Rational design of a thermostable Trichoderma reesei endo-1,4-xylanase II variant with improved resistance toward proteinaceous inhibitors

Abstract

Endo-1,4-xylanase II from Trichoderma reesei is highly sensitive toward grain proteinaceous inhibitors, which remains a major bottleneck for its industrial applications. In this study, the mutant TrXYNII_{DT_}TX1 (T2C-T28C, G21W, N124A) was engineered to gain resistance to two types of xylanase inhibitors, TAXI-I and XIP-I. Compared with the wild-type, the sensitivity of TrXYNII_DT_TX1 toward TAXI-I and XIP-I was decreased by 2.0-and 11.3-fold, respectively. After interaction with two types of inhibitors TAXI-I and XIP-I simultaneously, the residual activity of TrXYNII_DT_TX1 was 3.4-fold higher than that of the wild-type. During grain saccharification, the reducing sugar released by TrXYNII_DT_TX1 was 4.3-fold greater than that of the wild-type. In addition, TrXYNII_DT_TX1 was also more thermostable than the wild-type. All these properties make TrXYNII_DT_TX1 attractive for potential applications in the feed and brewing industries.

Graphical abstract

Keywords

Endo-1,4-xylanases / Trichoderma reesei / Rational design / Inhibitors / Thermostability

References

[1] BhardwajN, KumarB, VermaP. A detailed overview of xylanases: an emerging biomolecule for current and future prospective. Bioresour Bioprocessi, 2019, 6: 40.

[2] AlokikaB. Singh, Production, characteristics, and biotechnological applications of microbial xylanases. Appl Microbiol Biotechnol, 2019, 103: 8763-8784.

[3] YanS, XuY, YuXW. Rational engineering of xylanase hyper-producing system in Trichoderma reesei for efficient biomass degradation. Biotechnol Biofuels, 2021, 14: 90.

[4] JugeN. Plant protein inhibitors of cell wall degrading enzymes. Trends Plant Sci, 2006, 11: 359-367.

[5] Krogh MadsenC, PetterssonD, HjortshojR, KatholmA, Brinch-PedersenH. Superior growth rates in broilers fed wheat with low in vitro feed-xylanase inhibition. J Agric Food Chem, 2018, 66: 4044-4050.

[6] GebruersK, BrijsK, CourtinCM, FierensK, GoesaertH, RabijnsA, RaedscheldersG, RobbenJ, SansenS, SorensenJF, Van CampenhoutS, DelcourJA. Properties of TAXI-type endoxylanase inhibitors. Biochim Biophys Acta, 2004, 1696: 213-221.

[7] GoesaertH, ElliottG, KroonPA, GebruersK, CourtinCM, RobbenJ, DelcourJA, JugeN. Occurrence of proteinaceous endoxylanase inhibitors in cereals. Biochim Biophys Acta, 2004, 1696: 193-202.

[8] BonninE, DavietS, GebruersK, DelcourJA, GoldsonA, JugeN, SaulnierL. Variation in the levels of the different xylanase inhibitors in grain and flour of 20 French wheat cultivars. J Cereal Sci, 2005, 41: 375-379.

[9] PaesG, BerrinJG, BeaugrandJ. GH11 xylanases: Structure/function/properties relationships and applications. Biotechnol Adv, 2012, 30: 564-592.

[10] LiuM, WuX, HuoW, LiJ, WengX, LiuJ, FangZ. Differential inhibition of GH family 11 endo-xylanase by rice xylanase inhibitor and verification by a modified yeast two-hybrid system. Int J Biol Macromol, 2019, 132: 514-523.

[11] TahirTA, BerrinJG, FlatmanR, RousselA, RoepstorffP, WilliamsonG, JugeN. Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger. J Biol Chem, 2002, 277: 44035-44043.

[12] RasmussenLE, SorensenJF, MeyerAS. Kinetics and substrate selectivity of a Triticum aestivum xylanase inhibitor (TAXI) resistant D11F/R122D variant of Bacillus subtilis XynA xylanase. J Biotechnol, 2010, 146: 207-214.

[13] XiongH, FenelF, LeisolaM, TurunenO. Engineering the thermostability of Trichoderma reesei endo-1,4-beta-xylanase II by combination of disulphide bridges. Extremophiles, 2004, 8: 393-400.

[14] FierensK, BrijsK, CourtinCM, GebruersK, GoesaertH, RaedscheldersG, RobbenJ, Van CampenhoutS, VolckaertG, DelcourJA. Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins. FEBS Lett, 2003, 540: 259-263.

[15] ZhangXF, AiYH, XuY, YuXW. High-level expression of Aspergillus niger lipase in Pichia pastoris: characterization and gastric digestion in vitro. Food Chem, 2019, 274: 305-313.

[16] AnandakrishnanR, AguilarB, OnufrievV. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulation. Nucleic Acids Res, 2012, 40: 537-541.

