Vikramaditya Yadav

Clinical studies on cannabis reveal its importance in alleviating inflammation and counteracting certain neurodegenerative disorders. However, these benefits carry side effects induced by the hallucinogenic property of ∆⁹-tetrahydrocannabinol (THC). Investigation into cannabinoids has led to the discovery of cannabinol (CBN), a degradation product of THC that provides multiple benefits without effect on the perception, cognition, or behavior of the patient. Current methods of production rely on traditional farming and degradation of THC to produce CBN which are less sustainable and efficient than fermentation-based cannabinoid production. CBN formation is the result of oxidative degradation of THC, and currently, there’s no enzyme associated with this reaction. To create a biosynthetic route towards CBN, a multistep biosynthetic mechanism has been prepared utilizing CYP450 monooxygenases and dehydratases to facilitate the double bond formation on THC. Cytochrome P450 19A1, 2C9, and 3A4 were in-vitro assayed with THC and THCA to examine their metabolite products and provide a solution to achieving the first step in the proposed mechanism. THC and THCA were also docked to these enzymes to create predictions on expected metabolites. An alternative approach was to use the consensus sequence motif of Cytochrome P450 19A1 and BLAST search the C. sativa genome for candidate enzymes that could potentially convert ∆⁹-tetrahydrocannabinolic acid (THCA) to cannabinolic acid (CBNA) or present a candidate suitable for the formation of the first intermediate. The candidates were then modeled and docked with THCA to obtain the five models with the highest binding affinity. These candidates were further docked with THC. Out of these, the docking of THC and THCA with CsCYP84A1 provides the best models that allow the formation of the first intermediate. Similarly, CYP19A1 docking with THC and THCA, and CYP2C9 docking with THCA provided models that predict the formation of the first intermediate. Only the in-vitro assays of CYP2C9 show catalytic activity against THC to produce 11-OH-THC, while the assay with THCA produces 11-OH-THC, 11-OH-THCA, and an unknown metabolite that needs to be identified. The assays need to be re-analyzed with a GC-MS to identify finite metabolites that were not observed when THC and THCA decreased over time.

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Capsaicinoids are a class of compounds natively produced by pepper plants as secondary metabolites. They possess therapeutic properties and have been used for treating medical conditions such as chronic pain, overactive bladder and diabetes. Capsaicin, a well-known chemical in this class, is responsible for the spicy taste of chili peppers. The precursors for capsaicinoids include vanillylamine and an acyl-CoA. Capsinoids, a group of capsaicinoid analogues, use vanillyl alcohol as a precursor instead of vanillylamine, making them less pungent. Here, we describe preliminary work towards the development of an E. coli strain for the production of capsaicinoids and capsinoids using vanillin and glycerol as precursors. Successful implementation of this system could allow targeted production of compound libraries or individual compounds if required. Such flexibility can be achieved through the mixing and matching of different enzymes within the host system. This platform can lead to production of high-value low-volume chemical products that have pharmaceutical applications. However, a sufficiently productive platform could also provide a less resource intensive source of capsaicinoids, currently gathered from pepper plants at less than 1% yield. In this work, we evaluate E. coli as a host for the synthesis of capsaicinoids and capsinoids. Initially, we created a pathway and successfully demonstrated synthesis of C6 to C12 straight and branched-chain fatty acids. We then focused on candidate enzymes for acyl-CoA synthesis to activate the aforementioned fatty acids, and a number of potential candidates were identified based on literature review. Furthermore, we investigated vanillylamine and vanillyl alcohol production. Previous publications have demonstrated conversion of vanillin to vanillylamine in E. coli using transaminases. Vanillyl alcohol on the other hand can be synthesized from vanillin using the endogenous reductases in E. coli. Lastly, we attempted expression of capsaicin synthase in E. coli with and without the SUMO tag, however results were inconclusive. More investigation needs to be performed to determine whether capsaicin synthase, a key enzyme on our pathway, can be expressed in its active form.

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Chronic immune activation and inflammation are etiologically associated with the pathology of inflammatory bowel disorders, certain cancers, and persisting infections (eg. HIV)¹⁻³. Recently, a series of novel, microbe-derived, Site-Specific Immunomodulators (SSIs; Qu Biologics) have been described to repurpose the immune response and exert therapeutic effects at specific sites of pathology⁴⁻⁷. The mechanism for these effects are poorly understood, but may involve a phenomenon called innate immune memory⁴⁻⁷. Importantly, innate immune memory is distinct from classic adaptive immune memory, as it enhances immune responses against subsequent exposures to different, rather than the same pathogen⁸'⁹. Cells of the monocyte/macrophage (M/Ms) lineage are key innate immune memory cells¹⁰. Interestingly, it has been established that M/Ms exert many of their effects through autophagy (“self eating”), a critical regulator of cellular homeostasis, immune defense and inflammation¹¹⁻¹³. Therefore, it was hypothesized that autophagy is induced by and is required for the biologic effects of SSIs. U937 monocytic and HT-29 colonic epithelial cell lines were selected as in vitro models. First, a novel flow cytometry-based autophagy detection approach (CYTO-ID®) was validated and used to establish that SSIs may indeed act through their capacity to mobilize the autophagy pathway. Subsequently, the downstream effects of SSI-induced autophagy were investigated by analyzing the expression of cytokines, IL-1β and IL-18, both of which are reputed to be modulated by the autophagy pathway¹⁴. However, neither cytokine was detectable by the ELISA approach used. Finally, experiments to shed light on the molecular mechanisms by which SSIs induce autophagy were undertaken. Knowing that SSIs are derived from specific bacteria⁴⁻⁷, I examined whether SSI-induced autophagy was mediated by the cell’s bacterial lipopolysaccharide Pattern Recognition Receptors (PRRs) (Toll-like receptors (TLR)-2 and TLR4). Since TLR2/TLR4 signaling proceeds via the Myeloid differentiation factor 88 (MyD88) adaptor protein, a MyD88 antagonist was used to establish that SSIs induce autophagy through their bacterial components, and the MyD88 signaling pathway, specifically. Finally, the thesis provides a basis for further investigations into the role of autophagy in SSI-induced therapeutic effects in vivo using the Lewis Lung Carcinoma, spontaneous colitis (Muc2-/-), and other mouse models of disease.

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The development of organic photosensitive materials has opened up a breadth of new areas for advancement in photovoltaics and dye-sensitized solar cells (DSSCs). The approach of organic DSSCs is to use photo-excitable dyes over a conductive nanoparticle layer in the presence of an electrolyte to create a working electrode. There has been a large emphasis on the improvement of organic DSSCs in recent years, and there have been significant increases in their photovoltaic efficiencies. However, the fabrication process and extraction of the dyes involves complicated and costly methods that require the use of toxic chemicals and a tightly controlled clean-room environment. To alleviate these issues, a novel approach was developed that uses genetically engineered bacteria capable of producing lycopene, a photo-excitable dye, internally. Preliminary research of these genetically engineered cells implemented in organic solar cell production shows promising results, but significant improvements must be made in order to be comparable to conventional solar cells. The thesis focuses on improving the conductivity of the genetically engineered bacteria capable of synthesizing lycopene. The approach is to use the electroconductive properties found in another bacterial species, Shewanella Oneidensis MR-1 (SO MR-1), to increase the photovoltaic properties of the system. The conductive ability of SO MR-1 arises from its bacterial nanowires which are capable of extracellular electron transfer. The experimental methods include identifying the genes that are responsible for bacterial nanowires formation in SO MR-1, extracting and cloning the identified genes into the lycopene producing bacteria, verifying the expression of the bacterial nanowire genes, evaluating the photovoltaic characteristics, and comparing the measurements of the systems with and without bacterial nanowires. The results show successful implementation of the genes responsible for bacterial nanowire formation into the lycopene producing bacteria, but the expression level analysis revealed ambiguous results which could be addressed with more precise methods. The photovoltaic analysis had some issues with short-circuiting, which made it difficult to draw any significant conclusions. Although the main objective of the thesis might need to be further investigated, several integral objectives were achieved, which can be used as a stepping stone in future research.

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Bioprocesses based on metabolically engineered microbes have become tremendously important in recent decades as a platform for the synthesis of complex molecules. Substantial research effort has been devoted to the improvement of microbial strains involved, and while this has enhanced some metrics of strain performance dramatically, namely product yield with respect to substrate and biomass, achieving similar results with other aspects has remained elusive. Improving productivity, the rate at which a modified strain can synthesize a product of interest, in particular has presented an engineering challenge despite its obvious value to the economics of a process and has typically only been done through bioprocess optimization. A strategy that could yield the desired result is strain engineering to better integrate with the bioprocess context in which it is used. The work described in this thesis has sought to achieve that goal by providing a method to the operating engineer to dynamically control the induction of genes associated with product formation. More specifically, a T7 RNA polymerase was modified by the insertion of a mutant variant of the S cerevisiae Vacuolar membrane ATPase(VMA) intein. This mutant intein will only splice out of its host only under conditions of reduced temperature, which in effect makes the polymerase active only after a temperature shift from 37℃ to 18℃ degrees. This creates a strict demarcation between biomass accumulation and product synthesis, only allowing this transition to be made at an optimal point during fermentation, as chosen by the operating engineer. Using lycopene biosynthesis as a case study and applying this approach, it was found that a productivity improvement of approximately 15% over an uncontrolled strain was attained. It was also found that a remarkable degree of control stringency was conferred upon the system, with no premature product synthesis detected under any condition investigated.These results are expanded upon to generate a series of simple mathematical models, with the aim of describing how such a dynamic metabolic control element might be expected to perform in a more generalized context, and to provide a means by which to more quantitatively assess the strain’s performance.

