Phenolic polymers like polyphenols and polyphenylenes have several industrial applications including electrical insulation, specialty membranes, and packings but are typically synthesized under harsh reaction conditions and require hazardous chemicals like formaldehyde. Hydroxycinnamic acids, such as p-coumaric acid (p-CA), are aromatic derivatives of lignin hydrolysates, an underutilized and promising renewable feedstock for production of phenolics and phenolic polymers. Recently a strain of Corynebacterium glutamicum has been created by the Joint BioEnergy Institute (JBEI) which expresses phenolic acid decarboxylase (PAD), an enzyme which catalyzes the reaction of p-CA to 4-vinylphenol (4-VP). Further, a deletion of the phdA gene prevents assimilation of p-CA, thereby increasing 4-VP yield. 4-VP is a substituted phenol which can be polymerized to poly(4-vinylphenol) (PVP) in the presence of ligninolytic enzymes like laccases or peroxidases. This work explores in situ polymerization of 4-VP to PVP by supplementing ligninolytic enzymes during fermentation. Cultured in the presence of p-CA, the engineered C. glutamicum strain achieved a maximum 4-VP yield of 45.2%, 57.9%, and 34.7% when fed 2, 5, and 10 g/L p-CA, respectively. Low yield can be attributed to photodegradation of 4-VP and accumulation of the native laccase present in C. glutamicum which may form only dimers and trimers. To further investigate carbon utilization in the cell, the engineered strain was plasmid cured thus removing the PAD enzyme and fermentations for 13C pathway analysis was performed. Polymerization experiments were performed and the polymer was characterized using GPC.

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.

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.

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

Cyanobacteria and its complex photosynthetic systems have been a prime target for synthetic biologists and their molecular engineering tools for the last couple of decades. However, characterizing meaningful carbon dioxide (CO₂) removal performance has always been a struggle within the field. It is proposed that measuring changes in CO₂ gas concentration within a dynamic system can be accomplished with a simple automated Arduino-powered system. The system employs solenoids in parallel (one for each outlet stream) which are then connected to one large manifold which feeds into a single IR-based CO₂ probe. Since CO₂ probes are expensive, this approach allows for sample multiplexing while remaining affordable. The development of such a system allows for high resolution growth experiments between different strains of cyanobacteria. This approach provides continuous data collection over the entire life cycle of each individual culture, allowing differences in total CO₂ fixation between strains to be readily determined. From a culture of PCC 6803, it was found that the peak mg of CO₂ fixed per day is around 92 mg CO₂/day. In the future, the system can be modified to fit other simple dynamic gas systems, as well as testing similar gas utilization/production capabilities of other organisms.

The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular biological factories with individualized subunits that function in concert to create novel peptides.One element at the heart of environmental health debates today is plastics. Biodegradable alternatives for petroleum-based plastics is a necessity. One NRPS, cyanophycin synthetase (CphA), can produce cyanophycin grana protein (CGP), a polymer composed of a poly-aspartic acid backbone with arginine side chains. The aspartic backbone has the potential to replace synthetic polyacrylate, although current production costs are prohibitive. In Chapter 2, a CphA variant from Tatumella morbirosei is characterized, that produces up to 3x more CGP than other known variants, and shows high iCGP specificity in both flask and bioreactor trials. Another CphA variant, this one from Acinetobacter baylyi, underwent rational protein design to create novel mutants. One, G217K, is 34% more productive than the wild type, while G163K produces a CGP with shorter chain lengths. The current structure refined from 4.4Å to 3.5Å. Another exciting application of NRPSs is in healthcare. They can be used to generate novel peptides such as complex antibiotics. A recently discovered iterative polyketide synthase (IPTK), dubbed AlnB, produces an antibiotic called allenomycin. One of the modular subunits, a dehydratase named AlnB_DH, was crystallized to 2.45Å. Several mutations were created in multiple active site residues to help understand the functional mechanism of AlnB_DH. A preliminary holoenzyme AlnB structure at 3.8Å was generated although the large disorganized regions demonstrated an incomplete structure. It was found that chain length is the primary factor in driving dehydratase action within AlnB_DH, which helps lend understanding to this module.

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.

In this project, the potential of ferrous iron precipitation as an alternative for ground improvement applications is investigated. This study analyzes the potential of naturally occurring iron oxidation, which uses Fe2+ as an electron donor to produce Fe3+ precipitate. The goal of this study was to stimulate or accelerate the naturally occurring iron oxidation and precipitation process, to form a ferruginous crust in the subsurface, that would reduce hydraulic conductivity or increase soil strength. Iron precipitation can occur through aerobic or anaerobic iron oxidizers. Initial experimental test results in falcon tubes and a literature review showed that to obtain significant oxidation of ferrous iron and consequent precipitation of iron minerals required a buffer to prevent acidification. Experimental studies in which aerobic and anaerobic iron precipitation is stimulated in sand columns under various boundary conditions also leads to an optimization of conditions for mineralization. Mineralized zones are evaluated via permeability loss tests, extent of iron oxidized and characterization tests which show that the crust has the most concentration of precipitated iron, which can be used in targeting pollution mitigation, erosion control, etc. The results show a significant loss of permeability- by a factor of two, in high concentration of iron with a balanced buffer control. In this study, the knowledge on ground stabilization by studying the naturally occurring mechanism of iron precipitation, leading to possible industrially relevant geotechnical applications are successfully investigated.