Journal
ACS SYNTHETIC BIOLOGY
Volume 6, Issue 1, Pages 139-147Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00176
Keywords
Type I modular polyketide synthase; acyltransferase domain; substrate specificity; protein engineering; synthetic biology tool
Categories
Funding
- Defense Advanced Research Projects Agency (DARPA), U.S. Department of Defense [HR001148071]
- National Science Foundation [MCB-1341894, EEC-0540879]
- Joint BioEnergy Institute - U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research [DEAC02-05CH11231]
- NNF Center for Biosustainability [Synthetic Biology Tools for Yeast] Funding Source: researchfish
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1341894] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1341414] Funding Source: National Science Foundation
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Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.
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