Ketoreductase domain mutagenesis reprogrammes chain-length control in alternapyrone biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Alternapyrone is a decaketide-derived compound produced by alternapyrone polyketide synthase.
• Polyketide synthases (PKS) are large multi-modular enzymes crucial for producing diverse secondary metabolites.
• The ketoreductase (KR) domain is a key catalytic domain within PKS, involved in modifying nascent polyketide chains.

Purpose of the Study:

• To investigate the function of the ketoreductase (KR) domain in alternapyrone polyketide synthase (PKS).
• To determine the impact of KR domain mutation on alternapyrone biosynthesis and PKS product profile.
• To elucidate the role of the KR domain in controlling polyketide chain length and starter unit selection.

Main Methods:

• Site-directed mutagenesis was used to mutate the ketoreductase domain of alternapyrone PKS.
• Analysis of PKS products was performed using techniques such as HPLC and Mass Spectrometry.
• Investigated the utilization of different starter units (C2-C8) by the mutated PKS.

Main Results:

• Mutation of the KR domain abolished the production of decaketide-derived alternapyrone.
• Biosynthesis was redirected to produce non-reduced triketide-derived α-pyrones.
• The mutated PKS exhibited promiscuous utilization of starter units ranging from C2 to C8.

Conclusions:

• The ketoreductase domain plays a critical role in controlling polyketide chain-length programming during alternapyrone biosynthesis.
• Loss of KR domain function leads to the production of shorter, non-reduced polyketide products.
• The KR domain influences substrate specificity and chain termination events in PKS.