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

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

doi:10.1128/aem.01665-20

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

Journal Article · Fri Aug 21 00:00:00 EDT 2020 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.01665-20· OSTI ID:1826706

Thompson, Mitchell G. ^[1]; Incha, Matthew R. ^[2]; Pearson, Allison N. ^[2]; Schmidt, Matthias ^[3]; Sharpless, William A. ^[4]; Eiben, Christopher B. ^[2]; Cruz-Morales, Pablo ^[5]; Blake-Hedges, Jacquelyn M. ^[2]; Liu, Yuzhong ^[4]; Adams, Catharine A. ^[6]; Haushalter, Robert W. ^[4]; Krishna, Rohith N. ^[4]; Lichtner, Patrick ^[4]; Blank, Lars M. ^[7];

^[4]; Deutschbauer, Adam M. ^[8];

^[1];

^[9]

Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Davis, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); RWTH Aachen Univ. (Germany). Institute of Applied Microbiology (iAMB)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey (México). Centro de Biotecnología FEMSA
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
RWTH Aachen Univ. (Germany). Institute of Applied Microbiology (iAMB)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark); Shenzhen Institutes for Advanced Technologies, Shenzhen (China)

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.

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

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); German Research Foundation (DFG)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1826706

Alternate ID(s):: OSTI ID: 1826690

Journal Information:: Applied and Environmental Microbiology, Vol. 86, Issue 21; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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

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