Metabolic engineering has emerged as a highly effective approach to optimizing industrial fermentation processes by introducing purposeful genetic alterations using recombinant DNA technology. Successful metabolic engineering begins with a careful investigation of cellular function, and based on the outcomes of this analysis, an improved strain is created and then constructed using genetic engineering. By modifying the genetic makeup of cells, can increase the production of important chemicals, biofuels, medications, and agricultural products. The most often used genetic engineering tool is plasmid-based gene editing. In plasmid-based gene editing, the desired gene sequence is flanked by similar genome sequences, which encourages the foreign gene's integration into the genome. The main flaw of plasmid-based editing is the presence of selectable markers in the integrated DNA, which impacts cell stability as well as downstream applications that are critical to industries. Recently, with the growth of science, the new gene-editing technology CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the field of gene editing. It has been used to incorporate the foreign genes into the genome of the microbial host without any mark and has more efficiency than the plasmid-based gene editing technique. CRISPR is utilized to achieve markerless integration of genes in genomes of microbes, which promotes cell stability and is also especially beneficial for applications in industries. In this experiment successfully integrated two genes into the genome of C.glutamicum employing markerless integration via homologous recombination, allowing cells to metabolize acetate into acetyl-CoA and improve the conversion of pyruvate into lactate. Further, this strain of C.glutamicum can be utilized as a platform for producing ethyl lactate, a green solvent using a microbial host

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.

Clustered regularly interspace short palindromic repeats (CRISPR) and CRISPR associated (Cas) technologies have become integral to genome editing. Canonical CRISPR-Cas9 systems function as a ribonucleic acid (RNA)-guided nucleases. Single guide RNAs (sgRNA) can be easily designed to target Cas9’s nuclease activity towards protospacer deoxyribonucleic acid (DNA) sequences. The relatively ease and efficiency of CRISPR-Cas9 systems has enabled numerous technologies and DNA manipulations. Genome engineering in human cell lines is centered around the study of genetic contribution to disease phenotypes. However, canonical CRISPR-Cas9 systems are largely reliant on double stranded DNA breaks (DSBs). DSBs can induce unintended genomic changes including deletions and complex rearrangements. Likewise, DSBs can induce apoptosis and cell cycle arrest confounding applications of Cas9-based systems for disease modeling. Base editors are a novel class of nicking Cas9 engineered with a cytidine or adenosine deaminase. Base editors can install single letter DNA edits without DSBs. However, detecting single letter DNA edits is cumbersome, requiring onerous DNA isolation and sequencing, hampering experimental throughput. This document describes the creation of a fluorescent reporter system to detect Cytosine-to-Thymine (C-to-T) base editing. The fluorescent reporter utilizes an engineered blue fluorescent protein (BFP) that is converted to green fluorescent protein (GFP) upon targeted C-to-T conversion. The BFP-to-GFP conversion enables the creation of a strategy to isolate edited cell populations, termed Transient Reporter for Editing Enrichment (TREE). TREE increases the ease of optimizing base editor designs and assists in editing cell types recalcitrant to DNA editing. More recently, Prime editing has been demonstrated to introduce user defined DNA edits without the need for DSBs and donor DNA. Prime editing requires specialized prime editing guide RNAs (pegRNAs). pegRNAs are however difficult to manually design. This document describes the creation of a software tool: Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE). PINE-CONE rapidly designs pegRNAs based off basic edit information and will assist with synthetic biology and biomedical research.

Alkanolamines are useful as building blocks for a variety of applications, ranging from medical applications such as drug and gene delivery. In this work, Escherichia coli was investigated as a viable candidate for the production of 5-amino-1-pentanol (5-AP). Taking advantage of the existing L-lysine degradation pathway, a novel route to 5-AP was constructed by co-expressing the genes cadA (encoding lysine decarboxylase, responsible for the conversion of L-lysine to cadaverine) and patA (encoding putrescine aminotransferase, responsible for the conversion of cadaverine to 5-amino-1-pentanal), followed by the endogenous reduction of 5-amino-pentanal (5-APL) to 5-AP. To avoid the competing conversion of 5-APL to 5-amino-1-pentanoate and avoid accumulation of byproduct 1-Δ-piperideine, further host engineering was performed to delete the gene patD also known as prr (encoding 5-amino-pentanal dehydrogenase). Flask scale fermentation experiments in minimal medium of the newly constructed pathway was conducted where 62.6 mg/L 5-AP was observed to be produced. It was hypothesized that 5-AP production could be boosted by optimizing production medium to M10 media. However, change in the culture medium resulted in the production of just 51 mg/L 5-AP. Shifts observed in HPLC chromatogram peaks made it difficult to conclude exact titers of 5-AP and can be further improved by exploring different analysis methods and optimization of the method currently in place.

Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Recent efforts have sought to supplant these sugar-intensive processes through the metabolic engineering of cyanobacteria, which instead can directly utilize atmospheric carbon dioxide (CO2) and sunlight. One of the most promising among recently discovered photoautotrophic strains is Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973), which has been reported to have doubling times as low as 1.5 hours. While encouraging, there are still major challenges preventing the widespread industrial acceptance of engineered cyanobacteria, chief among them is the scarcity of genetic tools and parts with which to engineer production strains. Here, UTEX 2973 was engineered to overproduce L-lysine through the heterologous expression of feedback-resistant copies of aspartokinase lysC and the L-lysine exporter ybjE from Escherichia coli, as aided by the characterization of novel combinations of genetic parts and expression sites. At maximum, using a plasmid-based expression system, a L-lysine titer of 556 ± 62.3 mg/L was attained after 120 hours, surpassing a prior report of photoautotrophic L-lysine bioproduction. Modular extension of the pathway then led to the novel photosynthetic production of the corresponding diamine cadaverine (55.3 ± 6.7 mg/L by 96 hours) and dicarboxylate glutarate (67.5 ± 2.2 mg/L by 96 hours). Lastly, mass transfer experiments were carried out to determine if the solubility of CO2 in and its rate of mass transfer to BG-11 media could be improved by supplementing it with various amines, including cadaverine. Ultimately, however, cyanobacteria grown in the presence of all tested amines was worse than in BG-11 alone, demonstrating the need for additional tolerance engineering to successfully implement this strategy.

Cyanobacteria contribute to more than a quarter of the primary carbon fixation worldwide. They have evolved a CO2 concentrating mechanism (CCM) to enhance photosynthesis because inorganic carbon species are limited in the aqueous environment. Bicarbonate transporters SbtA and BicA are active components of CCM, and the determination of their structures is important to investigate the bicarbonate transport mechanisms. E. coli was selected as the expression host for these bicarbonate transporters, and optimization of expression and protein purification conditions was performed. Single particle electron cryomicroscopy (cryo-EM) or protein crystallography was carried out for each transporter. In this work, SbtA, BicA and SbtB, a regulator protein of SbtA, were heterologously expressed in E. coli and purified for structural studies. SbtB was highly expressed and two different crystal structures of SbtB were resolved at 2.01 Å and 1.8 Å, showing a trimer and dimer in the asymmetric unit, respectively. The yields of SbtA and BicA after purification reached 0.1 ± 0.04 and 6.5 ± 1.0 mg per liter culture, respectively. Single particle analysis showed a trimeric conformation of purified SbtA and promising interaction between SbtA and SbtB, where the bound SbtB was also possibly trimeric. For some crystallization experiments of these transporters, lipidic cubic phase (LCP) was used. In the case of LCP, often times the crystals grown are generally too tiny to withstand radiation damage from the X-ray beam during an X-ray diffraction experiment. As an alternative approach for this research, the microcrystal electron diffraction (MicroED) method was applied to the LCP-laden crystals because it is a powerful cryo-EM method for high-resolution structure determination from protein microcrystals. The new technique is termed as LCP-MicroED, however, prior to applying LCP-MicroED to the bicarbonate transporters, methods needed to be developed for LCP-MicroED. Therefore the model protein Proteinase K was used and its structure was determined to 2.0 Å by MicroED. Additionally, electron diffraction data from cholesterol and human A2A adenosine receptor crystals were collected at 1.0 Å and 4.5 Å using LCP-MicroED, respectively. Other applications of MicroED to different samples are also discussed.

Bioconversion of lignocellulosic sugars is often suboptimal due to global regulatory mechanisms such as carbon catabolite repression and incomplete/inefficient metabolic pathways. While conventional bioprocessing strategies for metabolic engineering have predominantly focused on a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting unique community features (i.e., modularity, division of labor, and facile tunability). In this dissertation, these features are leveraged to develop a suite of generalizable strategies and transformative technologies for engineering Escherichia coli coculture systems to more efficiently utilize lignocellulosic sugar mixtures. This was achieved by rationally pairing and systematically engineering catabolically-orthogonal Escherichia coli sugar specialists. Coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W, ethanologenic LY180, lactogenic TG114 or succinogenic KJ122. Net catabolic activities were then readily balanced by simple tuning of the inoculum ratio between sugar specialists, ultimately enabling improved co-utilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) under simple batch fermentation conditions. We next extended this strategy to a coculture-coproduction system capable of capturing and fixing CO2 evolved during biofuel production through inter-strain metabolic cooperation. Holistically, this work contributes to an improved understanding of the dynamic behavior of synthetic microbial consortia as enhanced bioproduction platforms and carbon conservation strategy for renewable fuels and chemicals from non-food carbohydrates

Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.

Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to

allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.