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.

Modular polyketide synthase ketosynthases collaborate with upstream dehydratases to install double bonds.

A VMYH motif was determined to help ketosynthases in polyketide assembly lines select α,β-unsaturated intermediates from an equilibrium mediated by an upstream dehydratase. Alterations of this motif decreased ketosynthase selectivity within a model tetraketide synthase, most significantly when replaced by the TNGQ motif of ketosynthases that accept D-β-hydroxy intermediates.

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.

Crystal structures reveal the framework of -acyltransferase modular polyketide synthases.

Although the domains of -acyltransferase ( -AT) modular polyketide synthases (PKS's) have been understood at atomic resolution for over a decade, the domain-domain interactions responsible for the architectures and activities of these giant molecular assembly lines remain largely uncharacterized. The multimeric structure of the α β fungal fatty acid synthase (FAS) provides 6 equivalent reaction chambers for its acyl carrier protein (ACP) domains to shuttle carbon building blocks and the growing acyl chain between surrounding, oriented enzymatic domains. The presumed homodimeric oligomerization of -AT assembly lines is insufficient to provide similar reaction chambers; however, the crystal structure of a ketosynthase (KS)+AT didomain presented here and three already reported show an interaction between the AT domains appropriate for lateral multimerization.

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.