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Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing
- Source :
- Appl Environ Microbiol, Applied and environmental microbiology, vol 86, iss 21
- Publication Year :
- 2020
- Publisher :
- American Society for Microbiology, 2020.
-
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.
- Subjects :
- DNA, Bacterial
transposon
Coenzyme A
Genetics and Molecular Biology
Computational biology
Biology
Microbiology
Applied Microbiology and Biotechnology
Metabolic engineering
03 medical and health sciences
chemistry.chemical_compound
Genetics
030304 developmental biology
RB–Tn-Seq
chemistry.chemical_classification
0303 health sciences
Ecology
Pseudomonas putida
030306 microbiology
Catabolism
Prevention
Human Genome
Fatty Acids
Bacterial
Substance Abuse
Fatty acid
DNA
Sequence Analysis, DNA
Isoprenol
biology.organism_classification
Metabolic pathway
chemistry
Alcohols
DNA Transposable Elements
Sequence Analysis
Flux (metabolism)
Metabolic Networks and Pathways
RB-Tn-Seq
Food Science
Biotechnology
Subjects
Details
- ISSN :
- 10985336 and 00992240
- Volume :
- 86
- Database :
- OpenAIRE
- Journal :
- Applied and Environmental Microbiology
- Accession number :
- edsair.doi.dedup.....52a67ed1b9fa7153746daab712661442