Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

Biotechnology for Biofuels

Timothy J Tschaplinski²  Robert F Standaert¹  Nancy L Engle²  Madhavi Z Martin²  Amandeep K Sangha¹  Jerry M Parks²  Jeremy C Smith¹  Reichel Samuel²  Nan Jiang²  Yunqiao Pu²  Arthur J Ragauskas²  Choo Y Hamilton²  Chunxiang Fu²  Zeng-Yu Wang²  Brian H Davison²  Richard A Dixon²  Jonathan R Mielenz² 

[1] Department of Biochemistry and Molecular & Cellular Biology, University of Tennessee, Knoxville, TN, 37996, USA

[2] BioEnergy Science Center, Oak Ridge, TN 38731, USA

关键词: Transgenic;    Caffeic acid O-methyltransferase;    Recalcitrance;    Bioenergy;    Switchgrass;    Monolignol;    iso-Sinapyl alcohol;    4-Dimethoxy-5-hydroxycinnamyl alcohol;    trans-3;   

Others  :  798223
DOI  :  10.1186/1754-6834-5-71

 received in 2012-06-11, accepted in 2012-09-05,  发布年份 2012

【 摘 要 】

Background

Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.

Results

GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.

Conclusions

Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

【 授权许可】

   
2012 Tschaplinski et al.; licensee BioMed Central Ltd.

【 图 表 】

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