Understanding and engineering the gladiostatin biosynthetic pathway

Bacteria belonging to the Burkholderia genus thrive in various ecological niches, ranging from the plant rhizosphere to the human lung, and play critical roles in ecological interactions often through the secretion of bioactive secondary metabolites. In recent years, various Burkholderia species have been shown to produce a wide range of specialised metabolites, many of which are assembled by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic assembly lines.
We recently discovered gladiostatin, a novel member of the glutarimide class of antibiotics as a metabolite of Burkholderia gladioli BCC0238. Genome mining and gene knockout experiments identified a trans-AT PKS that is responsible for gladiostation biosynthesis. This PKS contains a unique AsfA-like domain for chain release, in addition to putative trans-acting KR, ER and other catalytic domains.
In this project, gladiostatin will be purified and the remaining ambiguities in its structure will be clarified via derivatisation and comparisons with synthetic standards / total synthesis. The biological activity of galdiostatin towards a range of prokaryotic and eukaryotic cells will be investigated. Key enzymes and PKS catalytic domains involved in gladiostatin biosynthesis will be overproduced and purified and their activity will investigated via intact protein mass spectrometry. Once a clearer picture of the mechanisms underpinning gladiostatin assembly is available, biosynthetic engineering experiments aimed at producing novel gladiostatin analogues will be pursued.