Harnessing Microbial Biosynthetic Pathways for the Production of Complex Molecules
Creators: name: Hassan, Mohamed
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2020
Subjects: Synthetic Biology
Microbial Engineering

description: 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 (&gt; 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 (&lt; 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.