[17] ZhuD, LiuX, XieX, YangS, LinH, ChenH. Characteristics of a XIP-resistant xylanase from Neocallimastix sp. GMLF1 and its advantage in barley malt saccharification. Int J Food Sci Technol, 2019, 55: 2152-2160.

[18] TambelLIM, ZhouM, ChenY, ZhangX, ChenY, ChenD. Amino acids application enhances flowers insecticidal protein content in Bt cotton. J Cotton Res, 2019.

[19] Berlamont H, De Witte C, De Bruyckere S, Fox JG, Backert S, Smet A, Boyen F, Haesebrouck F. Differentiation of gastric helicobacter species using MALDI-TOF mass spectrometry, pathogens. 2021; 10. https://doi.org/10.3390/pathogens10030366.

[20] McClearyBV, McGeoughP. A comparison of polysaccharide substrates and reducing sugar methods for the measurement of endo-1,4-beta-xylanase. Appl Biochem Biotechnol, 2015, 177: 1152-1163.

[21] BourgoisTM, NguyenDV, SansenS, RomboutsS, BelienT, FierensK, RaedscheldersG, RabijnsA, CourtinCM, DelcourJA, Van CampenhoutS, VolckaertG. Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types. J Biotechnol, 2007, 130: 95-105.

[22] VardakouM, DumonC, MurrayJW, ChristakopoulosP, WeinerDP, JugeN, LewisRJ, GilbertHJ, FlintJE. Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases. J Mol Biol, 2008, 375: 1293-1305.

[23] TangF, ChenD, YuB, LuoY, ZhengP, MaoX, YuJ, HeJ. Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds. Electron J Biotechnol, 2017, 26: 52-59.

[24] FenelF, LeisolaM, JanisJ, TurunenO. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II. J Biotechnol, 2004, 108: 137-143.

[25] SansenS, De RanterCJ, GebruersK, BrijsK, CourtinCM, DelcourJA, RabijnsA. Structural basis for inhibition of Aspergillus niger xylanase by triticum aestivum xylanase inhibitor-I. J Biol Chem, 2004, 279: 36022-36028.

[26] FuG, WangY, WangD, ZhouC. Cloning, expression, and characterization of an GHF 11 xylanase from Aspergillus niger XZ-3S, Indian. J Microbiol, 2012, 52: 682-688.

[27] GuoB, ChenXL, SunCY, ZhouBC, ZhangYZ. Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-beta-1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl Microbiol Biotechnol, 2009, 84: 1107-1115.

[28] ShinJ-H, ChoiJ-H, LeeO-S, KimY-M, LeeD-S, KwakY-Y, KimW-C, RheeI-K. Thermostable xylanase from Streptomyces thermocyaneoviolaceus for optimal production of xylooligosaccharides. Biotechnol Bioprocess Eng, 2009, 14: 391-399.

[29] RaedscheldersG, FierensK, SansenS, RomboutsS, GebruersK, RobbenJ, RabijnsA, CourtinCM, DelcourJA, Van CampenhoutS, VolckaertG. Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity. Biochem Biophys Res Commun, 2005, 335: 512-522.

[30] LiuM, LiJ, RehmanAU, LuoS, WangY, WeiH, ZhangK. Sensitivity of family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the T33I mutant to Oryza sativa xylanase inhibitor protein (OsXIP): An experimental and computational study. Enzyme Microb Technol, 2022.

[31] Zouari AyadiD, Hmida SayariA, Ben HlimaH, Ben MabroukS, MezghaniM, BejarS. Improvement of Trichoderma reesei xylanase II thermal stability by serine to threonine surface mutations. Int J Biol Macromol, 2015, 72: 163-170.

[32] BhallaA, BischoffKM, UppugundlaN, BalanV, SaniRK. Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1. Bioresour Technol, 2014, 165: 314-318.

[33] GiridharPV, ChandraTS. Production of novel halo-alkali-thermo-stable xylanase by a newly isolated moderately halophilic and alkali-tolerant Gracilibacillus sp. TSCPVG Process Biochem, 2010, 45: 1730-1737.

[34] CollinsT, GerdayC, FellerG. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev, 2005, 29: 3-23.

[35] ByeongYJWJO, MinJ, SoonY, SeongP, KangW. Lignocellulose Biotechnology: present and future prospects. World J Microbiol Biotechnol, 2001, 17: 657.

[36] LiuXY. Properties of xylanase inhibitor protein from wheat and its influence on efficacy of exogenous xylanase. Microbiol China, 2020, 4772300-2308.

[37] FigueiredoR, AraújoP, LlerenaJPP, MazzaferaP. Suberin and hemicellulose in sugarcane cell wall architecture and crop digestibility: a biotechnological perspective. Food Energy Secur, 2019.

Funding

National Natural Science Foundation of China(32072162)

Key Research and Development Program of Hunan Province of China(2021YFC2100203)

RIGHTS & PERMISSIONS

Jiangnan University