Doctor of Philosophy in Chemical and Biological Engineering (PhD)
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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Taking this chance #GreatSupervisor occasion at #UBC to express my thanks to Vikramaditya Yadav @biofoundry. He is an incredible mentor, and brilliant person. I am so grateful for all of your support.
I am privileged to have the best and most supportive supervisor. I have learned a lot from you, whether it is dealing with stress or asking critical questions; whether it’s research or teaching. @biofoundry is #GreatSupervisor at #UBC also a great friend and mentor.
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.
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.
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.
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.
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.
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.