title: Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production.
abstract: 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.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yuzawa
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.|Sandia National Laboratories , Livermore, California 94551, United States.
authors.forename: Kai
authors.initials: K
authors.lastname: Deng
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Synthetic Biology Research Center , Emeryville, California 94608, United States.
authors.forename: Leonard
authors.initials: L
authors.lastname: Katz
authors.affiliation: Joint BioEnergy Institute , Emeryville, California 94608, United States.|Synthetic Biology Research Center , Emeryville, California 94608, United States.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé, DK2970-Hørsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D066298:In Vitro Techniques; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D058615:Synthetic Biology
chemical_list: D000214:Acyl Coenzyme A; D007659:Ketones; D011994:Recombinant Proteins; D048630:Polyketide Synthases; D000217:Acyltransferases
keywords: Type I modular polyketide synthase; acyltransferase domain; protein engineering; substrate specificity; synthetic biology tool