Nitrogen metabolism in Pseudomonas putida : functional analysis using random barcode transposon sequencing

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida’s diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.

Authors 11

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. Allison N. Pearson

   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 Plant and Microbial Biology, 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

   Department of Plant and Microbial Biology, University of California, Berkeley, CA 9720, USA

3. Matthew R. Incha

   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 Plant and Microbial Biology, 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

   Department of Plant and Microbial Biology, University of California, Berkeley, CA 9720, USA

4. Mitchell G. Thompson

   Lawrence Berkeley National Laboratory · Joint BioEnergy Institute

   Affiliation as printed

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

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

5. Edward E. K. Baidoo

   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

6. Ramu D. Kakumanu

   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

7. Aindrila Mukhopadhyay

   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

8. Patrick M. Shih

   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 Plant and Microbial Biology, 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

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

   Department of Plant and Microbial Biology, University of California, Berkeley, CA 9720, 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, Berkeley, CA 94720, USA

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

   Department of Plant and Microbial Biology, University of California, Berkeley, CA 9720, USA

10. 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

11. 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

    Institute for Quantitative Biosciences, University of California, Berkeley, California, USA

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

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

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