Manipulating the Sleeping Beauty Mutase Operon in Engineered Escherichia Coli for Controlled Biosynthesis of 1-Propanol and Other Value-Added Chemicals

A great fraction of the world’s energy requirements are presently met through the unrestricted use of fossil-derived fuels. However, due to the anticipated demise of these energy sources and the environmental and socioeconomic concerns associated with their use, a recent paradigm shift is to displace conventional fuels with renewable energy sources. Although most resources in biofuels have been directed towards the implementation of bioethanol platforms, the advanced alcohol 1-propanol has recently received significant attention as a promising alternative biofuel. Compared to that of ethanol, 1-propanol has an energy density that is more comparable to gasoline and is far less hygroscopic and volatile. Nevertheless, no microorganism has been identified as a natural 1-propanol producer. Accordingly, in this thesis, we manipulated a novel metabolic pathway for the synthesis of 1-propanol in the genetically tractable bacterium Escherichia coli. E. coli strains capable of producing 1-propanol were engineered by extending the dissimilation of the tricarboxylic acid intermediate succinate to the C3 biogenic precursor propionyl-CoA. This was accomplished by activation of the dormant yet extant Sleeping beauty mutase operon genes (i.e. sbm-ygfD-ygfG). In our initial studies, we developed propanogenic E. coli strains by episomally expressing selection of key genes, i.e. (1) three native genes in the sleeping beauty mutase (Sbm) operon (2) the genes encoding bifunctional aldehyde/alcohol dehydrogenases (ADHs) from various microbial sources, and (3) the sucCD gene encoding succinyl-CoA synthetase from E. coli. Using these triple-plasmid expression systems in E. coli, production titers up to 150 mg/L of 1-propanol were obtained in laboratory shake-flask growths under strict anaerobic conditions using glucose as the major carbon source. Following the development of these plasmid-haboring propanogenic E. coli hosts, we systematically explored various biochemical, genetic and metabolic/physiological factors to potentially enhance 1-propanol production and productivity. It was found that 1-propanol production can be significantly improved in a bioreactor under anaerobic conditions by using glycerol as a carbon source using a single-plasmid system solely expressing the Sbm operon genes. This may in part be due to the high reductance degree of glycerol compared with the microbial cell biomass. Equally important, we also alleviated plasmid-induced metabolic burden by chromosomally activating the Sbm operon genes. This plasmid-free propanogenic strain allowed high-level coproduction of 1-propanol and ethanol (accounting for 85 % of dissimilated carbon) under anaerobic fed-batch cultivation using glycerol as the major carbon source. To expand the chemical diversity and utility of our plasmid-free propanogenic E. coli strains, we explored the possibility of producing other value-added chemicals of biotechnological relevance derived propionyl-CoA. We first examined the possibility of producing butanone, an important commodity ketone. To produce butanone, we developed a modular CoA-dependent chain elongation platform to fuse Sbm-derived propionyl-CoA and endogenous acetyl-CoA to form the C5 biogenic precursor 3-ketovaleryl-CoA. Next, 3-ketovaleryl-CoA was channeled into the clostridial acetone-formation pathway for thioester hydrolysis and subsequent decarboxylation. In also manipulating initial glycerol dissimilation in the engineered ketogenic E. coli strains, we achieved co-production of 1.3 g/L butanone and 2.9 g/L acetone under semi-aerobic batch cultivation with glycerol as the major carbon source. In our final study we investigated the feasibility of using our developed propanogenic strains for the production of the bio(co)polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using unrelated carbon sources glycerol or glucose. (i.e. without exogenous supplementation of propionate or valerate). P(3HB-co-3HV) producing propanogenic strains were developed by first fusing two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA generate the C4 and C5 thioesters 3-hydoxybutyryl-CoA and 3-ketovaleryl-CoA, respectively via a CoA-dependent chain elongation platform. Next, the resulting C4 and C5 thioesters intermediates were channeled into a polyhydroxyalkanoate biosynthetic pathway for subsequent thioester reduction and polymerization. In modulating various carbon sources, aeration regimes, and host-gene deletions, copolymers with 3HV fractions ranging from ~3 mol% to ~19 mol% were obtained. Taken together, we have demonstrated that activating the Sbm operon not only transforms E. coli to be propanogenic, but also introduces an intracellular “flux competition” between the traditional C2-fermentative pathway (i.e. acetate and ethanol) and the novel C3-fermentative pathway (i.e. propionate and 1-propanol). Harnessing this flux and employing various modular chain elongation and pathway enzymes can open the avenue for the controlled production of various odd-chain organic acids, medium chain ketones, bio(co)polymers and other oleochemicals. Accordingly, the developed propanogenic E. coli strains and associated genetic and metabolic tools reported here expands the classes of chemicals that can be produced microbially via propionyl-CoA

