Role of Ganoderma lucidum Rho GTPase in polysaccharide synthesis by specifically affecting β-1,3-glucosyltransferase isoenzymes

Abstract

β-1,3-Glucosyltransferase is a key glycosyltransferase that affects the biosynthesis of polysaccharides in Ganoderma lucidum, an edible and medicinal fungal polysaccharide. Its isoenzymes, GL20535 and GL24465, serve dominant and auxiliary roles, respectively. Although the catalytic activity of β-1,3-glucosyltransferase is regulated by Rho GTPases, the mechanisms by which these regulators affect GL20535 and GL24465 and consequently influence polysaccharide synthesis remain unclear. In the present study, three members of the G. lucidum Rho GTPase (GLRho) family—GLRho1/GLRho2/GLRho3—were identified by gene mining. Through AlphaFold3 modeling and protein–protein interaction analysis, it was found that GLRho1 and GLRho3 possess similar structures, binding primarily to GL20535. However, GLRho exhibited stronger binding capacity to GL24465 due to differences in secondary structure by computational predictions. Polysaccharide metabolism and transcription analysis showed that GLRho1 and GLRho3 exerted positive effects on growth and polysaccharide synthesis in G. lucidum, with the overexpression of both genes upregulating the expression of gl20535, leading to significant increases of 27.65% and 24.26% in exopolysaccharide yield, respectively, compared with the wild-type strain. In contrast, glrho2 gene overexpression upregulated the expression of isoenzyme gene gl24465 but did not influence gl20535 expression. Consequently, the polysaccharide yield was unaffected. Moreover, glrho gene regulation did not exert significant influence on the intracellular polysaccharide yield or monosaccharide composition. Overall, this study revealed that the Rho GTPase family influences the molecular network of polysaccharide synthesis by specifically binding to isoenzymes. These results provide theoretical guidance for enhancing the biomanufacturing of polysaccharides in edible and medicinal fungi.

Graphical abstract

Keywords

Rho GTPase / β-1,3-glucosyltransferase / Polysaccharide biosynthesis / Ganoderma lucidun

References

[1] Calonge TM, Nakano K, Arellano M, Arai R, Katayama S, Toda T, Mabuchi I, Perez P. Schizosaccharomyces pombe Rho2p GTPase regulates cell wall α-glucan biosynthesis through the protein kinase Pck2p. Mol Biol Cell, 2000, 11(12): 4393-4401

[2] Chen S, Xu J, Liu C, Zhu Y, Nelson DR, Zhou S, Li C, Wang L, Guo X, Sun Y, et al. . Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat Commun, 2012, 3 1923

[3] Chen H, Zhao L, You C, Liu J, Chen L, Gu Z, Shi G, Li J, Ding Z. Roles of α-1,3-glucosyltransferase in growth and polysaccharides biosynthesis of Ganoderma lucidum. Int J Biol Macromol, 2024, 276 134031

[4] Cui FJ, Wu XH, Tao TL, Zan XY, Sun WJ, Mu DS, Yang Y, Wu D. Functions of a glucan synthase gene GFGLS in mycelial growth and polysaccharide production of Grifola frondosa.. J Agric Food Chem, 2019, 67(32): 8875-8883

[5] Cui FJ, Fu X, Sun L, Zan XY, Meng LJ, Sun WJ. Recent insights into glucans biosynthesis and engineering strategies in edible fungi. Crit Rev Biotechnol, 2024, 44(7): 1262-79

[6] Deng Y, Lei J, Luo X, Wang SP, Tan HM, Zhang JY, Wu DT. Prospects of Ganoderma polysaccharides: structural features, structure-function relationships, and quality evaluation.. Int J Biol Macromol, 2025, 309 142836

[7] Deshavath NN, Mukherjee G, Goud VV, Veeranki VD, Sastri CV. Pitfalls in the 3, 5-dinitrosalicylic acid (DNS) assay for the reducing sugars: interference of furfural and 5-hydroxymethylfurfural. Int J Biol Macromol, 2020, 156: 180-5

