The recently discovered fast growing cyanobacterium Synechococcus sp. PCC 11901 has high industrial potential due to its quick doubling time, ability to grow on various carbon substrates and unique metabolism. Since its discovery, little work has been done to model the metabolic pathways present in the organism. In order to accurately model such an organism, experimentally determined steady state biomass flux constraints are necessary. These constraints will influence the design of a flux balance analysis model & provide realistic restrictions on the model’s outputs. The construction of such a metabolic model will assist metabolic engineers in their genetic design. By modeling the thousands of reactions and each metabolite present in the organism, engineers can gain deep insights into the complex nature of metabolism. By designing new reaction pathways, and changing the model, metabolic engineers can use this work to predict the result of various genetic manipulations on the organism. This serves as the experimental basis for building such a model.

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

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.

Phenolic polymers such as polyphenols and polyphenylenes are generated industrially for several applications but are typically associated with harsh reaction conditions and environmentally hazardous chemicals, such as formaldehyde. Additionally, hydroxycinnamic acids, such as p-coumaric acid (CA), are found in high concentrations in underutilized lignin-derived hydrolysates and represent a renewable and sustainable feedstock for the production of various aromatics and phenolics. To that end, recently a strain of Corynebacterium glutamicum has been developed by the Joint Bioenergy Institute to express a Phenolic Acid Decarboxylase (PAD), which can convert CA into 4-vinylphenol (4VP). 4VP is cytotoxic but can be polymerized by ligninolytic enzymes such as laccases or peroxidases into less-toxic poly(4-vinylphenol) (PVP). This work investigates the potential of polymerizing 4VP in situ by adding ligninolytic enzymes into the fermentation media to polymerize 4VP into PVP as it is produced, while reducing cellular toxicity to aid in chemical conversion. The engineered C. glutamicum strain was cultured in the presence of CA to produce 4VP, with a maximum yield of 80.75%. Simultaneously, two ligninolytic enzymes, laccase and horseradish peroxidase (HRP), were explored in an in vitro experiment for their ability to polymerize 4VP, with laccase achieving full polymerization within 45 minutes and HRP able to polymerize 54.06% of 4VP in 24 hours. The resulting polymers were further analyzed by using gas permeation chromatography - nuclear magnetic resonance, validating the synthesis of PVP from 4VP with the addition of laccase or HRP. Finally, the C. glutamicum strain was evaluated for its ability to grow in the presence of hydrogen peroxide, which is a necessary reagent for HRP functionality, and it was able to reach an optical density of 3.69 within 36 hours. These findings suggest that in situ polymerization may be possible. Further work is underway to explore the enzyme kinetics at different pH, validate the potential of polymerization in situ, and study the fermentative benefits associated with in situ polymerization. This will be followed by additional analytical studies to characterize the resulting PVP.

The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as PET, constitutes a large percentage of the waste that ends up in landfills. Material and chemical/thermal methods for recycling are both costly, and inefficient, which necessitates a more sustainable and cheaper alternative. The current study aims at fulfilling that role through genetic engineering of Bacillus subtilis with integration of genes from LCC, Ideonella sakaiensis, and Bacillus subtilis. The plasmid construction was done through restriction cloning. A recombinant plasmid for the expression of LCC was constructed, and transformed into Escherichia coli. Future experiments for this study should include redesigning of primers, with possible combination of signal peptides with genes during construct design, and more advanced assays for effective outcomes.

The development of Corynebacterium glutamicum for the microbial production of high-value products has made this bacterium an industrial workhorse. This metabolically engineered microbe is capable of accumulating and secreting flavonoids, a class of high functioning compounds found in plants. In human health, flavonoids are known to have powerful antioxidant, anti-inflammatory, anticancer, and antiviral properties which has led the growing interest to produce these compounds commercially. Recent literature seeks to overcome potential pathway bottlenecks to optimize flavonoid production by regulating protein expression within the central carbon, shikimate, chorismate, and fatty acid synthesis pathways. This paper reviews engineering strategies performed to increase the precursor titers of malonyl-CoA, phenylalanine, and tyrosine for increased flavonoid production.

Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.