Abstract：In plant cell wall, the ferulic acid can form covalent cross-linking in lignin-ferulic acid-arabia xylan complexes with hemicellulose and lignin, which is an important molecular basis for the formation of anti-degradation barrier in lignocellulose. Ferulic acid esterase (FAE) can break the ester bond between hemicelluloses as well as hemicellulose and lignin. In this study, the codon of a thermophilic ferulic acid esterase gene from Clostridium thermocellum was optimized and then constructed into the plant binary expression vector containing different signal peptide coding sequence which the thermophilic FAE protein expressed in Arabidopsis thaliana targeting to the cytoplasm, apoplast, endoplasmic reticulum, chloroplast or mitochondria, respectively. The results indicated that the thermophilic FAE was successfully expressed in different transgenic lines, and with the highest value when expressed in the cytoplasm, its enzyme activity at 70 °C was much higher than that at 25 °C (P<0.05). The plant height, fresh weight and 100-seed weight between different transgenic lines and wild type were not significantly different, but an early flowering was observed in the lines with FAE proteins expressed in chloroplasts or mitochondria. This study can provide new ideas and methods for efficient resource utilization of energy plants, pastures and crop straws.
1 蒋建雄, 孙建中, 李霞, 等. 2015. 我国草本纤维素类能源作物产业化发展面临的主要挑战与策略[J]. 生物产业技术, (2): 22-31. (Jiang J X, Sun J Z, Li X, et al. 2015. Main challenges and strategies faced by industrialization development of herb cellulose energy crops in China[J]. Biotechnology & Business, (2): 22-31.) 2 李齐东, 高璐, 蒋建雄, 等. 2018. 嗜热内切葡聚糖酶(E1) 植物亚细胞定位表达载体的构建及验证[J]. 江苏农业学报, 34(6): 1247-1253. (Li Q D, Gao L, Jiang J X, et al.2018.Construction and verification of the thermophilic endoglucanase (E1) subcellular plant expression vector[J]. Jiangsu Journal of Agricultural Sciences, 34(6): 1247-1253.) 3 刘奕彤, 孙建中, 蒋建雄. 2017. 嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析[J]. 微生物学通报, 44(6): 1331-1338. (Liu Y T, Sun J Z, Jiang J X.2017. Cloning, heterologous expression and characterization of a ferulic acid esterase gene from thermophilic Clostridium thermocellum[J]. Microbiology China, 44(6): 1331-1338.) 4 曾薇, 陈洪章. 2009. 阿魏酸酯酶和纤维素酶在水解汽爆稻草中的协同作用[J]. 生物工程学报, 25(1): 49-54. (Zeng W, Chen H Z.2009. Synergistic effect of feruloyl esterase and cellulase in hydrolyzation of steam-exploded rice straw[J]. Chinese Journal of Biotechnology, 25(1): 49-54.) 5 Badhan A, Jin L, Wang Y, et al.2014. Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility[J]. Biotechnology for Biofuels, 7(1): 39-53. 6 Buanafina M M D O, Dalton S, Langdon T, et al.2015. Functional co-expression of a fungal ferulic acid esterase and a β-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction[J]. Planta, 242(1): 97-111. 7 Buanafina M M D O, Fescemyer H W.2012. Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm[J]. Planta, 236(2): 513-523. 8 Buanafina M M D O, Langdon T, Hauck B, et al.2006. Manipulating the phenolic acid content and digestibility of Italian ryegrass (Lolium multiflorum) by vacuolar-targeted expression of a fungal ferulic acid esterase[J]. Applied Biochemistry Biotechnology, 130(1-3): 416-426. 9 Buanafina M M D O, Langdon T, Hauck B, et al.2008. Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea)[J]. Plant Biotechnology, 6(3): 264-280. 10 Buanafina M M D O, Langdon T, Hauck B, et al.2010. Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea)[J]. Plant Biotechnology Journal, 8(3): 316-331. 11 Chong S L, Derba-Maceluch M, Koutaniemi S, et al.2015. Active fungal GH115 α-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans[J]. BMC Biotechnology, 15(1): 56-68. 12 Damm T, Commandeur U, Fischer R, et al.2016. Improving the utilization of lignocellulosic biomass by polysaccharide modification[J]. Process Biochemistry, 51(2): 288-296. 13 Guerriero G, Hausman J F, Strauss J, et al.2015. Destructuring plant biomass: Focus on fungal and extremophilic cell wall hydrolases[J]. Plant Science, 234(3): 180-193. 14 Harholt J, Bach I C, Lindbouquin S, et al.2010. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm[J]. Plant Biotechnology Journal, 8(3): 351-362. 15 Harrison M D, Geijskes R J, Robyn L, et al.2014. Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane[J]. Molecular Biotechnology, 56(9): 795-802. 16 Harrison M D, Zhang Z Y, Kylie S, et al.2014. The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse[J]. Biotechnology for Biofuels, 7(1): 131-145. 17 Khare S K, Pandey A, Larroche C, et al.2015. Current perspectives in enzymatic saccharification of lignocellulosic biomass[J]. Biochemical Engineering Journal, 102(2015): 38-44. 18 Klinger J, Fischer R, Commandeur U.2015. Comparison of Thermobifida fusca cellulases expressed in Escherichia coli and Nicotiana tabacum indicates advantages of the plant system for the expression of bacterial cellulases[J]. Frontiers in Plant Science, 6(26): 1-9. 19 Klose H, Usadel B, Fischer R, et al.2015. Cell wall modification in tobacco by differential targeting of recombinant endoglucanase from Trichoderma reesei[J]. BMC Plant Biology, 15(1): 54-64. 20 Lambertz C, Garvey M, Klinger J, et al.2014. Challenges and advances in the heterologous expression of cellulolytic enzymes: A review[J]. Biotechnology for Biofuels, 7(1): 135-149. 21 Li Q, Jian S, Peng S, et al.2014. Plant biotechnology for lignocellulosic biofuel production[J]. Plant Biotechnology Journal, 12(9): 1174-1192. 22 Park S H, Ong R G, Sticklen M.2015. Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production[J]. Plant Biotechnology Journal, 14(6): 1329-1344. 23 Pawar P M, Derbamaceluch M, Chong S L, et al.2016. Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose[J]. Plant Biotechnology Journal, 14(1): 387-397. 24 Pereira E O, Kolotilin I, Conley A J, et al.2014. Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags[J]. BMC Biotechnology, 14(1): 59-69. 25 Pogorelko G, Fursova O, Lin M, et al.2011. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast[J]. Plant Molecular Biology, 77(4-5): 433-445. 26 Várnai A, Costa T H, Faulds C B, et al.2014. Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions[J]. Biotechnology for Biofuels, 7(1): 153-163. 27 Zhang S B, Zhai H C, Wang L, et al.2013. Expression, purification and characterization of a feruloyl esterase A from Aspergillus flavus[J]. Protein Expression and Purification, 92(1): 36-40.