[8] Fu X, Zan XY, Sun L, Tan M, Cui FJ, Liang YY, Meng LJ, Sun WJ. Functional characterization and structural basis of the β-1,3-glucan synthase CMGLS from mushroom Cordyceps militaris. J Agric Food Chem, 2022, 70(28): 8725-37

[9] Guo Y, Xu M, Zhang J, Ma Z, Cui J, Zhao L, Chen L, Shi G, Ding Z. Refined regulation of polysaccharide biosynthesis in edible and medicinal fungi: from pathways to production.. Carbohydr Polym, 2025, 358 123560

[10] Hu X, Yang P, Chai C, Liu J, Sun H, Wu Y, Zhang M, Zhang M, Liu X, Yu H. Structural and mechanistic insights into fungal ß-1,3-glucan synthase FKS1. Nature, 2023, 616(7955): 190-8

[11] Hu Y, Li M, Wang S, Yue S, Shi L, Ren A, Zhao M. Ganoderma lucidum phosphoglucomutase is required for hyphal growth, polysaccharide production, and cell wall integrity. Appl Microbiol Biotechnol, 2018, 102(4): 1911-1922

[12] Jiang LH, Li XF, Zan XY, Fu X, Cui FJ, Zhu HA, Sun WJ, Tao TL. The β-1,3-glucan synthase gene GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis in Grifola frondosa.. Appl Microbiol Biotechnol, 2022, 106(2): 563-578

[13] Krull R, Wucherpfennig T, Esfandabadi ME, Walisko R, Melzer G, Hempel DC, Kampen I, Kwade A, Wittmann C. Characterization and control of fungal morphology for improved production performance in biotechnology. J Biotechnol, 2013, 163(2): 112-23

[14] Kumawat SK, Parsad BD, Sahni S. Rapid method for genomic DNA isolation of mungbean. Curr J Appl Sci Technol, 2023, 42(11): 10-4

[15] Li J, Liu H, Li J, Liu J, Dai X, Zhu A, Xiao Q, Qian W, Li H, Guo L, Yan C, Deng D, Luo Y, Wang X. Cryo-EM structure of the β-1,3-glucan synthase FKS1-Rho1 complex. Nat Commun, 2025, 16(1): 57152

[16] Li M, Chen T, Gao T, Miao Z, Jiang A, Shi L, Ren A, Zhao M. UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose. Fungal Genet Biol, 2015, 82: 251-263

[17] Li Q, Zhang C, Zhou J, Li Z, Du G, Chen J, Zhang G. Morphological regulation of filamentous fungi improves industrial production. Biotechnol Adv, 2026, 87 108793

[18] Liu J, Xu M, Shen M, Li J, Chen L, Gu Z, Shi G, Ding Z. Mechanism underlying Ganoderma lucidum polysaccharide biosynthesis regulation by the β-1,3-glucan gene gl20535.. J Fungi, 2025, 11(7): 532

[19] Liu J, Xu M, Guo Y, Li J, Chen L, Gu Z, Shi G, Ding Z. Unique synergistic and compensatory interaction between β-1,3-glucosyltransferase isoenzyme in Ganoderma lucidum polysaccharide biosynthesis. Int J Biol Macromol, 2026, 351 151053

[20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆CT method. Methods, 2001, 25(4): 402-8

[21] Ma Z, Zhao L, Wang Q, Shen Y, Xu M, Chen L, Shi G, Ding Z. Artificially created UDP-glucose 2-epimerase enables concise UDP/GDP-mannose production via the synthase-epimerase route. ACS Catal, 2024, 14(23): 18072-84

[22] Nakano K, Imai J, Arai R, Toh-e A, Matsui Y, Mabuchi I. The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast. J Cell Sci, 2002, 115(23): 4629-4639

[23] Okuda N, Nakazawa T, Horii M, Wu H, Kawauchi M, Sakamoto M, Honda Y. Overexpressing Pleurotus ostreatus rho1b results in transcriptional upregulation of the putative cellulolytic enzyme-encoding genes observed in ccl1 disruptants. Environ Microbiol, 2021, 23(11): 7009-7027

[24] Ornelas-Llamas MM, Perez-Mozqueda LL, Callejas-Negrete OA, Castro-Longoria E. RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa. Fungal Genet Biol, 2024, 171 103873

