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Title: Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Abstract

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step inmore » coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1579601
Alternate Identifier(s):
OSTI ID: 1633296
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Name: Metabolic Engineering Communications Journal Volume: 10 Journal Issue: C; Journal ID: ISSN 2214-0301
Publisher:
Elsevier
Country of Publication:
Netherlands
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Incha, Matthew R., Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Liu, Yuzhong, Pearson, Allison N., Schmidt, Matthias, Gin, Jennifer W., Petzold, Christopher J., Deutschbauer, Adam M., and Keasling, Jay D.. Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida. Netherlands: N. p., 2020. Web. https://doi.org/10.1016/j.mec.2019.e00119.
Incha, Matthew R., Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Liu, Yuzhong, Pearson, Allison N., Schmidt, Matthias, Gin, Jennifer W., Petzold, Christopher J., Deutschbauer, Adam M., & Keasling, Jay D.. Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida. Netherlands. https://doi.org/10.1016/j.mec.2019.e00119
Incha, Matthew R., Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Liu, Yuzhong, Pearson, Allison N., Schmidt, Matthias, Gin, Jennifer W., Petzold, Christopher J., Deutschbauer, Adam M., and Keasling, Jay D.. Mon . "Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida". Netherlands. https://doi.org/10.1016/j.mec.2019.e00119.
@article{osti_1579601,
title = {Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida},
author = {Incha, Matthew R. and Thompson, Mitchell G. and Blake-Hedges, Jacquelyn M. and Liu, Yuzhong and Pearson, Allison N. and Schmidt, Matthias and Gin, Jennifer W. and Petzold, Christopher J. and Deutschbauer, Adam M. and Keasling, Jay D.},
abstractNote = {Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.},
doi = {10.1016/j.mec.2019.e00119},
journal = {Metabolic Engineering Communications},
number = C,
volume = 10,
place = {Netherlands},
year = {2020},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
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https://doi.org/10.1016/j.mec.2019.e00119

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