Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Mitchell G Thompson; Matthew R Incha; Allison N Pearson; Matthias Schmidt; William A Sharpless; Christopher B Eiben; Pablo Cruz-Morales; Jacquelyn M Blake-Hedges; Yuzhong Liu; Catharine A Adams; Robert W Haushalter; Rohith N Krishna; Patrick Lichtner; Lars M Blank; Aindrila Mukhopadhyay; Adam M Deutschbauer; Patrick M Shih; Jay D Keasling

doi:10.1128/AEM.01665-20

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Appl Environ Microbiol. 2020 Oct 15;86(21):e01665-20. doi: 10.1128/AEM.01665-20. Print 2020 Oct 15.

Authors

Mitchell G Thompson^#^{1

2

3}, Matthew R Incha^#^{1

2

4}, Allison N Pearson^#^{1

2

4}, Matthias Schmidt^{1

2

5}, William A Sharpless^{1

2}, Christopher B Eiben^{1

2

6}, Pablo Cruz-Morales^{1

2

7}, Jacquelyn M Blake-Hedges^{1

2

8}, Yuzhong Liu^{1

2}, Catharine A Adams², Robert W Haushalter^{1

2}, Rohith N Krishna^{1

2}, Patrick Lichtner^{1

2}, Lars M Blank⁵, Aindrila Mukhopadhyay^{1

2}, Adam M Deutschbauer^{4

9}, Patrick M Shih^{10

2

3

11}, Jay D Keasling^{10

2

6

12

13

14

15}

Affiliations

¹ Joint BioEnergy Institute, Emeryville, California, USA.
² Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
³ Department of Plant Biology, University of California, Davis, California, USA.
⁴ Department of Plant and Microbial Biology, University of California, Berkeley, California, USA.
⁵ Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany.
⁶ Joint Program in Bioengineering, University of California, Berkeley, California, USA.
⁷ Centro de Biotecnología FEMSA, Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, México.
⁸ Department of Chemistry, University of California, Berkeley, California, USA.
⁹ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
¹⁰ Joint BioEnergy Institute, Emeryville, California, USA pmshih@lbl.gov jdkeasling@lbl.gov.
¹¹ Environmental and Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
¹² Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA.
¹³ Institute for Quantitative Biosciences, University of California, Berkeley, California, USA.
¹⁴ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
¹⁵ Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China.

^# Contributed equally.

Abstract

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Keywords: Fatty acid; Pseudomonas putida; RB–Tn-Seq; transposon.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Alcohols / metabolism*
DNA Transposable Elements*
DNA, Bacterial*
Fatty Acids / metabolism*
Metabolic Networks and Pathways
Pseudomonas putida / metabolism*
Sequence Analysis, DNA

Substances

Alcohols
DNA Transposable Elements
DNA, Bacterial
Fatty Acids