[25] Peng L, Qiao S, Xu Z, Guan F, Ding Z, Gu Z, Zhang L, Shi G. Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes. Carbohydr Polym, 2015, 133: 104-109

[26] Popolo L, Gilardelli D, Bonfante P, Vai M. Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae. J Bacteriol, 1997, 179(2): 463-469

[27] Roumanie O, Wu H, Molk JN, Rossi G, Bloom K, Brennwald P. Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex. J Cell Biol, 2005, 170(4): 583-94

[28] Sorais F, Barreto L, Leal JA, Bernabé M, San-Blas G, Niño-Vega GA. Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis. Med Mycol, 2010, 48(1): 35-47

[29] Tao TL, Cui FJ, Chen XX, Sun WJ, Huang DM, Zhang J, Yang Y, Wu D, Liu WM. Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc. Microb Cell Fact, 2018, 17(1): 1

[30] Wagner R, Mitchell DA, Sassaki GL, Amazonas M. Links between morphology and physiology of Ganoderma lucidum in submerged culture for the production of exopolysaccharide. J Biotechnol, 2004, 114(1–2): 153-164

[31] Wang Z, Chen J, Wang L, Ding J, Zhao M, Liu R. GlPP2C1 silencing increases the content of Ganoderma lingzhi polysaccharide (GL-PS) and enhances Slt2 phosphorylation. J Fungi, 2022, 8(9): 949

[32] Wang Q, Xu M, Zhao L, Chen L, Ding Z. Novel insights into the mechanism underlying high polysaccharide yield in submerged culture of Ganoderma lucidum revealed by transcriptome and proteome analyses. Microorganisms, 2023, 11(3): 772

[33] Wen TT, Yang YM, Zhang YX, Liu MQ, Qian ZY, Zhang ZY, Dong CH, Sun L, Xu L, Sun WJ, Cui FJ. CRISPR-Cas9/safe harbor-targeted overexpression of glucan synthase gene CmGls in edible mushroom Cordyceps militaris. J Agric Food Chem, 2025, 73(17): 10456-10469

[34] Xu YL, Yuan H, Li N, Xiao JH, Xu JW. Increased production and anti-senescence activity of exopolysaccharides in Ganoderma lingzhi by co-overexpression of β-1,3-glucan synthase and UDP-glucose pyrophosphorylase.. Int J Biol Macromol, 2023, 253 126778

[35] Xu Y, Zhao YJ, Wang ZX, Xu JW. Improved production and rheological properties of exopolysaccharides by co-overexpression of β-1,3-glucan synthase and UDP-glucose pyrophosphorylase genes in Ganoderma lingzhi.. J Agric Food Chem, 2025, 73(14): 8567-8577

[36] Xu JW, Ji SL, Li HJ, Zhou JS, Duan YQ, Dang LZ, Mo MH. Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous α-phosphoglucomutase gene. Bioprocess Biosyst Eng, 2015, 38(2): 399-405

[37] Yang YM, Fu X, Cui FJ, Sun L, Zan XY, Sun WJ. Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize β-1,3-glucan. Biotechnol Biofuels Bioprod, 2023, 16(1): 23806

[38] Zan XY, Zhu HA, Jiang LH, Liang YY, Sun WJ, Tao TL, Cui FJ. The role of Rho1 gene in the cell wall integrity and polysaccharides biosynthesis of the edible mushroom Grifola frondosa. Int J Biol Macromol, 2020, 165: 1593-1603

[39] Zeng C, Ye G, Li G, Cao H, Wang Z, Ji S. RID serve as a more appropriate measure than phenol sulfuric acid method for natural water-soluble polysaccharides quantification. Carbohydr Polym, 2022, 278 118928

[40] Zhang ZY, Sun L, Lei J, Yang YM, Sun WJ, Zan XY, Meng LJ, Cui FJ. Structural and catalytic characterization of β-1,3-glucan synthases of mushroom Ganoderma lucidum. Appl Biochem Biotechnol, 2025, 197(7): 4665-4684

Funding

National Natural Science Foundation of China(32272283)

RIGHTS & PERMISSIONS

Jiangnan University