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Title: Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomassmore » saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.« less
Authors:
 [1] ;  [2] ;  [3] ;  [1] ;  [4] ;  [1] ;  [1] ;  [2] ;  [3] ;  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering & Dept. of Chemical & Biomolecular Engineering
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; San Francisco State Univ., CA (United States)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Plant and Cell Physiology
Additional Journal Information:
Journal Volume: 57; Journal Issue: 3; Journal ID: ISSN 0032-0781
Publisher:
Japanese Society of Plant Physiologists
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS; Arabidopsis; Bioenergy; Cell wall; HCT; Lignin; Saccharification
OSTI Identifier:
1379120

Eudes, Aymerick, Pereira, Jose H., Yogiswara, Sasha, Wang, George, Teixeira Benites, Veronica, Baidoo, Edward E. K., Lee, Taek Soon, Adams, Paul D., Keasling, Jay D., and Loqué, Dominique. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin. United States: N. p., Web. doi:10.1093/pcp/pcw016.
Eudes, Aymerick, Pereira, Jose H., Yogiswara, Sasha, Wang, George, Teixeira Benites, Veronica, Baidoo, Edward E. K., Lee, Taek Soon, Adams, Paul D., Keasling, Jay D., & Loqué, Dominique. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin. United States. doi:10.1093/pcp/pcw016.
Eudes, Aymerick, Pereira, Jose H., Yogiswara, Sasha, Wang, George, Teixeira Benites, Veronica, Baidoo, Edward E. K., Lee, Taek Soon, Adams, Paul D., Keasling, Jay D., and Loqué, Dominique. 2016. "Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin". United States. doi:10.1093/pcp/pcw016. https://www.osti.gov/servlets/purl/1379120.
@article{osti_1379120,
title = {Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin},
author = {Eudes, Aymerick and Pereira, Jose H. and Yogiswara, Sasha and Wang, George and Teixeira Benites, Veronica and Baidoo, Edward E. K. and Lee, Taek Soon and Adams, Paul D. and Keasling, Jay D. and Loqué, Dominique},
abstractNote = {Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.},
doi = {10.1093/pcp/pcw016},
journal = {Plant and Cell Physiology},
number = 3,
volume = 57,
place = {United States},
year = {2016},
month = {2}
}