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.

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.

Bio-based production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated monomeric fraction in Escherichia coli.

In this study, we applied metabolic engineering and bioprocessing strategies to enhance heterologous production of an important biodegradable copolymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a modulated 3-hydroxyvalerate (3-HV) monomeric fraction from structurally unrelated carbon of glycerol in engineered Escherichia coli under different oxygenic conditions. We used our previously derived propanologenic (i.e., 1-propanol-producing) E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon as a host for heterologous expression of the phaCAB operon. The 3-HV monomeric fraction was modulated by regulating dissimilated carbon flux channeling from the tricarboxylic acid (TCA) cycle into the Sbm pathway for biosynthesis of propionyl-CoA, which is a key precursor to (R)-3-hydroxyvaleryl-CoA (3-HV-CoA) monomer. The carbon flux channeling was regulated either by manipulating a selection of genes involved in the TCA cycle or varying oxygenic condition of the bacterial culture. With these consolidated strategies being implemented, we successfully achieved high-level PHBV biosynthesis with a wide range of 3-HV monomeric fraction from ~ 4 to 50 mol%, potentially enabling the fine-tuning of PHBV mechanical properties at the biosynthesis stage. We envision that similar strategies can be applied to enhance bio-based production of chemicals derived from succinyl-CoA. KEY POINTS: • TCA cycle engineering was applied to enhance 3-HV monomeric fraction in E. coli. • Effects of oxygenic conditions on 3-HV incorporation into PHBV in E. coli were investigated. • Bacterial cultivation for high-level PHBV production in engineered E. coli was performed.

The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome. The ensuing clone selection relying on empirical screens and cell line characterization is extensive and time-consuming. An emerging paradigm in CHO cell line development is the use of site-specific recombinases to enable the integration of therapeutic transgenes into pre-marked chromosomal locations with defined expression characteristics. Recombinase-mediated cassette exchange (RMCE) provides a sophisticated alternative to conventional CHO cell line development, leading to the generation of more consistent and reliable clones and may ultimately shorten the "time-to-clinic" of recombinant therapeutics. Herein, we review the recent advances in the use of site-specific recombination systems and their associated cassette exchange technologies for the rapid generation of stable CHO clones with predictable growth, stability, quality and productivity characteristics. Particular emphasis is placed on cassette exchange technologies currently used in the industry. We also discuss the technical hurdles associated with uses of site-specific recombinase systems in CHO cells, illustrate how these problems can be mitigated and provide a perspective on future work concerning these systems.

Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli.

3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l-1 with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.

Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli.

Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L-1 butyrate, 8.95 g L-1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.

Recent advances in engineering propionyl-CoA metabolism for microbial production of value-added chemicals and biofuels.

Diminishing fossil fuel reserves and mounting environmental concerns associated with petrochemical manufacturing practices have generated significant interests in developing whole-cell biocatalytic systems for the production of value-added chemicals and biofuels. Although acetyl-CoA is a common natural biogenic precursor for the biosynthesis of numerous metabolites, propionyl-CoA is unpopular and non-native to most organisms. Nevertheless, with its C3-acyl moiety as a discrete building block, propionyl-CoA can serve as another key biogenic precursor to several biological products of industrial importance. As a result, engineering propionyl-CoA metabolism, particularly in genetically tractable hosts with the use of inexpensive feedstocks, has paved an avenue for novel biomanufacturing. Herein, we present a systematic review on manipulation of propionyl-CoA metabolism as well as relevant genetic and metabolic engineering strategies for microbial production of value-added chemicals and biofuels, including odd-chain alcohols and organic acids, bio(co)polymers and polyketides. [Formula: see text].

Engineering of Escherichia coli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources.

While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of ß-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of E. coli host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered E. coli strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB-co-3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum.

UNLABELLED: Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter(-1)). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter(-1)), yielding a high n-butanol selectivity of 0.83 g n-butanol g(-1) of solvents compared to 0.51 g n-butanol g(-1) of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCE: Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.

Engineering Escherichia coli for Microbial Production of Butanone.

To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohol genesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter(-1)glycerol as the carbon source, we achieved coproduction of 1.3 g liter(-1)butanone and 2.9 g liter(-1)acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.

Engineering Escherichia coli for high-level production of propionate.

Mounting environmental concerns associated with the use of petroleum-based chemical manufacturing practices has generated significant interest in the development of biological alternatives for the production of propionate. However, biological platforms for propionate production have been limited to strict anaerobes, such as Propionibacteria and select Clostridia. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli. Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e., propionate-producing) but also introduced an intracellular "flux competition" between the traditional C2-fermentative pathway and the novel C3-fermentative pathway. Dissimilation of the major carbon source of glycerol was identified to critically affect such "flux competition" and, therefore, propionate synthesis. As a result, the propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For optimal propionate production with high C3:C2-fermentative product ratios, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titers of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for ~50 % of dissimilated glycerol) were achieved, demonstrating the potential for industrial application. To our knowledge, this represents the most effective engineered microbial system for propionate production with titers and yields comparable to those achieved by anaerobic batch cultivation of various native propionate-producing strains of Propionibacteria.

Biochemical, genetic, and metabolic engineering strategies to enhance coproduction of 1-propanol and ethanol in engineered Escherichia coli.

We recently reported the heterologous production of 1-propanol in Escherichia coli via extended dissimilation of succinate under anaerobic conditions through expression of the endogenous sleeping beauty mutase (Sbm) operon. In the present work, we demonstrate high-level coproduction of 1-propanol and ethanol by developing novel engineered E. coli strains with effective cultivation strategies. Various biochemical, genetic, metabolic, and physiological factors affecting relative levels of acidogenesis and solventogenesis during anaerobic fermentation were investigated. In particular, CPC-PrOH3, a plasmid-free propanogenic E. coli strain derived by activating the Sbm operon on the genome, showed high levels of solventogenesis accounting for up to 85 % of dissimilated carbon. Anaerobic fed-batch cultivation of CPC-PrOH3 with glycerol as the major carbon source produced high titers of nearly 7 g/L 1-propanol and 31 g/L ethanol, implying its potential industrial applicability. The activated Sbm pathway served as an ancillary channel for consuming reducing equivalents upon anaerobic dissimilation of glycerol, resulting in an enhanced glycerol dissimilation and a major metabolic shift from acidogenesis to solventogenesis.

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.

Biotechnological advances on penicillin G acylase: pharmaceutical implications, unique expression mechanism and production strategies.

In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of ß-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform.

Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria.

As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems.