bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906529; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1 The synergistic actions of hydrolytic genes in coexpression networks reveal the potential 2 of Trichoderma harzianum for cellulose degradation 3 4 Déborah Aires Almeida1,2, Maria Augusta Crivelente Horta1,2,#, Jaire Alves Ferreira Filho1,2, 5 Natália Faraj Murad1 and Anete Pereira de Souza1,3,* 6 7 1Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas 8 (UNICAMP), Campinas, SP, Brazil 9 2Graduate Program in Genetics and Molecular Biology, Institute of Biology, UNICAMP, 10 Campinas, SP, Brazil 11 3Department of Plant Biology, Institute of Biology, UNICAMP, Campinas, SP, Brazil 12 13 # Present Address: Holzforshung München, TUM School of Life Sciences Weihenstephan, 14 Technische Universität München, Freising, Germany 15 16 *Corresponding author 17 Profa Anete Pereira de Souza 18 Dept. de Biologia Vegetal, Universidade Estadual de Campinas, CEP 13083-875, Campinas, 19 São Paulo, Brazil 20 Tel.: +55-19-3521-1132 21 E-mail:[email protected] 22 23 Abstract 24 Background: Bioprospecting key genes and proteins related to plant biomass degradation is 25 an attractive approach for the identification of target genes for biotechnological purposes, bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906529; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 26 especially genes with potential applications in the biorefinery industry that can enhance 27 second-generation ethanol production technology. Trichoderma harzianum is a potential 28 candidate for cellulolytic enzyme production. Herein, the transcriptome, exoproteome, 29 enzymatic activities of extracts, and coexpression networks of the T. harzianum strain 30 CBMAI-0179 under biomass degradation conditions were examined. 31 Results: We used RNA-Seq to identify differentially expressed genes (DEGs) and 32 carbohydrate-active enzyme (CAZyme) genes related to plant biomass degradation and 33 compared them with genes of strains from congeneric species (T. harzianum IOC-3844 and 34 T. atroviride CBMAI-0020). T. harzianum CBMAI-0179 harbors species- and treatment- 35 specific CAZyme genes, transporters and transcription factors. Additionally, we detected 36 important proteins related to biomass degradation, including β-glucosidases, endoglucanases, 37 cellobiohydrolases, lytic polysaccharide monooxygenases (LPMOs), endo-1,4-β-xylanases 38 and β-mannanases, in the exoproteome under cellulose growth conditions. Coexpression 39 networks were constructed to explore the relationships among the genes with corresponding 40 secreted proteins that act synergistically for cellulose degradation. An enriched cluster with 41 degradative enzymes was described, and the subnetwork of CAZymes showed linear 42 correlations among secreted proteins (AA9, GH6, GH10, GH11 and CBM1) and 43 differentially expressed CAZyme genes (GH45, GH7, AA7 and GH1). 44 Conclusions: The coexpression network revealed genes with strong correlations acting 45 synergistically to hydrolyze cellulose. Our results provide valuable information for future 46 studies on the genetic regulation of plant cell wall-degrading enzymes. This knowledge can 47 be exploited for the improvement of enzymatic reactions to degrade plant biomass, which is 48 useful for bioethanol production. 49 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906529; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3 50 Keywords: Trichoderma harzianum, Cellulose, RNA-Seq, CAZymes, Exoproteome, 51 Coexpression networks 52 53 Background 54 The expanding worldwide demand for renewable and sustainable energy sources has 55 increased the interest in alternative energy sources, and the production of second-generation 56 biofuels seems to be the most viable option to confront these issues [1, 2]. Lignocellulosic 57 biomass is the most abundant renewable organic carbon resource on earth, consisting of three 58 major polymers, cellulose, hemicellulose, and lignin [2]. However, due to its recalcitrant 59 characteristics that prevent enzyme access, degrading this complex matrix is still a major 60 challenge [3]. For the complete hydrolysis of lignocellulose, a variety of enzymes acting in 61 synergy are required, and much research has focused on this topic in recent decades [4]. 62 Interactions between different enzymes have been investigated to identify optimal 63 combinations and ratios of enzymes for efficient biomass degradation, which are highly 64 dependent on the properties of the lignocellulosic substrates and the surface structure of 65 cellulose microfibrils [4, 5]. Due to their abundance in nature, microorganisms are considered 66 natural producers of enzymes, and many of them, including members of both bacteria and 67 fungi, have evolved to digest lignocellulose [6, 7]. The search for microorganisms that are 68 able to efficiently degrade lignocellulosic biomass is pivotal for the establishment of the 69 sustainable production of bioethanol [8]. 70 Filamentous fungi, including the genera Trichoderma, Aspergillus, Penicillium and 71 Neurospora, produce extracellular proteins that act synergistically to degrade plant cell walls 72 and are widely used in the enzymatic industry [9]. Species in the filamentous ascomycete 73 genus Trichoderma are among the most commonly isolated saprotrophic fungi [10] and are 74 important from a biotechnological perspective [7]. Trichoderma species are widely used in bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906529; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4 75 agriculture as biocontrol agents due their ability to antagonize plant-pathogenic fungi and in 76 industry as producers of plant cell wall-degrading enzymes [11-14]. In addition, Trichoderma 77 species are easily isolated from soil and decomposing organic matter [15]. Within the 78 Trichoderma genus, T. reesei is the most intensively studied species [16]. T. reesei is a well- 79 known producer of cellulase and hemicellulase, and due to the high effectiveness of the 80 synergistic cellulases in this species, it is widely employed in industry, as technologies for its 81 use and handling are based on seventy years of experience [8, 16-19]. However, studies on T. 82 harzianum strains have shown their potential to produce a set of enzymes that can degrade 83 lignocellulosic biomass [20-23]; therefore, T. harzianum strains are being investigated as 84 potentially valuable sources of industrial cellulases [6]. 85 The identification of carbohydrate-active enzymes (CAZymes) that act synergistically 86 under biodegradation conditions [4] has the potential to improve the enzymatic hydrolysis 87 process by optimizing and reducing bioethanol costs. The CAZy database (www.cazy.org) 88 classifies CAZymes into six major groups: glycoside hydrolases (GHs), glycosyltransferases 89 (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (AAs), 90 and carbohydrate-binding modules (CBMs) [24]. CAZymes are extensively used for the 91 genetic classification of important hydrolytic enzymes [22, 25]. 92 The conversion of cellulose to glucose involves the synergistic action of three 93 principal groups of enzymes: endo-β-1,4-glucanases (EC 3.2.1.4), β-glucosidases (EC 94 3.2.1.21), and cellobiohydrolases (EC 3.2.1.91/176) [20, 26]. For hemicellulose hydrolysis, 95 several enzymes are needed, such as endo-1,4-β-xylanases (EC 3.2.1.8), β-xylosidases (EC 96 3.2.1.37), β-mannanases (EC 3.2.1.78), arabinofuranosidases (EC 3.2.1.55), and acetylxylan 97 esterases (EC 3.1.1.72) [26, 27]. In addition, a number of auxiliary enzymes are involved in 98 this process, such as lytic polysaccharide monooxygenases (LPMOs), cellulose-induced bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906529; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5 99 protein 1 and 2 (CIP1 and CIP2) and swollenin, which can increase the hydrolytic 100 performance of enzymatic cocktails used in industry for bioethanol production [6, 28-30]. 101 As genetic variation occurs within species [31, 32], understanding and exploring the 102 genetic mechanisms of different T. harzianum strains can provide valuable information for 103 industrial applications. In the present study, we analyzed the enzymatic activity, 104 transcriptome and exoproteome of T. harzianum CBMAI-0179 and compared