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. Here, tandem repeats in the DNA encoding 949 modules within 129 cis-acyltransferase PKSs were cataloged, and the locations of the corresponding amino acids within the module were identified. The most frequently inserted interdomain loop corresponds with the updated module boundary immediately downstream of the ketosynthase (KS), while the loops bordering the dehydratase are nearly intolerant to such insertions. From the 949 modules, no repetitive sequence loop insertions are located within ACP, and only 2 reside within KS, indicating the sensitivity of these domains to alteration.

How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates.

With the redefinition of polyketide synthase (PKS) modules, a new appreciation of their most downstream domain, the ketosynthase (KS), is emerging. In addition to performing its well-established role of generating a carbon-carbon bond between an acyl-CoA building block and a growing polyketide, it may gatekeep against incompletely processed intermediates. Here, we investigate 739 KSs from 92 primarily actinomycete, cis-acyltransferase assembly lines. When KSs were separated into 16 families based on the chemistries at the α- and ß-carbons of their polyketide substrates, a comparison of 32 substrate tunnel residues revealed unique sequence fingerprints. Surprisingly, additional fingerprints were detected when the chemistry at the γ-carbon was considered. Representative KSs were modeled bound to their natural polyketide substrates to better understand observed patterns, such as the substitution of a tryptophan by a smaller residue to accommodate an l-α-methyl group or the substitution of four smaller residues by larger ones to make better contact with a primer unit or diketide. Mutagenesis of a conserved glutamine in a KS within a model triketide synthase indicates that the substrate tunnel is sensitive to alteration and that engineering this KS to accept unnatural substrates may require several mutations.

Preparative production of an enantiomeric pair by engineered polyketide synthases.

Using the updated module boundary of polyketide assembly lines, modules from the pikromycin synthase were recombined into engineered synthases that furnish an enantiomeric pair of 2-stereocenter triketide lactones at >99% ee with yields up to 0.39 g per liter of E. coli K207-3 in shake flasks.