Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Schmidt, Matthias; Pearson, Allison N.; Incha, Matthew R.; Thompson, Mitchell G.; Baidoo, Edward K.; Kakumanu, Ramu; Mukhopadhyay, Aindrila; Shih, Patrick M.; Deutschbauer, Adam M.; Blank, Lars M.; Keasling, Jay D.

doi:10.1128/aem.02430-21

Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Journal Article · Mon Mar 14 00:00:00 EDT 2022 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.02430-21· OSTI ID:1969645

Schmidt, Matthias ^[1]; Pearson, Allison N. ^[2]; Incha, Matthew R. ^[2]; Thompson, Mitchell G. ^[3]; Baidoo, Edward K. ^[3]; Kakumanu, Ramu ^[4]; ^[3]; Shih, Patrick M. ^[5]; Deutschbauer, Adam M. ^[6]; Blank, Lars M. ^[7]; ^[8]

Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); RWTH Aachen University (Germany)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Joint BioEnergy Institute, Emeryville, California, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Joint BioEnergy Institute and Environmental Genomics and Systems Biology Division
University of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint BioEnergy Institute and Environmental Genomics and Systems Biology Division
RWTH Aachen University (Germany)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States). Joint Program in Bioengineering; Technical University of Denmark, Lyngby (Denmark); Shenzhen Institutes for Advanced Technologies, Shenzhen (China)

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. So 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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); German Research Foundation (DFG); Simons Foundation

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1969645

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 7 Vol. 88; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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

Journal Article · Thu Oct 15 00:00:00 EDT 2020 · Applied and Environmental Microbiology · OSTI ID:1826690

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59 BASIC BIOLOGICAL SCIENCES
BarSeq
RB-TnSeq
amino acid
aminotransferase
aminotransferases
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biosensors
lactam
metabolism
nitrate
nitrite
nitrogen
nitrogen metabolism
nucleotide
polyamine
pseudomonas putida
t-SNE
transposon

Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

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