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.

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.

Heterotrophs such as E. coli contain metabolic pathways with enzymes called carboxylases that are capable of fixing CO2 gas to form metabolites, which has implications for aiding with CO2’s role in climate change. The reductive branch of the tricarboxylic acid (TCA) cycle serves as an important pathway for NAD+ regeneration in enteric bacteria in anaerobic conditions and leads to the production of succinate, a useful industrial product. The enzyme phosphoenolpyruvate carboxykinase is responsible for fixing CO2 in the conversion of PEP to OAA within this pathway and has potential to be a significant carbon fixation module in heterotrophic organisms. This project explored pck genes from select organisms by transforming plasmids to test if these variants have improved kinetics compared to the native E. coli Pck and to investigate their ability to improve succinate bioproduction.

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.

Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis. However, to create industrially competitive strains to efficiently convert lignocellulose to high-value chemicals, current challenges must be addressed. Redox constraints, allosteric regulation, and transport-related limitations are important bottlenecks limiting the commercial production of renewable chemicals from lignocellulose. Advances in metabolic engineering techniques have enabled researchers to engineer microbial strains that overcome some of these challenges but new approaches that facilitate the commercial viability of lignocellulose valorization are needed. Biological systems are complex with a plethora of regulatory systems that must be carefully modulated to efficiently produce and excrete the desired metabolites. In this work, I explore metabolic engineering strategies to address some of the biological constraints limiting bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective lignocellulose bioconversion.

Environmentally harmful byproducts from solid waste’s decomposition, including methane (CH4) emissions, are managed through standardized landfill engineering and gas-capture mechanisms. Yet only a limited number of studies have analyzed the development and composition of Bacteria and Archaea involved in CH4 production from landfills. The objectives of this research were to compare microbiomes and bioactivity from CH4-producing communities in contrasting spatial areas of arid landfills and to tests a new technology to biostimulate CH4 production (methanogenesis) from solid waste under dynamic environmental conditions controlled in the laboratory. My hypothesis was that the diversity and abundance of methanogenic Archaea in municipal solid waste (MSW), or its leachate, play an important role on CH4 production partially attributed to the group’s wide hydrogen (H2) consumption capabilities. I tested this hypothesis by conducting complementary field observations and laboratory experiments. I describe niches of methanogenic Archaea in MSW leachate across defined areas within a single landfill, while demonstrating functional H2-dependent activity. To alleviate limited H2 bioavailability encountered in-situ, I present biostimulant feasibility and proof-of-concepts studies through the amendment of zero valent metals (ZVMs). My results demonstrate that older-aged MSW was minimally biostimulated for greater CH4 production relative to a control when exposed to iron (Fe0) or manganese (Mn0), due to highly discernable traits of soluble carbon, nitrogen, and unidentified fluorophores found in water extracts between young and old aged, starting MSW. Acetate and inhibitory H2 partial pressures accumulated in microcosms containing old-aged MSW. In a final experiment, repeated amendments of ZVMs to MSW in a 600 day mesocosm experiment mediated significantly higher CH4 concentrations and yields during the first of three ZVM injections. Fe0 and Mn0 experimental treatments at mesocosm-scale also highlighted accelerated development of seemingly important, but elusive Archaea including Methanobacteriaceae, a methane-producing family that is found in diverse environments. Also, prokaryotic classes including Candidatus Bathyarchaeota, an uncultured group commonly found in carbon-rich ecosystems, and Clostridia; All three taxa I identified as highly predictive in the time-dependent progression of MSW decomposition. Altogether, my experiments demonstrate the importance of H2 bioavailability on CH4 production and the consistent development of Methanobacteriaceae in productive MSW microbiomes.

Single and double deletion strains of Escherichia coli were grown in paired co-cultures with an intent to identify examples of metabolite exchange and cooperative interactions between strains. The essential genes pheA, argA, tyrA, and trpC, as well as the non- essential genes pykF, pykA, mdh, ppc, and nuoN were deleted from Escherichia coli strains Bw25113 and ATCC 9637. Cultures were paired at three different initial ratios and grown at plate and flask scale. Optical density measurements were used to observe the performance of tested co-cultures, with changes in maximum optical density and growth rate used as indicators of interaction or lack thereof between tested pairs. Auxotrophic strains unable to produce essential amino acids were observed to grow in co-culture but not in monoculture, indicative of metabolite exchange facilitating growth. An increase in optical density for non-essential pairs when compared to the prototrophic parent and precursor monocultures was indicative of metabolite exchange. The initial frequency of paired mutants with non-essential deletions appeared to have an impact on growth performance, but whether this was indicative of any beneficial exchange was not able to be determined from data.

The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a sustainable future. One potential option is the utilization of metabolically engineered bacteria to produce value-added chemicals during fermentation. Currently, numerous strains of metabolically engineered Escherichia coli have shown great capacity to specialize in the production of high titers of a desired chemical. These metabolic systems, however, are constrained by the biological limits of E. coli itself. During fermentation, E. coli grows to less than one twentieth of the density that aerobically growing cultures can reach. I hypothesized that this decrease in growth during fermentation is due to cellular stress associated with fermentative growth, likely caused by stress related genes. These genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress response, may have an impact on fermentative growth constraints. Through transcriptional analysis, I identified that the genes pspC and relE are highly expressed in fermenting strains of both wild type and metabolically engineered E. coli. Fermentation of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX, yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ, yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN significantly arrested cell growth in both tube and fermentation tests, thus proving these toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli fermentation

The purpose behind this research was to identify unknown transport proteins involved in lactate export. Lactate bioproduction is an environmentally beneficial alternative to petroleum-based plastic production as it produces less toxic waste byproduct and can rely on microbial degradation of otherwise wasted biomass. Coupled with appropriate product refinement, industrial microbial producers can be genetically engineered to generate quantities of bioplastic approaching 400 million metric tons each year. However, this process is not entirely suitable for large investment, as the fermentative bottlenecks, including product export and homeostasis control, limit production metrics. Previous studies have based their efforts on enhancing cellular machinery, but there remain uncharacterized membrane proteins involved in product export yet to be determined. It has been seen that deletion of known lactate transporters in Escherichia coli resulted in a decrease in lactate production, unlike the expected inhibition of export. This indicates that there exist membrane proteins with the ability to export lactate which may have another similar substrate it primarily transports.To identify these proteins, I constructed a genomic library of all genes in an engineered lactate producing E. coli strain, with known transporter genes deleted, and systematically screened for potential lactate transporter proteins. Plasmids and their isolated proteins were compared utilizing anaerobic plating to identify genes through sanger sequencing. With this method, I identified two proteins, yiaN and ybhL-ybhM, which did not show any significant improvement in lactate production when tested. Attempts were made to improve library diversity, resulting in isopropyl-β-D-1-thiogalactopyranoside induction as a likely factor for increased expression of potential fermentation-associated proteins. A genomic library from Lactobacillus plantarum was constructed and screened for transport proteins which could improve lactate production. Results showed that isolated plasmids contained no notable inserts, indicating that the initial transformation limited diversity. Lastly, I compared the results from genomic screening with overexpression of target transporter genes by computational substrate similarity search. Induced expression of ttdT, citT and dcuA together significantly increased lactate export and thus production metrics as well as cell growth. These positive results indicate an effective means of determining substrate promiscuity in membrane proteins with similar organic acid transport capacity.