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Title: Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds in Novosphingobium aromaticivorans

Abstract

Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here we show that, when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously-hypothetical enzymes in an unbiased genome-scale search. We use this technique to identify two genes, which we name desC and desD, are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the non-model bacterium Novosphingobium aromaticivorans. We show that DesC is a methyl-esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. Here, this approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering.

Authors:
 [1];  [2]; ORCiD logo [3]; ORCiD logo [3]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Arizona, Tucson, AZ (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1471938
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 84; Journal Issue: 22; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES

Citation Formats

Cecil, Jacob H., Garcia, David C., Giannone, Richard J., and Michener, Joshua K.. Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds in Novosphingobium aromaticivorans. United States: N. p., 2018. Web. https://doi.org/10.1128/AEM.01185-18.
Cecil, Jacob H., Garcia, David C., Giannone, Richard J., & Michener, Joshua K.. Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds in Novosphingobium aromaticivorans. United States. https://doi.org/10.1128/AEM.01185-18
Cecil, Jacob H., Garcia, David C., Giannone, Richard J., and Michener, Joshua K.. Fri . "Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds in Novosphingobium aromaticivorans". United States. https://doi.org/10.1128/AEM.01185-18. https://www.osti.gov/servlets/purl/1471938.
@article{osti_1471938,
title = {Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds in Novosphingobium aromaticivorans},
author = {Cecil, Jacob H. and Garcia, David C. and Giannone, Richard J. and Michener, Joshua K.},
abstractNote = {Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here we show that, when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously-hypothetical enzymes in an unbiased genome-scale search. We use this technique to identify two genes, which we name desC and desD, are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the non-model bacterium Novosphingobium aromaticivorans. We show that DesC is a methyl-esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. Here, this approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering.},
doi = {10.1128/AEM.01185-18},
journal = {Applied and Environmental Microbiology},
number = 22,
volume = 84,
place = {United States},
year = {2018},
month = {9}
}

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