PhD Defence Michal Poborsky

Title of thesis: Engineering Escherichia coli towards production of plant specialized metabolites

Abstract

Plant specialized metabolites are an incredibly diverse group of natural products that are historically valuable to medicine and industry. The native plant producers often pose a challenge as they accumulate specialized metabolites at low amounts and through a tightly-regulated, complex biosynthetic pathways. To overcome these hurdles, the intersection of metabolic engineering and synthetic biology aims to realize the production of natural products in microbial cell factories. Glucosinolates are amino acid-derived and sulfur-rich specialized metabolites characteristic of brasicaceous plants. Recognized as promoters of human health, some glucosinolates attract attention as potential nutraceuticals. In this thesis, we present a systematic engineering of the complex glucosinolate biosynthetic pathway in Escherichia coli.

The following chapters address the major challenges associated with glucosinolate production in bacteria. We start by engineering the expression of two ER-bound cytochrome P450 enzymes, CYP79 and CYP83, that control the entrance to the glucosinolate pathway. Attempting to accommodate the demanding glucosinolate core structure pathway, we then focus on establishing sufficient cofactor supply to support high product titers. Finally, we screen natural homologs of the pathway enzymes with improved substrate specificity to increase pathway throughput and overcome by-product formation.

Following these principles, we show the first successful bacterial production of indolyl-3-methyl and p-hydroxybutyl glucosinolates. We engineer the biosynthesis of the most attractive glucosinolate product, glucoraphanin, increasing its production >5000-fold and report the biosynthesis of indolyl-3-methyl glucosinolate reaching 561 mg/L, the highest glucosinolate titer in microbes to date.

Assessment committee
Professor Marjan De Mey, Ghent University, Belgium
Senior Researcher, Morten Nørholm, DTU Biosustain, Denmark
Associate Professor Fernando Geu-Flores, PLEN, KU

Reception afterwards in the M117, Thorvaldsensvej 40, 1st floor

Details
Time: 5 July 2022, 13:00-16:00

Place: Festauditoriet A1-01.01

Organizer: DynaMo Center