Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase

Journal of the American Chemical Society, vol. 145, pp. 8822–8832

Abstract

High Resolution Image Download MS PowerPoint Slide Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro .

Authors 20

1. Elias Englund

   Science for Life Laboratory · Joint BioEnergy Institute · KTH Royal Institute of Technology

   Affiliation as printed

   Joint BioEnergy Institute, Emeryville, California 94608, United States

   Science for Life Laboratory, KTH - Royal Institute of Technology, 17165 Stockholm, Sweden

2. Matthias Schmidt Aachen Institute of Applied Microbiology

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute · RWTH Aachen University

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

   Institute of Applied Microbiology, Aachen Biology and Biotechnology, RWTH Aachen University, 52074 Aachen, Germany

   Joint BioEnergy Institute, Emeryville, California 94608, United States

3. Alberto A. Nava

   Lawrence Berkeley National Laboratory · University of California, Berkeley

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

   Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States

4. Anna Lechner

   University of California, Berkeley

   Affiliation as printed

   Department of Bioengineering, University of California, Berkeley, California 94720, United States

   Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States

5. Kai Deng

   Sandia National Laboratories California · Joint BioEnergy Institute

   Affiliation as printed

   Department of Biomaterials and Biomanufacturing, Sandia National Laboratory, Livermore, California 94550, United States

   Joint BioEnergy Institute, Emeryville, California 94608, United States

6. Renee Jocic

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

   Joint BioEnergy Institute, Emeryville, California 94608, United States

7. Yingxin Lin

   University of California, Berkeley

   Affiliation as printed

   Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States

8. Jacob B. Roberts

   Lawrence Berkeley National Laboratory · University of California, Berkeley

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

   Department of Bioengineering, University of California, Berkeley, California 94720, United States

9. Veronica Teixeira Benites

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

   Joint BioEnergy Institute, Emeryville, California 94608, United States

10. Ramu D. Kakumanu

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

11. Jennifer W. Gin

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

12. Yan Chen

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

13. Yuzhong Liu

    University of California, Berkeley

    Affiliation as printed

    Department of Bioengineering, University of California, Berkeley, California 94720, United States

    Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States

14. Christopher J. Petzold

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

15. Edward E. K. Baidoo

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

16. Trent R. Northen

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

17. Paul D. Adams

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute · University of California, Berkeley

    Affiliation as printed

    Department of Bioengineering, University of California, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

    Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

18. Leonard Katz

    QB3 · Joint BioEnergy Institute · University of California, Berkeley

    Affiliation as printed

    Joint BioEnergy Institute, Emeryville, California 94608, United States

    QB3 Institute, University of California, Berkeley, California 94720, United States

19. Satoshi Yuzawa corresponding

    Lawrence Berkeley National Laboratory · Keio University · Keio University Shonan Fujisawa · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Institute of Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan

    Joint BioEnergy Institute, Emeryville, California 94608, United States

    Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-0882, Japan

20. Jay D. Keasling corresponding

    QB3 · Lawrence Berkeley National Laboratory · Sandia National Laboratories California · Novo Nordisk Foundation · Shenzhen Institutes of Advanced Technology · Joint BioEnergy Institute · University of California, Berkeley · Technical University of Denmark

    Affiliation as printed

    Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States

    Department of Bioengineering, University of California, Berkeley, California 94720, United States

    Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States

    Joint BioEnergy Institute, Emeryville, California 94608, United States

    QB3 Institute, University of California, Berkeley, California 94720, United States

    Center for Synthetic Biochemistry, Shenzhen Institutes of Advanced Technologies, Shenzhen, Guangdong 518055, China

    Department of Biomaterials and Biomanufacturing, Sandia National Laboratory, Livermore, California 94550, United States

    Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark