Dissection and Engineering of Modular Polyketide Synthase Extender Unit Specificity Motifs

Area of Science:

• Biochemistry
• Synthetic Biology
• Natural Product Discovery

Background:

• Polyketide synthases (PKSs) are crucial for natural product biosynthesis.
• Acyltransferase (AT) domains dictate extender unit specificity in PKSs.
• Understanding AT domain sequence-function relationships is key for engineering PKSs.

Purpose of the Study:

• To identify sequence elements governing extender unit specificity in the EryAT6 acyltransferase domain.
• To explore the modularity and engineering potential of conserved motifs within AT domains.
• To develop strategies for accessing diverse polyketides through PKS redesign.

Main Methods:

• Site-directed mutagenesis of conserved motifs (LSM and SSM) in EryAT6.
• Functional reconstitution of engineered EryAT6 variants in the Ery6TE system.
• Analysis of extender unit incorporation using non-native substrates like butylmalonyl-CoA.

Main Results:

• Specific residue substitutions within LSM and SSM significantly altered extender unit selectivity.
• A triple-residue mutation enhanced butylmalonyl-CoA incorporation by 42-fold.
• Exchanging LSM and SSM motifs reprogrammed AT selectivity, enabling the formation of butyl-substituted pyrones.

Conclusions:

• Compact sequence motifs within AT domains are critical determinants of extender unit specificity.
• Motif-level engineering of PKS AT domains is a viable strategy for generating novel polyketides.
• This work provides insights into the rational design of PKS for diverse natural product synthesis.