Site directed mutagenesis as a precision tool to enable synthetic biology with engineered modular polyketide synthases

Deciphering and engineering the polyunsaturated fatty acid synthase pathway from eukaryotic microorganisms

“…Utilizing random mutagenesis and screening, Hayashi et al demonstrated that the amino acid mutation of F65L, F230L and I231T, located in the KS B of Aurantiochytrium eukaryotic PUFA synthase, can change the main final product from the main DHA (EPA/DHA = 0) to the main EPA (EPA/DHA=1.07) in E. coli ( Hayashi et al, 2019a ). In addition, site-directed mutagenesis is also a precise way to introduce molecular diversity with less potential for global disruption of the protein architecture ( Drufva et al, 2020 ). For instance, targeted point mutagenesis to residues in the PKS could alter domain specificity or selectivity, affect protein stability and interdomain communication, and promote more complex catalytic reactivity ( Drufva et al, 2020 ).…”

“…In addition, site-directed mutagenesis is also a precise way to introduce molecular diversity with less potential for global disruption of the protein architecture ( Drufva et al, 2020 ). For instance, targeted point mutagenesis to residues in the PKS could alter domain specificity or selectivity, affect protein stability and interdomain communication, and promote more complex catalytic reactivity ( Drufva et al, 2020 ). There are also examples in the study of PUFA synthase.…”

Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics

“…1 The core structures of macrolides are biosynthesized by large, modular enzymes known as polyketide synthases (PKSs). 2,3 The prototypical macrolide antibiotic, erythromycin A (ErA), while clinically useful, has the limitations of acid instability and limited bioavailability. 4 Clarithromycin (CLA), a secondgeneration macrolide antibiotic derived from ErA (Figure 1A), effectively addresses these limitations, while also expanding the scope of activity to other microorganisms not impacted by ErA.…”

“…Macrolides are a large class of natural products that display potent biological activities that span many therapeutic areas . The core structures of macrolides are biosynthesized by large, modular enzymes known as polyketide synthases (PKSs). , The prototypical macrolide antibiotic, erythromycin A (ErA), while clinically useful, has the limitations of acid instability and limited bioavailability . Clarithromycin (CLA), a second-generation macrolide antibiotic derived from ErA (Figure A), effectively addresses these limitations, while also expanding the scope of activity to other microorganisms not impacted by ErA. − Moreover, CLA has served as a platform for the synthesis and derivatization of ketolide antibiotics including telithromycin and solithromycin (Figure B). − CLA and ErA differ at the C6-position of the macrolactone core: the C6-hydroxyl group in ErA is replaced with a methoxy group in CLA.…”

Natural products research in the modern age

“…Drufva and colleagues reviewed the potential of using a site-directed mutagenesis approach to enable synthetic biology with engineered modular polyketide synthases. Reviewed and discussed are a number of examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity, affect protein stability and interdomain communication, and promote more complex catalytic reactivity [ 7 ].…”