Manipulating the sleeping beauty mutase operon for the production of 1-propanol in engineered Escherichia coli

Background: While most resources in biofuels were directed towards implementing bioethanol programs, 1-propanol has recently received attention as a promising alternative biofuel. Nevertheless, no microorganism has been identified as a natural 1-propanol producer. In this study, we manipulated a novel metabolic pathway for the synthesis of 1-propanol in the genetically tractable bacterium Escherichia coli. Results: E. coli strains capable of producing heterologous 1-propanol were engineered by extending the dissimilation of succinate via propionyl-CoA. This was accomplished by expressing a selection of key genes, i.e. (1) three native genes in the sleeping beauty mutase (Sbm) operon, i.e. sbm-ygfD-ygfG from E. coli, (2) the genes encoding bifunctional aldehyde/alcohol dehydrogenases (ADHs) from several microbial sources, and (3) the sucCD gene encoding succinyl-CoA synthetase from E. coli. Using the developed whole-cell biocatalyst under anaerobic conditions, production titers up to 150 mg/L of 1-propanol were obtained. In addition, several genetic and chemical effects on the production of 1-propanol were investigated, indicating that certain host-gene deletions could abolish 1-propanol production as well as that the expression of a putative protein kinase (encoded by ygfD/argK) was crucial for 1-propanol biosynthesis. Conclusions: The study has provided a novel route for 1-propanol production in E. coli, which is subjected to further improvement by identifying limiting conversion steps, shifting major carbon flux to the productive pathway, and optimizing gene expression and culture conditions.Natural Sciences and Engineering Research Council (NSERC); Canada Research Chair (CRC) program of Canad

Helping Québec Pharmacists Seize the Vaccination Service Opportunity: The Pharmacy Best Practice Workshops

Vaccinations are a safe and effective way to protect against infectious diseases. The World Health Organization estimates vaccines have saved more lives than any other interventions and every year about two to three million deaths are averted worldwide through immunization. To improve vaccination coverage, pharmacists have been increasingly involved in immunization roles in their communities—as advocates, educators, and immunizers. Community pharmacy-based vaccination services have increased both in the number of immunization providers and the number of sites where patients can receive immunizations. In Canada, health care is under provincial legislation—and so, there are distinct differences in scope of pharmacist practice across the country. Prior to the COVID-19 outbreak in early 2020, in Québec, Canada’s second-largest province, pharmacists did not have the authority to administer vaccines. To help prepare pharmacists in Québec to become immunizers, we developed and deployed a series of accredited workshops. In these facilitated workshops, pharmacists were able to share best practices that may lead to providing effective vaccination services, identify common competency gaps, discuss effective patient communication skills, and determine how to target the most vulnerable population groups. Participants were also asked to evaluate the workshop. Our results indicate the evaluation was very reliable in measuring participant satisfaction (Cronbach’s α = 0.94) and pharmacists commented that the workshops’ learning outcomes exceeded their expectations, and the topics covered were relevant and applicable. The evaluation also asked participants to identify weaknesses of training, so future educational interventions can be planned accordingly. We believe this work will contribute to the continual growth and advancement of the pharmacy profession in Canada

Development and Implementation of Workshops to Optimize the Delivery of Vaccination Services in Community Pharmacies: Thinking beyond COVID-19

Vaccines are widely recognized as the most economically efficient strategy to combat infectious diseases. Community pharmacists, being highly accessible healthcare professionals, have the potential to significantly contribute to the promotion and facilitation of vaccination uptake. In Canada, the jurisdiction of healthcare falls under provincial legislation, leading to variations in the extent of pharmacist practice throughout the country. While some pharmacists in Canada already functioned as immunizers, Québec pharmacists gained the authority to prescribe and administer vaccines in March 2020 amidst the COVID-19 pandemic. Our workshop aimed to equip pharmacists in Québec with the necessary guidance to optimize vaccinations, emphasizing the importance of maintaining and expanding immunization services beyond influenza and COVID-19 vaccines in the future. During the workshop, pharmacists had the opportunity to exchange valuable insights and best practices regarding workflow optimization, identifying areas for improvement in competency, effectively reaching vulnerable population groups, and integrating allied team members into their practice. Participants were also asked to develop a plan of action to help implement practice change beyond the workshop. Interactive workshops centered around discussions like these serve as catalysts for advancing the pharmacy profession, uniting professionals with a collective aim of enhancing patient care

for the production of 1-propanol in engineered Escherichia coli

Manipulating the sleeping beauty mutase opero