Skeletal editing via multi-step engineering of a modular polyketide synthase

Area of Science:

• Biochemistry
• Synthetic Biology
• Metabolic Engineering

Background:

• Assembly line biosynthesis via modular polyketide synthases (PKS) produces diverse natural products.
• The collinearity of PKS architecture and product structure suggests potential for designer biosynthesis.
• Reliable strategies for reprogramming PKS without activity loss are needed.

Purpose of the Study:

• To establish best practices for PKS engineering.
• To demonstrate reprogramming of the mediomycin PKS for novel compound synthesis.
• To develop a rational strategy for accessing complex organic molecules via PKS engineering.

Main Methods:

• In vitro CRISPR/Cas9 gene editing for PKS modification.
• Heterologous expression of engineered PKS constructs.
• Module editing using an evolution-supported cut site downstream of the acyltransferase domain.
• Thioesterase swapping for accessing different molecular architectures.

Main Results:

• Reconstruction of tetrafibricin, a drug lead for the fibrinogen receptor, at a yield of 82 ± 3 mg/L.
• Retained 26% productivity after a five-step module edit in the PKS.
• Successful synthesis of a macrocyclic aminopolyol via thioesterase swapping.
• Demonstrated reprogramming of the mediomycin PKS without significant loss of productivity.

Conclusions:

• Established a best practice for PKS engineering through successful reprogramming.
• Validated CRISPR/Cas9 gene editing and specific cut sites for efficient PKS module editing.
• Paved the way for rational PKS reprogramming to access diverse and complex natural products for drug discovery.