Dataset Information

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

ABSTRACT: Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.

SUBMITTER: Eudes A

PROVIDER: S-EPMC6680230 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Eudes Aymerick A Sathitsuksanoh Noppadon N Baidoo Edward E K EE George Anthe A Liang Yan Y Yang Fan F Singh Seema S Keasling Jay D JD Simmons Blake A BA Loqué Dominique D

Plant biotechnology journal 20150113 9

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering ...[more]

PMID: 25583257

Similar Datasets

Project description:BackgroundLignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading.ResultsThe 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass (Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene (pShOMT) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process.ConclusionThis study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.

Project description:BackgroundNon-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.ResultsIn this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.ConclusionsOur findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Project description:BACKGROUND:One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants. RESULTS:Consistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis. CONCLUSIONS:Our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in above ground biomass, selective down-regulation of individual components of C1 metabolism is an approach that should be explored further for the improvement of lignocellulosic feedstocks.

Dataset Information

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Publications

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets