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

Autor:	Thompson MG; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Department of Plant Biology, University of California, Davis, California, USA., Incha MR; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Department of Plant and Microbial Biology, University of California, Berkeley, California, USA., Pearson AN; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Department of Plant and Microbial Biology, University of California, Berkeley, California, USA., Schmidt M; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany., Sharpless WA; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Eiben CB; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Joint Program in Bioengineering, University of California, Berkeley, California, USA., Cruz-Morales P; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Centro de Biotecnología FEMSA, Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, México., Blake-Hedges JM; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Department of Chemistry, University of California, Berkeley, California, USA., Liu Y; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Adams CA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Haushalter RW; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Krishna RN; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Lichtner P; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Blank LM; Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany., Mukhopadhyay A; Joint BioEnergy Institute, Emeryville, California, USA.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Deutschbauer AM; 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., Shih PM; Joint BioEnergy Institute, Emeryville, California, USA pmshih@lbl.gov jdkeasling@lbl.gov.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Department of Plant Biology, University of California, Davis, California, USA.; Environmental and Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Keasling JD; Joint BioEnergy Institute, Emeryville, California, USA pmshih@lbl.gov jdkeasling@lbl.gov.; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.; Joint Program in Bioengineering, University of California, Berkeley, California, USA.; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA.; Institute for Quantitative Biosciences, University of California, Berkeley, California, USA.; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.; Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China.
Jazyk:	angličtina
Zdroj:	Applied and environmental microbiology [Appl Environ Microbiol] 2020 Oct 15; Vol. 86 (21). Date of Electronic Publication: 2020 Oct 15 (Print Publication: 2020).
DOI:	10.1128/AEM.01665-20
Abstrakt:	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. (Copyright © 2020 American Society for Microbiology.)
Databáze:	MEDLINE
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