Thiotemplated Polyketide Chain Fusion and Reductive Cyclization Build the Reactive Butenolide Core of Malleicyprol

Area of Science:

• Biochemistry
• Synthetic Biology
• Microbiology

Background:

• Malleicyprol is a critical virulence factor in Burkholderia pseudomallei (BP) group pathogens.
• Its complex structure, featuring a cyclopropanol warhead and butenolide core, suggested a unique biosynthetic pathway involving two polyketide chains, but the mechanism remained unclear.

Purpose of the Study:

• To elucidate the precise mechanism of malleicyprol biosynthesis.
• To understand the role of the PKS-NRPS hybrid synthetase BurF in this process.
• To explore potential applications in synthetic biology and antivirulence strategies.

Main Methods:

• Chemical synthesis of malleicyprol.
• In vitro reconstitution of the biotransformation pathway.
• Mutational analysis of the BurF synthetase.
• Biochemical assays and substrate surrogate trapping.
• Biomimetic synthesis for mechanism confirmation.

Main Results:

• Two distinct polyketide chains are joined by a noncanonical condensation domain of BurF, forming an ester bond.
• The terminal reductase domain mediates reductive chain release and subsequent ring formation.
• A Knoevenagel-type intramolecular cyclization forms the butenolide moiety.
• The biosynthesis involves an unprecedented thiotemplated, head-to-head fusion of polyketide chains.

Conclusions:

• The study reveals an unprecedented thiotemplated butenolide biosynthesis pathway.
• This mechanism involves the head-to-head fusion of two polyketide chains.
• Findings expand the synthetic biology toolbox and offer insights for developing antivirulence strategies against BP infections.