Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Type I polyketide synthases (T1PKSs) hold an enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite the great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum , Escherichia coli , and Pseudomonas putida . Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enables the production of an unnatural polyketide in each of the hosts. Furthermore, we developed a free online tool ( https://basebuddy.lbl.gov ) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. Here, we not only highlight the significance of codon optimization but also establish the groundwork for high-throughput assembly and characterization of PKS pathways in alternative hosts.

Authors 12

1. Matthias Schmidt Aachen Institute of Applied Microbiology

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

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

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

2. Namil Lee

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

3. Chunjun Zhan

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

4. Jacob B. Roberts

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Joint Program in Bioengineering, University of California, Berkeley/San Francisco, CA 94720, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

5. Alberto A. Nava

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

6. Leah S. Keiser

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

7. Aaron A. Vilchez

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

8. Yan Chen

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

9. Christopher J. Petzold

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

   Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

10. Robert W. Haushalter

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

    Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

11. Lars M. Blank Aachen Institute of Applied Microbiology

    RWTH Aachen University

    Affiliation as printed

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

12. Jay D. Keasling corresponding

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

    Affiliation as printed

    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 9470, USA

    Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China

    Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

    Department of Chemistry, University of California, Berkeley, CA 94720, USA

    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA

    Joint Program in Bioengineering, University of California, Berkeley/San Francisco, CA 94720, USA

    The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark