Synthetic biology approach for polyketide engineering and enzyme function elucidation

Abstract

For decades, actinomycetes have been a major source of antibacterial and antiproliferative compounds, providing medicine with drugs invaluable for the treatment of a variety of diseases and types of cancer. The discovery of antibiotics allowed humanity to combat infections that would have been lethal otherwise, which puts them among the most transformative medical innovations in human history. Nowadays, widespread antibiotic use in agriculture and farming has led to the rise in bacterial antibiotic resistance, which, together with a rising number of cancer cases worldwide, creates a critical need for the discovery and synthesis of new bioactive compounds. Synthetic biology offers a comprehensive toolbox for the development of new, biologically active natural products, presenting a compelling alternative to conventional drug discovery methods.

The research presented in this thesis demonstrates how a synthetic biology approach, particularly the BioBrick technique, can facilitate the discovery and production of secondary metabolites in Streptomyces strains. First, we developed a BioBrick-based synthetic biology toolbox for the de novo construction of biosynthetic pathways, which proves to be especially effective in stepwise pathway building. We assembled eight deoxysugar pathways and expressed them in a heterologous Streptomyces host carrying 8-demethyl-tetracenomycin C aglycone, which resulted in the production of eight compounds, including four new glycosylated tetracenomycins.

Secondly, we characterised the functions of key enzymes in the chartreusin pathway using a BioBrick-based synthetic biology platform. We discovered a three-enzyme cascade responsible for converting an intermediate aglycone, auramycinone, into resomycin C through a double dehydration reaction. We proved that the 9,10-dehydration of auramycinone is catalysed by an enzyme pair ChaX/ChaU, while 7,8-dehydration is catalysed by the ChaJ enzyme.

Together, these findings establish synthetic biology as a valuable tool in both pathway assembly and enzyme function elucidation. They shed a new light on the elloramycin glycodiversification as well as the process of double dehydration of auramycinone in chartreusin biosynthesis. The synthetic biology approach used here can help expand our ability to engineer and manipulate biosynthetic pathways for the future production of valuable compounds.