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Efforts to rejuvenate the under-exploited, but high-value, natural product space has focusedon wiring nature’s biochemical reactions into microorganisms and features as a sustainablealternative to the chemical synthesis. However, for industrial relevance, the metrics of strainperformance, yield, titer, and productivity, need to be improved, to achieve a better economicvalue. To tackle this, most metabolic engineering strategies have focused on rational deletions oroverexpression of genes across metabolic pathways and given little thought to metabolic fluxesand how their channeling could affect biomass accumulation and product formation. Consideringthis, we propose two approaches: one for increasing the flux across metabolic pathways throughthe creation of fusion enzyme complexes; and the second to control flux, by tweaking thedistribution of resources between biomass and product formation, through an optogenetic circuit.For the first, we have focused on increasing the flux through the non-mevalonate pathway,which is a precursor for the biosynthesis of several terpenoids. Taking inspiration from naturallyoccurring fusion or bifunctional enzymes of this pathway, we constructed several artificial fusionsbetween rate limiting enzymes, by varying the catalytic domains and linkers, and tested theirability to improve flux, by enabling better substrate channeling. From our data, we found the fusionbetween the enzymes of IspD and IspE, with a flexible linker, outperformed the other strainsespecially in terms of lycopene titer. For controlling flux, we created an optogenetic circuit, which provides fine spatial control over individual cells and has advantages over chemical inducers. On exposure to red-light at 660 nm, the circuit activates T7 RNA polymerase and allocates resources between biomass accumulation and product formation. Design elements of the circuit include: plant-based phytochromes, that function as optical dimers; and yeast-based split-inteins, that can trigger a trans-splicing reaction. Using this circuit and external optical systems, we have dynamicallycontrolled the expression of T7 RNA polymerase, which controls expression of the secondarymetabolite, lycopene in our case. We expressed this circuit in bacteria and observed roughly a fivefold increase in lycopene titer with light, versus no light illumination, providing proof-of-conceptof the approach.

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Biocatalyst discovery is integral to bioeconomy development, enabling design of scalable bioprocesses that can compete with the resource-intensive petrochemical industry. Uncultivated microbial communities within natural and engineered ecosystems provide a near-infinite reservoir of genomic diversity and metabolic potential that can be harnessed for this purpose. To bridge the cultivation gap, functional metagenomic screens have been developed to recover active genes directly from environmental samples. In this thesis, a pipeline for recovery of biomass-deconstructing biocatalysts sourced from pulp and paper mill sludge (PPS) metagenome is described. This environment is targeted given its high composition of cellulose that is hypothesized to direct enrichment of enzymes capable of hydrolysing it. The resulting oligosaccharides represent platform molecules that can be fed to downstream applications using consolidated process design for converting biological waste streams into value-added products. High-molecular weight DNA was extracted from sludge and used to construct a fosmid library containing 15,000 clones using the copy control system in EPI300™-T1 R E.coli. Extracted DNA was also used in whole genome shotgun sequencing to compare the metabolic potential of the sludge community with fosmid screening outcomes as well as other waste biomass environments using MetaPathways v2.5 software pipeline, with specific emphasis on carbohydrate-active enzymes (CAZymes). Metagenomic assembling, open reading frame (ORF) prediction, binning and taxonomic assignment approaches were also used to bring out correlations between function and taxonomy. In total, 32,232 ORF’s were mapped to the CAZy database predicted to encode glycoside hydrolases, glycosyl transferases, and carbohydrate binding module families. The fosmid library was screened for glycosidase hydrolase activities using a pool of sensitive fluorogenic glycosides of 6-chloro-4-methylumbelliferone (CMU). A total of 744 clones capable of converting pooled substrates were recovered indicating an extremely high hit rate (1 hit per 43 clones). Following fosmid sequencing and annotation, two of the most promising hits with defined single GH family loci were sub-cloned and overexpressed in E.coli BL21 DE3 strain to conduct basic biochemical characterization. Activity of purified enzymes was demonstrated on model lignocellulosic substrates to evaluate the potential of implementing the proposed circular bioprocess with waste PPS as both the feedstock and source of enriched biocatalysts.

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The nucleocapsids of the baculovirus have been observed to undergo intracellular trafficking driven by actin polymerization. Propelled by an actin tail through the cytoplasm, the baculovirus nucleocapsid finds its way to the nucleus of the host cell. Then it docks to the cytoplasmic filaments of the nuclear pore complex (NPC), and manages to enter the nucleus intact. These interesting experimental observations inspired the current research. We first focus on the actin polymerization mechanism and the propulsive force generated at the back of the virus. Then, at the NPC interface, we integrate the mechanism for opening the central channel and passage of the virus. For the first part, using a microscopic approach and implementing an elastic Brownian ratchet model, we suggest a biphasic force-velocity relationship for baculovirus riding on the actin comet tail, which stalls at an external force of around 50 pN. Then, having this force value as the key parameter, we evaluate the idea of mechanical breakthrough into the NPC channel. For this purpose, we model the central channel of the NPC as saturated hydrogel. A mechanical fracture model shows that in order for the actin force to affect a purely mechanical breakthrough, the gel must be exceedingly soft. Although our results do not offer direct support for the hypothesis of a purely mechanical entry, they do not disprove the idea, either. Possibly the homogeneous hydrogel model for the NPC is inadequate, and more complex models (e.g. polymer brushes and forest) need to be examined. It is also possible that the mechanical entry of the virus is aided by biochemical signals that soften or partially remove the NPC barrier.

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