Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

Nature Communications, vol. 14, pp. 4871

Abstract

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

Authors 9

1. Elias Englund

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

   Affiliation as printed

   Joint BioEnergy Institute, Emeryville, CA, USA

   School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, KTH - Royal Institute of Technology, 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, CA, USA

   Institute of Applied Microbiology, Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany

   Joint BioEnergy Institute, Emeryville, CA, USA

3. Alberto A. Nava

   Joint BioEnergy Institute · University of California, Berkeley

   Affiliation as printed

   Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

4. Sarah H. Klass

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

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA

   Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

5. Leah S. Keiser

   Joint BioEnergy Institute · University of California, Berkeley

   Affiliation as printed

   Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

6. Qingyun Dan

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

7. Leonard Katz

   Joint BioEnergy Institute · University of California, Berkeley

   Affiliation as printed

   Joint BioEnergy Institute, Emeryville, CA, USA

   QB3, University of California, Berkeley, Berkeley, CA, USA

8. Satoshi Yuzawa

   Lawrence Berkeley National Laboratory · Keio University · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA

   Graduate school of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan

   Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan

   Joint BioEnergy Institute, Emeryville, CA, USA

9. Jay D. Keasling corresponding

   Lawrence Berkeley National Laboratory · Shenzhen Institutes of Advanced Technology · Shenzhen Institute of Synthetic Biology · Joint BioEnergy Institute · University of California, Berkeley · Technical University of Denmark

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA. jdkeasling@lbl.gov

   Center for Biosustainability, Danish Technical University, Lyngby, Denmark. jdkeasling@lbl.gov

   Center for Synthetic biochemistry, Institute for Synthetic biology, Shenzhen Institute of Advanced Technology, Shenzhen, China. jdkeasling@lbl.gov

   Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. jdkeasling@lbl.gov

   Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA. jdkeasling@lbl.gov

   Joint BioEnergy Institute, Emeryville, CA, USA. jdkeasling@lbl.gov

   QB3, University of California, Berkeley, Berkeley, CA, USA. jdkeasling@lbl.gov

   Biological Systems and Engineering Division, Lawrence Berkeley National laboratory, Berkeley, CA, USA

   Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

   QB3, University of California, Berkeley, Berkeley, CA, USA