cationic surfactants. Total FFA concentrations in Synechocystis supernatants quantified by the novel, Nile Red fluorescence-based assay are demonstrated herein to be highly correlative to total FFA concentrations quantified by LC-MS; this correlation was seen in supernatant samples of wild type Synechocystis and Synechocystis FFA secretion strains, both in 96-well plates and 30-mL, aerated culture tubes. This work also establishes the expression of a cytochrome P450 fusion enzyme, CYP153A-CPRmut, or a monooxygenase system from Pseudomonas putida GPo1, AlkBGT, in FFA secretion strains of Synechocystis for the generation of ω-hydroxy laurate from laurate. After finding greatly increased ω-hydroxylation activity of CYP153A-CPRmut with concurrent superoxide dismutase and catalase overexpression, 55 or 1.5 µM of ω-hydroxy laurate were produced over five days by Synechocystis strains expressing CYP153A-CPRmut or AlkBGT, respectively. As further indication of the presence of reactive oxygen species affecting ω-hydroxy laurate production with Synechocystis strains expressing CYP153A-CPRmut, concentrations of ω-hydroxy laurate in the supernatant increased over two-fold in the presence of 250 µM of the anti-oxidant, methionine, in bench-scale cultures and in 96-well plate cultures. Additionally, a mutation at the 55th amino acid position in AlkB (tryptophan to cysteine; AlkBW55C), resulted in a more than two-fold shift in AlkB’s substrate preference from decanoate towards the desired substrate, laurate. As a result, Synechocystis expressing AlkBW55C could produce 5.9 µM ω-hydroxy laurate and 2.0 µM dodecanedioic acid over five days of growth.

I studied the molecular mechanisms of ultraviolet radiation mitigation (UVR) in the terrestrial cyanobacterium Nostoc punctiforme ATCC 29133, which produces the indole-alkaloid sunscreen scytonemin and differentiates into motile filaments (hormogonia). While the early stages of scytonemin biosynthesis were known, the late stages were not. Gene deletion mutants were interrogated by metabolite analyses and confocal microscopy, demonstrating that the ebo gene cluster, was not only required for scytonemin biosynthesis, but was involved in the export of scytonemin monomers to the periplasm. Further, the product of gene scyE was also exported to the periplasm where it was responsible for terminal oxidative dimerization of the monomers. These results opened questions regarding the functional universality of the ebo cluster. To probe if it could play a similar role in organisms other than scytonemin producing cyanobacteria, I developed a bioinformatic pipeline (Functional Landscape And Neighbor Determining gEnomic Region Search; FLANDERS) and used it to scrutinize the neighboring regions of the ebo gene cluster in 90 different bacterial genomes for potentially informational features. Aside from the scytonemin operon and the edb cluster of Pseudomonas spp., responsible for nematode repellence, no known clusters were identified in genomic ebo neighbors, but many of the ebo adjacent regions were enriched in signal peptides for export, indicating a general functional connection between the ebo cluster and biosynthetic compartmentalization. Lastly, I investigated the regulatory span of the two-component regulator of the scytonemin operon (scyTCR) using RNAseq of scyTCR deletion mutants under UV induction. Surprisingly, the knockouts had decreased expression levels in many of the genes involved in hormogonia differentiation and in a putative multigene regulatory element, hcyA-D. This suggested that UV could be a cue for developmental motility responses in Nostoc, which I could confirm phenotypically. In fact, UV-A simultaneously elicited hormogonia differentiation and scytonemin production throughout a genetically homogenous population. I show through mutant analyses that the partner-switching mechanism coded for by hcyA-D acts as a hinge between the scytonemin and hormogonia based responses. Collectively, this dissertation contributes to the understanding of microbial adaptive responses to environmental stressors at the genetic and regulatory level, highlighting their phenomenological and mechanistic complexity.

Following the journey through the sewerage system, wastewater is subject to a series of purification procedures, prior to water reuse and disposal of the resultant sewage sludge. Biosolids, also known as treated sewage sludge, deemed fit for application on land, is a nutrient-rich, semisolid byproduct of biological wastewater treatment. Technological progression in metagenomics has allowed for large-scale analysis of complex viral communities in a number of samples, including wastewater. Members of the Microviridae family are non-enveloped, ssDNA bacteriophages, and are known to infect enterobacteria. Members of the Genomoviridae family similarly are non-enveloped, ssDNA viruses, but are presumed to infect fungi rather than eubacteria. As these two families of viruses are not relatively documented and their diversity poorly classified, this study aimed to analyze the presence of genomoviruses and the diversity of microviruses in nine samples representative of wastewater in Arizona and other regions of the United States. Using a metagenomic approach, the nucleic acids of genomoviruses and microviruses were isolated, assembled into complete genomes, and characterized through visual analysis: a heat chart showing percent coverage for genomoviruses and a circular phylogenetic tree showing diversity of microviruses. The heat map results for the genomoviruses showed a large presence of 99 novel sequences in all nine wastewater samples. Additionally, the 535 novel microviruses displayed great diversity in the cladogram, both in terms of sub-family and isolation source. Further research should be conducted in order to classify the taxonomy of microviruses and the diversity of genomoviruses. Finally, this study suggests future exploration of the viral host, prior to entering the wastewater system.

Each year, more and more multi-drug resistant bacterial strains emerge, thus complicating treatment and increasing the average stay in the intensive care unit. As antibiotics are being rendered inefficient, there is a need to look into ways of weakening the internal state of bacterial cells to make them more susceptible to antibiotics. For this, we first need to understand what methods bacteria employ to fight against antibiotics. In this work, we have reviewed how bacteria respond to antibiotics. There is a similarity in response to antibiotic exposure and starvation (stringent stress) which changes the metabolic state. We have delineated what metabolism changes take place and how they are associated with oxidative stress. For example, there is a common change in NADH concentration that is tied to both metabolism and oxidative stress. Finally, we have compared the findings in literature with our research on an antibiotic-resistant RNA polymerase mutant that alters the gene expression profile in the general areas of metabolism and oxidative stress. Based on this thesis, we have suggested a couple of strategies to make antibiotics more efficient; however, as antibiotic-mediated killing is very complex, researchers need to delve deeper to understand and manipulate the full cellular response.

Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.