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Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

ABSTRACT: BACKGROUND:Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. RESULTS:In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. CONCLUSION:Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

SUBMITTER: Badhan A

PROVIDER: S-EPMC3999942 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

Badhan Ajay A Jin Long L Wang Yuxi Y Han Shuyou S Kowalczys Katarzyna K Brown Daniel Cw DC Ayala Carlos Juarez CJ Latoszek-Green Marysia M Miki Brian B Tsang Adrian A McAllister Tim T

Biotechnology for biofuels 20140320 1

<h4>Background</h4>Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production.<h4>Results</h4>In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides ...[more]

PMID: 24650274

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Project description:Background:Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates. Results:In this study, transgenic hybrid poplar (Populus alba?×?grandidentata) was generated to express a hyperthermophilic endoglucanase from Thermotoga neapolitana with an optimal temperature over 100 °C. Functional hyperthermoactive endoglucanase was successfully produced in the transgenic events, and altered phenotypic growth was observed in transgenic lines. Moreover, the line with the highest TnCelB expression in both leaf and developing xylem had reduced lignin content and cellulose crystallinity, resulting in a more digestible cell wall. The activation of TnCelB by a post-harvest heat treatment resulted in enhanced saccharification efficiencies of transgenic poplar lines with moderate TnCelB expression and without alteration of cellulose and lignin when not heat-treated. In planta high-level overexpression of a hyperthermophilic endoglucanase paired with heat treatment following harvest, resulted in biomass that was comparable with wild-type lines that underwent a traditional pretreatment for saccharification. Conclusions:Overexpression of hyperthermophilic endoglucanase in feedstock had impacts on plant growth and cell wall composition, especially when the enzyme was highly expressed. Improved glucan saccharification efficiencies from transgenic lines before and after heat treatment could reduce both the economic and environmental costs associated with ethanol production from lignocellulosic biomass.

Project description:Background:Alfalfa (Medicago sativa) is an important forage material widely used for animal feed production. Ensiling is an effective method for preserving alfalfa, but it has shown some limitations in the production of high-quality alfalfa silage due to its low water soluble carbohydrates (WSC) content and high buffering capacity. Lactic acid bacteria (LAB) and cellulase are often used as silage additives to promote the ensiling process and enhance fermentation quality. Methods:Experiments were conducted to investigate the effects of ferulic acid esterase (FAE)-producing Lactobacillus fermentum 17SD-2 (LF) and cellulase (CE) on the fermentation quality and microbial community of alfalfa silage. After 60 days of ensiling, analysis of fermentation quality and bacterial diversity in alfalfa silages were conducted using high-performance liquid chromatography and high-throughput sequencing methods. Results:Alfalfa was ensiled with additives (LF, CE, and LF+CE) or without additives for 60 days. All additives increased lactic acid and decreased pH values and ammonia-N contents compared to control. Among all treatments, the combined addition of LF and CE showed lowest pH (4.66) and ammonia-N (NH3-N, 0.57% DM) content, highest contents of lactic acid (LA, 10.51% DM), dry matter (DM, 22.54%) and crude protein (CP, 24.60% DM). Combined addition of LF and CE performed better in reducing neutral detergent fiber (NDF, 29.76% DM) and acid detergent fiber (ADF, 22.86% DM) contents than the addition of LF (33.71, 27.39% DM) or CE (32.07, 25.45% DM) alone. Moreover, the microbial analysis indicated that LF+CE treatments increased the abundance of desirable Lactobacillus and inhibited the growth of detrimental Enterobacter and Clostridia in alfalfa silage. Discussion:Combined addition of FAE-producing LF and CE is more effective than treatments of LF or CE alone in improving fermentation quality and nutrition values of alfalfa silage. This is likely due to a synergistic effect of CE and FAE produced by LF on plant cell wall degradation, indicating that these additives promote each other to improve fiber degradation and silage fermentation. In conclusion, combined addition of FAE-producing LF and CE could be a feasible way to improve alfalfa silage quality.

Dataset Information

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

Publications

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility.

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