Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. The recently updated module boundary has been successful for engineering short synthases, yet larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases to test every module combination of the pikromycin synthase.

Mutagenesis Supports AlphaFold Prediction of How Modular Polyketide Synthase Acyl Carrier Proteins Dock With Downstream Ketosynthases.

The docking of an acyl carrier protein (ACP) domain with a downstream ketosynthase (KS) domain in each module of a polyketide synthase (PKS) helps ensure accurate biosynthesis. If the polyketide chain bound to the ACP has been properly modified by upstream processing enzymes and is compatible with gatekeeping residues in the KS tunnel, a transacylation reaction can transfer it from the 18.1-Å phosphopantetheinyl arm of the ACP to the reactive cysteine of the KS.

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase.

Insights into modular polyketide synthase loops aided by repetitive sequences.

The loops of modular polyketide synthases (PKSs) serve diverse functions but are largely uncharacterized. They frequently contain amino acid repeats resulting from genetic events such as slipped-strand mispairing. Determining the tolerance of loops to amino acid changes would aid in understanding and engineering these multidomain molecule factories.