Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains.

Area of Science:

• Synthetic biology
• Biochemistry
• Enzyme engineering

Background:

• Engineering assembly line polyketide synthases (PKSs) for novel bioactive compound production is a long-standing goal.
• Previous engineering efforts focused on module or domain exchange, often disrupting evolutionarily optimized domain-domain interactions and reducing PKS turnover rates.

Purpose of the Study:

• To define the least invasive interface for PKS engineering and expand the available engineering toolbox.
• To compare different types of chimeric PKSs, focusing on the impact of intermodular and intramodular interfaces on enzyme activity.

Main Methods:

• Generation of bimodular chimeric PKSs with either covalent linkers or noncovalent SYNZIP domain-mediated interfaces between modules.
• Introduction of noncovalent interfaces within modules, between condensing and processing domains.
• Comparison of chimeric PKS systems to assess their impact on catalytic cycle steps like chain translocation, acylation, and elongation.

Main Results:

• SYNZIP domains can efficiently substitute for natural PKS docking domains.
• A novel noncovalent interface between condensing and processing domains within a module can be utilized for PKS engineering.
• Established SYNZIP domains as a new tool for engineering PKSs by bridging non-native interfaces without perturbing PKS activity.

Conclusions:

• SYNZIP domains offer a versatile and effective method for engineering PKSs.
• The study expands the PKS engineering toolbox by demonstrating the utility of SYNZIP domains for creating functional chimeric enzymes.
• This work provides a foundation for designing more complex and efficient PKS systems for novel compound synthesis.