RB-TnSeq elucidates dicarboxylic-acid-specific catabolism in β-proteobacteria for improved plastic monomer upcycling

Applied and Environmental Microbiology, vol. 91, pp. e0092425

Abstract

ABSTRACT Dicarboxylic acids are key components of many polymers and plastics, making them a target for both engineered microbial degradation and sustainable bioproduction. In this study, we generated a comprehensive data set of functional evidence for the genetic basis of dicarboxylic and fatty acid metabolism using randomly barcoded transposon sequencing (RB-TnSeq). We identified four β-proteobacteria that displayed robust growth with dicarboxylic acid sole carbon source and cultured their mutant libraries with dicarboxylic and fatty acids with carbon chain lengths from C3 to C12. The resulting fitness data suggested that dicarboxylic and fatty acid metabolisms are largely distinct, and different sets of β-oxidation genes are required for catabolizing dicarboxylic versus fatty acids of the same carbon chain lengths. In addition, we identified transcriptional regulators and transporters with strong fitness phenotypes related to dicarboxylic acid utilization. In Ralstonia sp . UNC404CL21Col ( R. CL21 ), we deleted two transcriptional repressors to improve its utilization of short-chain dicarboxylic acids. We exploited the diacid-utilizing catabolism of R. CL21 to upcycle a mock mixture of the dicarboxylic acids produced when polyethylene is oxidized. After introducing a heterologous indigoidine production pathway, this engineered Ralstonia produced 0.56 ± 0.02 g/L indigoidine from a mixture of dicarboxylic acids as a carbon source, demonstrating the potential of R. CL21 to upcycle plastic wastes to products derived from tricarboxylic acid (TCA) cycle intermediates. IMPORTANCE Upcycling the carbon in plastic wastes to value-added products is a promising approach to address the plastic waste and climate crises, and dicarboxylic acid metabolism is an important facet of several approaches. Improving our understanding of the genetic basis of this metabolism has the potential to uncover new enzymes and genetic parts for engineered pathways involving dicarboxylic acids. Our data set is the most comprehensive interrogation of dicarboxylic acid catabolism to date, and this work will be of utility to researchers interested in both plastics bioproduction and upcycling applications.

Authors 12

1. Allison N. Pearson

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Department of Plant and Microbial Biology, University of California

   Joint BioEnergy Institute

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

   Department of Plant and Microbial Biology, University of California, Berkeley, California, USA

   Joint BioEnergy Institute, Emeryville, California, USA

2. Graham A. Hudson

   Lawrence Berkeley National Laboratory · University of California, Berkeley · QB3 · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   California Institute for Quantitative Biosciences (QB3 Institute), University of California

   Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory

   Joint BioEnergy Institute

   California Institute for Quantitative Biosciences (QB3 Institute), University of California, Berkeley, California, USA

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

   Joint BioEnergy Institute, Emeryville, California, USA

3. Jacob B. Roberts

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Joint BioEnergy Institute

   Joint Program in Bioengineering, University of California

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

   Joint BioEnergy Institute, Emeryville, California, USA

   Joint Program in Bioengineering, University of California, Berkeley, California, USA

4. Javier Menasalvas

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Joint BioEnergy Institute

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

   Joint BioEnergy Institute, Emeryville, California, USA

5. Aaron A. Vilchez

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Department of Chemical and Biomolecular Engineering, University of California

   Joint BioEnergy Institute

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

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

   Joint BioEnergy Institute, Emeryville, California, USA

6. Matthew R. Incha

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

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Department of Plant and Microbial Biology, University of California

   Joint BioEnergy Institute

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

   Department of Plant and Microbial Biology, University of California, Berkeley, California, USA

   Joint BioEnergy Institute, Emeryville, California, USA

7. Matthias Schmidt Aachen Institute of Applied Microbiology

   RWTH Aachen University · Lawrence Berkeley National Laboratory · University of California, Berkeley · QB3 · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   California Institute for Quantitative Biosciences (QB3 Institute), University of California

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

   Joint BioEnergy Institute

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

   California Institute for Quantitative Biosciences (QB3 Institute), University of California, Berkeley, California, USA

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

   Joint BioEnergy Institute, Emeryville, California, USA

8. Aindrila Mukhopadhyay

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

   Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

   Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory

   Joint BioEnergy Institute

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

   Joint BioEnergy Institute, Emeryville, California, USA

9. Adam M. Deutschbauer

   Lawrence Berkeley National Laboratory · University of California, Berkeley

   Affiliation as printed

   Department of Plant and Microbial Biology, University of California

   Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory

   Department of Plant and Microbial Biology, University of California, Berkeley, California, USA

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

10. Mitchell G. Thompson

    Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

    Affiliation as printed

    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory

    Joint BioEnergy Institute

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

    Joint BioEnergy Institute, Emeryville, California, USA

11. Patrick M. Shih

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

    Affiliation as printed

    Department of Plant and Microbial Biology, University of California

    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory

    Innovative Genomics Institute, University of California

    Joint BioEnergy Institute

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

    Innovative Genomics Institute, University of California, Berkeley, California, USA

    Joint BioEnergy Institute, Emeryville, California, USA

12. Jay D. Keasling

    Lawrence Berkeley National Laboratory · University of California, Berkeley · Technical University of Denmark · Novo Nordisk Foundation · University of California System · Joint BioEnergy Institute

    Affiliation as printed

    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory

    Department of Chemical and Biomolecular Engineering, University of California

    Joint BioEnergy Institute

    Joint Program in Bioengineering, University of California

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

    Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Joint BioEnergy Institute, Emeryville, California, USA

    Joint Program in Bioengineering, University of California, Berkeley, California, USA

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