Acidothermus Cellulolyticus E1 Endoglucanase Expressed in Planta Undergoes Extensive Hydroxyproline-O-glycosylation and Exhibits Enhanced Impact on Biomass Digestibility

Overview

Journal Plant Cell Rep

Publisher Springer

Date 2024 Jul 29

PMID 39073636

Authors

Hong Fang

Berry Dickey

Daniela PerezLaguna

Jacqueline Vargas Ulloa

Paula PerezSanchez

Jianfeng Xu

Affiliations

Soon will be listed here.

Abstract

E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-β-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

References

Kieliszewski M . The latest hype on Hyp-O-glycosylation codes. Phytochemistry. 2001; 57(3):319-23. DOI: 10.1016/s0031-9422(01)00029-2. View

Xu J, Tan L, Lamport D, Showalter A, Kieliszewski M . The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion. Phytochemistry. 2008; 69(8):1631-40. DOI: 10.1016/j.phytochem.2008.02.006. View

Herve C, Rogowski A, Blake A, Marcus S, Gilbert H, Knox J . Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects. Proc Natl Acad Sci U S A. 2010; 107(34):15293-8. PMC: 2930570. DOI: 10.1073/pnas.1005732107. View

Cosgrove D . Growth of the plant cell wall. Nat Rev Mol Cell Biol. 2005; 6(11):850-61. DOI: 10.1038/nrm1746. View

Kieliszewski M, SHPAK E . Synthetic genes for the elucidation of glycosylation codes for arabinogalactan-proteins and other hydroxyproline-rich glycoproteins. Cell Mol Life Sci. 2001; 58(10):1386-98. PMC: 11337274. DOI: 10.1007/PL00000783. View

Zhang N, Wright T, Caraway P, Xu J . Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture. Bioengineered. 2019; 10(1):87-97. PMC: 6527068. DOI: 10.1080/21655979.2019.1604037. View

Showalter A, Basu D . Extensin and Arabinogalactan-Protein Biosynthesis: Glycosyltransferases, Research Challenges, and Biosensors. Front Plant Sci. 2016; 7:814. PMC: 4908140. DOI: 10.3389/fpls.2016.00814. View

Mir B, Mewalal R, Mizrachi E, Myburg A, Cowan D . Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion. Trends Biotechnol. 2014; 32(5):281-9. DOI: 10.1016/j.tibtech.2014.03.003. View

Yennamalli R, Rader A, Kenny A, Wolt J, Sen T . Endoglucanases: insights into thermostability for biofuel applications. Biotechnol Biofuels. 2013; 6(1):136. PMC: 3856469. DOI: 10.1186/1754-6834-6-136. View

10.

Marzol E, Borassi C, Bringas M, Sede A, Rodriguez Garcia D, Capece L . Filling the Gaps to Solve the Extensin Puzzle. Mol Plant. 2018; 11(5):645-658. DOI: 10.1016/j.molp.2018.03.003. View

11.

Obembe O, Jacobsen E, Timmers J, Gilbert H, Blake A, Knox J . Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants. J Plant Res. 2007; 120(5):605-17. PMC: 2039807. DOI: 10.1007/s10265-007-0099-7. View

12.

Payne C, Resch M, Chen L, Crowley M, Himmel M, Taylor 2nd L . Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci U S A. 2013; 110(36):14646-51. PMC: 3767562. DOI: 10.1073/pnas.1309106110. View

13.

Dai Z, Hooker B, Anderson D, Thomas S . Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects. Transgenic Res. 2000; 9(1):43-54. DOI: 10.1023/a:1008922404834. View

14.

Tan L, Varnai P, Lamport D, Yuan C, Xu J, Qiu F . Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains. J Biol Chem. 2010; 285(32):24575-83. PMC: 2915693. DOI: 10.1074/jbc.M109.100149. View

15.

Zhang N, Wright T, Wang X, Karki U, Savary B, Xu J . Engineering 'designer' glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline-O-glycosylation process in plants. Plant Biotechnol J. 2018; 17(6):1130-1141. PMC: 6523594. DOI: 10.1111/pbi.13043. View

16.

Xu J, Okada S, Tan L, Goodrum K, Kopchick J, Kieliszewski M . Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life. Transgenic Res. 2010; 19(5):849-67. DOI: 10.1007/s11248-010-9367-8. View

17.

Bernardes A, Pellegrini V, Curtolo F, Camilo C, Mello B, Johns M . Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate. Carbohydr Polym. 2019; 211:57-68. DOI: 10.1016/j.carbpol.2019.01.108. View

18.

Sun Y, Cheng J, Himmel M, Skory C, Adney W, Thomas S . Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627. Bioresour Technol. 2006; 98(15):2866-72. DOI: 10.1016/j.biortech.2006.09.055. View

19.

Ziegelhoffer T, Raasch J, Austin-Phillips S . Expression of Acidothermus cellulolyticus E1 endo-beta-1,4-glucanase catalytic domain in transplastomic tobacco. Plant Biotechnol J. 2009; 7(6):527-36. DOI: 10.1111/j.1467-7652.2009.00421.x. View

20.

Tan L, Showalter A, Egelund J, Hernandez-Sanchez A, Doblin M, Bacic A . Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans. Front Plant Sci. 2012; 3:140. PMC: 3384089. DOI: 10.3389/fpls.2012.00140. View