Athanasios Kritharis
Doctor of Philosophy in Chemical and Biological Engineering (PhD)
Research Topic
A new technological paradigm for low-cost, decentralized vaccine manufacture
My research group utilizes metabolic & enzyme engineering to investigate and customize novel biosynthetic enzymes that can convert biomass-derived feedstocks into value-added chemicals. We have published highly acclaimed papers on model-guided enzyme engineering, process development, enzyme discovery using metagenomics and engineering metabolic control schemes that bridge with bioprocess control and improve productivity. Each of these works represents a critical advance in our ability to employ engineered microorganisms as a manufacturing platform. We also extend the principles of metabolic engineering to the design and development of unique bioremediation strategies to rehabilitate the water quality in and around industrial zones and new mining technologies and we are currently collaborating with Suncor and Jetti Resources, respectively, to deploy novel biotechnologies in the field. In addition to green engineering, my research group also pursues medical biotechnology research, and focuses on three stages in the drug discovery life cycle – (1) bioengineering for assay development, (2) biosynthetic engineering for lead generation, and (3) pharmaceutical product development. Our work on bioengineered assays aims to assemble three-dimensional, structured brain organoids from human pluripotent stem cells for use in pre-clinical screening of hits against Alzheimer’s disease. Through this work, we have established a formal collaboration with STEMCELL Technologies. Our work on pharmaceutical product development is advancing a concept that we dub ‘medicine-by-design’, a fast and low-cost methodology to advance a drug molecule from concept to formulated product based on the synergistic application of bioinformatics and data analysis, metabolic engineering and formulation science. We work closely with an industrial partner, InMed Pharmaceuticals, and have successfully advanced two projects to clinical testing. Our work on development of a ‘smart’ contact lens for treating glaucoma is among the most read scientific articles of 2018. Similarly, our work on the development of a printable bandage for healing damaged skin in patients suffering from Epidermolysis Bullosa Simplex (EBS) is currently under consideration for publication. Both works are the subjects of patent filings. We have recently initiated a new line of research in the group that fuses biology and materials science to develop better materials and transcend current limitations in manufacturing. The synergistic combination of biological systems with abiotic, functional materials that greatly improves the properties of the original host, and the resulting systems can be applied to a wealth of manufacturing, energy and environmental remediation applications. We laid the intellectual foundations of this paradigm in a forum article in Trends in Biotechnology and subsequently published a proof-of-concept study on a biohybrid photovoltaic cell that is the best in its class and could be used in bioorganic optoelectronics. My research group currently collaborates with 7 companies – STEMCELL Technologies, InMed Pharmaceuticals, Jetti Resources, Metabolik Technologies, Sanofi Pasteur, Reliance Industries Limited and Phytonix Corporation.
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Brain organoids are self-assembled, three-dimensionally structured tissues that are typically derived from pluripotent stem cells. They are multicellular aggregates that can more accurately recapitulate the tissue microenvironment compared to other cell culture systems and also reproduce organ function. These stem cell-derived 3D tissues can be excellent models for investigating mechanisms of tissue formation and responses to physiological and mechanical cues. However, understanding about their mechanical properties pales in comparison, which is all the more galling in light of newfound insights about how mechanical stimuli trigger the onset of neurodegenerative conditions. Herein, formative steps are taken to fill this knowledge gap, using neurospheres were generated from murine neural stem cells and subjected to compressive forces. I generated neurospheres that exhibit stress relaxation under static compression and viscoelastic behavior at low strains. The suitability of the Tatara model for characterizing the mechanical properties of neurospheres was also evaluated. Besides, the utility of cantilevered capillary force apparatus as a broadly applicable tool to evaluate tissue mechanics by quantifying the effect that oxidative stress has on the mechanical properties of neurospheres was demonstrated. Neurospheres exhibit viscoelasticity consistent with neural tissue and document a size-dependence of their elastic moduli. Oxidative stress altered the composition, architecture and signaling within the tissues. I observed a clear correlation between oxidative stress, the chemical state of the tissues and their biophysical properties. My results confirms that non-cytotoxic oxidative stress can modulate the differentiation of neural progenitor cells. This is the first study of its kind to investigate the mechanical properties of in vitro 3D tissues. Moreover, the methodologies developed can also be used to improve the quality and safety of cell and tissue biomanufacturing processes and yields insights for establishing rheological measurements as biomarkers.
