Heterologous biosynthetic pathway expression is an essential tool for natural products biochemists. It has provided a powerful methodology for elucidating and characterizing bacterial biosynthetic pathways. In this thesis I will discuss methods to harness biosynthetic pathways for the heterologous production of a monosaccharide natural product, Legionaminic acid (Leg5,7Ac2). This carbohydrate belongs to a family of sugars called nonulosonic acids (nine carbon α-keto acids) and is a 5,7-diamino derivative of sialic acid (Neu5Ac). It is found in cell surface glycoconjugates of bacteria including pathogens such as Helicobacter pylori, Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. Their presence on bacteria has been correlated with virulence in humans by mechanisms that likely involve subversion of the host’s immune system or interactions with host cell surfaces due to its similarity to sialic acid. Further investigation into their role in bacterial physiology and pathogenicity is limited as there are no effective methods to produce sufficient quantities of these carbohydrates. Herein, I harness microbial biosynthetic pathways via metabolic and genetic engineering to produce these complex nonulosonic acids. Leg5,7Ac2 is produced from N-acetylglucosamine using the Escherichia coli strain BRL04, which results in substantial over-production (> 100 mg L-1 of culture). Pure Leg5,7Ac2 could be readily isolated and converted into CMP-activated Leg5,7Ac2 for biochemical applications as well as the phenyl thioglycoside for chemical synthesis applications. A similar strategy was employed to access the related nonulosonic acid pseudaminic acid (Pse5,7Ac2). A biosynthetic pathway for production of Pse5,7Ac2 was constructed from H. pylori and C. jejuni and expressed in E. coli BRL04. Unlike Leg5,7Ac2, Pse5,7Ac2 was produced in low yields (< 20 mg L-1). A number of modifications were made to the biosynthetic constructs in an effort to enhance production levels yet improved titers were not obtained.Additionally, this thesis will look at the development of a new strategy for the heterologous expression of biosynthetic pathways in a number of diverse hosts. I will highlight a flexible in vivo heterologous expression system that was inspired by viral protein packaging, processing and cleavage to produce violacein, a bright purple pigment with anti-tumor properties. A de novo polyprotein design possessing the violacein biosynthetic pathway was shown to work effectively in prokaryotic hosts such as E. coli and S. typhimurium. Expression of the polyprotein design in eukaryotic hosts like mammalian cells and S. cerevisiae were less successful. The ultimate goal of the work presented herein is to highlight the flexibility and powerful nature of synthetic biology for the in vivo production of natural products in addition to contributing to the vast arsenal of techniques and strategies that are currently available to researchers in this field.

Un des enjeux en génie microbien est de produire des quantités élevées de protéines. Parmi les hôtes disponibles pour la production hétérologue de protéines, Lactococcus lactis est une bactérie à fort potentiel. Son innocuité et son caractère gram positif en fait un hôte de choix pour la production de protéines d'intérêt. Parmi ces protéines figurent des bactériocines, qui sont des proteines antibactériennes pouvant être utilisées à des fins de sécurité sanitaires des aliments voire comme antibiotique. Ces travaux ont pour ambition d'améliorer la production hétérologue de la bactériocine pédiocine chez L. lactis au niveau quantitatif et qualitatif en ciblant respectivement les étapes de sécrétion et de maturation post-traductionnelle. La sécrétion a été améliorée en insérant les pro-peptides SD ou LEISSTCDA entre le peptide signal de sécrétion et la séquence de la bactériocine. Il a été montré que cette insertions n’affectent pas l’activité antibactérienne. L’analyse in silico de l’opéron responsable de la production de pédiocine chez la bactérie productrice sauvage a révélé que le gène pedC code une protéine prédite comme thiol-disulfide oxydoréductase, suggérant un rôle de cette protéine dans le statut redox des cystéines de la pédiocine. La co-expression de PedC avec la pédiocine recombinante a permis d’augmenter son pouvoir antibactérien. Les résultats obtenus pendant cette thèse ont ainsi montré que la production de pédiocine par L. lactis peut être améliorée par fonctionnalisation de l’extrémité N-terminale sans effet significatif sur le potentiel antibactérien et que PedC joue un rôle majeur dans le potentiel antibactérien de la pédiocine / Lactococcus lactis is considered an efficient cell factory for recombinant protein production. It is able to produce and secrete class IIa bacteriocins such as pediocin PA-1 via the general secretion (Sec) pathway. However, the positive charges at the N-terminus of pediocin PA-1 might impair secretion via the Sec secretion pathway and the obtained recombinant pediocin has been described as less potent than the pediocin from the natural producer. The impact of two propeptides on the production yield and on the potency of recombinant pediocins was investigated. The nucleotide sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. Both propeptides improved secretion of the recombinant pediocins. Although no major impact on the antibacterial activity of recombinant pediocins was observed, all recombinant bacteriocins produced in L. lactis were less potent than wildtype pediocin. Co-expression of the putative thiol-disulfide oxidoreductase PedC, which is encoded by the pediocin PA-1 operon, with the recombinant pediocins allowed to significantly decrease the minimal inhibitory concentration of the produced bacteriocins. To our knowledge, this report shows for the first time that the propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC plays a major role in the potency of pediocin