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Naphthenic acid fraction compounds (NAFCs) are highly recalcitrant contaminants of oil sands process-affected water (OSPW). Classic naphthenic acids (NAs), with an empirical formula of CcHhO2, have been determined as the most toxic NAFCs. The concentration of classic NAs in the OSPW collected for this study was determined as 28.2 mg/L using high-performance liquid chromatography (HPLC) Orbitrap mass spectrometry. An Innovation Ecosystem for the commercialization of engineered bioremediation platforms was defined and connected to a detailed risk framework including technological risks, regulatory risks, and risk perception. Using enrichment culture techniques with NAFCs as the only carbon source, seven distinct cultures were isolated from OSPW. NAs treatability study with the seven isolated cultures resulted in selecting four Pseudomonas cultures for further characterization. These four cultures were identified as Pseudomonas protegens, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas sp. following the whole genome sequencing and assembly. A comparative genomic analysis of these cultures detected genomic islands and positively selected genes (PSGs). For the transcriptional response, P. protegens, P. putida, and a 1:1 co-culture of the isolates were examined in the presence of NAFCs, as the only carbon source. The RNA-Seq and HPLC-Orbitrap data were combined into custom Python scripts and revealed the identity of enzymes and pathways associated with different NAFC groups. Compared with individual cultures, co-cultures of these two isolates demonstrated a completely different degradation mechanism. Furthermore, the co-culture degraded a greater quantity of NAFCs (~30%) than pure cultures (~11%). A pilot-scale test was conducted in 5 m³ mesocosms with 1:1 co-culture of P. protegens and P. putida. Two experimental factors, aeration and inoculation, were tested with three replicates of each factor. Twelve mesocosms were divided into four groups: control, aeration, inoculation, and aeration and inoculation treatments. The genome-resolved metagenomics and metatranscriptomics analysis were performed to characterize the phylogenetic and metabolic diversity of OSPW. In general, the inoculated treatment demonstrated the most over-represented degradation pathways compared to the other two treatments. In the metatranscriptomic data, benzoate degradation and degradation of aromatic compounds pathways were only over-represented in inoculated and aerated treatments even though they were over-represented in all treatments in the metagenomic data.
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Polyketides are a natural chemical class of complex, frequently bioactive molecules, from which several therapeutics have been derived. Large enzyme complexes, polyketide synthases, are responsible for constructing the carbon backbone of polyketides from simple metabolic precursors. For modular polyketide synthases (mPKSs), the chemical tailoring of each precursor incorporated is controlled by a distinct cluster of domains. These modules are organized as an assembly line. Because the chemistry of the final polyketide chain reflects the organization of modules on the assembly line, mPKSs have become targets for rational protein engineering. If module types could be functionally rearranged in any order, this would expand control over polyketide biosynthetic pathways, enabling access to libraries of novel polyketides from which new antibiotics or therapeutics may be derived. However, combinatorial use of individual mPKS modules has not surpassed 3-module assembly lines. Interactions between the acyl carrier protein (ACP) and ketosynthase (KS) module domains have been particularly sensitive to mismatched interfaces, and these largely constrain use of engineered modules to their specific locations in their natural mPKS assembly lines. ACP-KS interactions remain challenging to model and engineer. In this thesis, I propose an alternative mPKS platform using modules from Mycobacterium tuberculosis PKS12. This bimodular PKS uses repeating, identical KS-ACP interfaces to form 10-module multimers which build long, saturated carbon chains. This makes PKS12 modules attractive templates for creating more interchangeable modules. A prerequisite to developing a PKS12-derived combinatorial platform is the affixing of chain-onloading and chain-offloading domains onto PKS12 modules, enabling it to produce fatty acids. I show that a fatty-acid-producing mPKS can be created from PKS12 parts, and identified an unexpected fusion point for the offloading domain. I identified a plasmid system and culture conditions enabling detectable and reproducible output from PKS12-derived assembly lines in E. coli. I then used this system to demonstrate that antiparallel SYNZIP domains can functionally reattach saturating module halves, providing a new principle for combinatorial mPKS engineering. Finally, I investigate the structural basis for the unexpected C-terminal fusion point, and identify a particular ACP region, helix 2, as a likely contributor.
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The biomanufacturing of terpenoids is limited by the low yields of heterologously expressed biosynthetic pathway and challenges associated with recovering these products at commercial scales. To enhance the flux through methylerythritol phosphate (MEP) pathway for terpenoid biosynthesis, I screened soil metagenomes for more active and stable orthologs of the rate-limiting enzymes. I successfully identified three entirely novel, natural fusions of IspD and IspF, one of which improved production of lycopene from 235 mg/L to 275 mg/L and production of isoprene from 3.6 mg/L to 6.3 mg/L when compared to the native enzyme overexpression. A comprehensive study of the role of the linking domain revealed the higher activity of each of the catalytic domains and the absence of substrate channeling. Moreover, the non-natural fusions of E. coli enzymes catalyzing consecutive steps were constructed. One such a fusion of IspD and IspE yielded 281 mg/L of lycopene, whereas the best performing fusion of IspE and IspF only yielded 39 mg/L of lycopene. Further investigation of the sequence of this biocatalytic cascade concluded the commencement with the activity of IspE, followed IspD and IspF suggesting the reactive plasticity in MEP pathway. I probed the promiscuous nature of terpene synthases (TSs) through the systematic study of monoterpene synthases from Picea abies in vivo and in vitro. I uncovered the influence of intracellular expression and oxygen supply on the promiscuity of TSs. Computational analysis revealed the putative roles of the amino acid residues within the active sites and their evolutionary trajectory. Finally, the fermentation of engineered E. coli strains for carene and myrcene were scaled up to 1 L and a newer technique was developed for efficient product capture using a fluidized bed capture device (FBCD) using a hydrophobic resin. The device was easy to integrate into the existing bioreactor set up. It yielded 2-fold higher carene titers and 17-fold higher myrcene titers.In conclusion, the three aspects of the terpenoid biomanufacture studied in this work address some of the biggest challenges facing the industry and lay strong foundations for commercialization of terpenoid biomanufacturing processes that employ genetically engineered microorganisms.
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Malaria presents a severe economic and healthcare burden for the developing world. Recent efforts to reduce the incidents of malaria-associated deaths achieved some success, but drug resistance is increasing and the number of drugs available to treat the diseases is thinning. The current thesis seeks to advance two complementary strategies to develop platform solutions to treat and prevent malaria. First, we applied cheminformatics to assess the chemical space of anti-malarial drugs to identify promising scaffolds. Open-source tools were used to analyze the scaffolds of candidates and approved anti-malarial drugs. Our scaffold-centric analysis reveals that the anti-malarial chemical space is disjointed and segregated into few dominant structural groups with these structures being distributed according to Paretos’ principle. This structural convergence can potentially be exploited for future drug discovery by incorporating it into bioinformatics workflows. This could be used to predict new combination therapies and areas for the development of new molecules.Our second strategy seeks to develop a better tool for repellent discovery; repellent usage to prevent mosquito bites is a safe way to control these infections. The current methods for repellent screening are time consuming. The olfactory pathway involves odorant receptors that form a heterodimeric ion channel with an odorant receptor co-receptor (Orco) and a switching odorant receptor (OR). The heterologous expression of these proteins in Xenopus-oocytes and HEK293 cells, have suggested that the Orco-OR complex is functional. While these hosts have permitted significant discoveries, they have slow growth rates and extensive handling requirements, which make them unwieldy for high-throughput screens. We sought to develop high-throughput repellent screens by reconstructing this olfactory pathway into a simpler host (Pichia pastoris) with the Orco receptor. The Anopheles gambiae Orco protein was successfully expressed, being able to discriminate between compounds (VUAA1, citronella and oct-1-en-3-ol) and doses (0.125 mM to 2 mM for VUAA1) when coupled with a reporter signal. In the future this system could be used to screen chemical libraries. Moreover, the heterologous expression of Orco protein could lead to future structural and functional investigation of OR compounds as well as the development of newer repellents and behavior-modifying compounds.
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Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Microbial Fuel Cells (MFC) are an emerging research field due to their ability to simultaneously tackle two prominent environmental concerns. MFCs are a clean waste valorization biotechnology that utilize microorganisms as biocatalysts to metabolize substrates available in wastewater, yielding renewable energy and clean water as a by-product. Despite their potential, MFCs face significant hurdles that hinder their practical application in industry. Consequently, research efforts are largely directed towards enhancing MFC performance by refining various components and operational parameters. This research is often labour intensive, time consuming and costly. This thesis aims to expedite MFC research through the development of a high-throughput biofilm formation assay tailored for MFC performance screening. The developed high-throughput plate reader assay enables real-time quantification of biofilm attachment to electrodes, to determine the cell load ratio. This assay is a novel achievement in biofilm quantification, as no other known method can numerically quantify living biofilm on an electrode surface. It serves as a versatile tool for comparing various MFC components such as electrode materials or treatments, bacterial strains, media and operating conditions like temperature, agitation rates or additives. The developed assay offers simplicity, cost-effectiveness, and scalability, thereby streamlining the evaluation process. To validate its efficacy, the assay was utilized to compare three distinct electrode conditions: untreated, ethanol-treated, and autoclaved electrodes. Results obtained from the plate-reader assay were corroborated using traditional electrochemical methods, including chronoamperometry to find current outputs and electrochemical impedance spectroscopy to determine overall MFC impedance.It was observed that a slower biofilm development rate proved more beneficial than achieving a higher biofilm attachment loading ratio, relative to the planktonic cell culture. A correlation was established indicating that the longer it took to achieve the maximum load ratio, the less impedance and greater current outputs exhibited by the MFC. This implies that a gradual cell attachment process enhances the electroactivity of the biofilm, thereby enhancing MFC performance. This trend was consistent across all three electrode test conditions. Notably, ethanol-treated electrodes exhibited the most promising performance, characterized by the longest peak load ratio time, lowest impedance, and highest current outputs, followed by autoclaved and untreated electrodes, respectively.
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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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