Posted on Authorea 1 Sep 2022 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.166030463.37247755/v2 | This a preprint and has not been peer reviewed. Data may be preliminary. Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus. Julieta Barchiesi1 , Marı́a B. Velazquez1 , Marı́a Busi1 , Diego F. Gomez-Casati1 , and Chitralekha Nag-Dasgupta2 1 2 Centro de Estudios Fotosinteticos y Bioquimicos Research Cell Lucknow Amity University Uttar Pradesh India September 1, 2022 Abstract Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce the necessary enzymes to metabolize it. In this work we detected the presence of extracellular cellulases in the genome of five species of Scenedesmus. These microalgae grow in both, freshwater and saltwater regions as well as in soils, displaying highly flexible metabolic properties. The comparison of sequences of the different cellulases with hydrolytic enzymes from other organisms by means of multi-sequence alignments and phylogenetic trees showed that these enzymes belong to the families of glycosyl hydrolases 1, 5, 9 and 10. In addition, most of these presented a greater similarity of sequence with enzymes from invertebrates, fungi, bacteria and other microalgae than with cellulases from plants; and the 3D modeling data obtained showed that both the main structures of the modeled proteins and the main amino acid residues implicated in catalysis and substrate binding are well conserved in Scenedesmus enzymes. We propose that these cellulase-producing phototrophic microorganisms could act as catalysts for the hydrolysis of cellulosic biomass fueled by sunlight. Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus. Marı́a B. Velazqueza , Marı́a V. Busia , Diego F. Gomez-Casatia , Chitralekha Nag-Dasguptab * and Julieta Barchiesia *. a Centro de Estudios Fotosintéticos y Bioquı́micos (CEFOBI-CONICET), Universidad Nacional de Rosario, Argentina. b Research Cell, Lucknow, Amity University Uttar Pradesh, India. Running title: Cellulose degradation by Scenedesmus. Julieta Barchiesi* Corresponding autor at: Centro de Estudios Fotosintéticos y Bioquı́micos (CEFOBI-CONICET), Suipacha 570, Rosario 2000, Argentina. E-mail address :barchiesi@cefobi-conicet.gov.ar Chitralekha Nag Dasgupta* Co-corresponding autor at: Research Cell, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida, 201313, India. E-mail address : cndasgupta@lko.amity.edu 1 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Abstract Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce the necessary enzymes to metabolize it. In this work we detected the presence of extracellular cellulases in the genome of five species ofScenedesmus . These microalgae grow in both, freshwater and saltwater regions as well as in soils, displaying highly flexible metabolic properties. The comparison of sequences of the different cellulases with hydrolytic enzymes from other organisms by means of multi-sequence alignments and phylogenetic trees showed that these enzymes belong to the families of glycosyl hydrolases 1, 5, 9 and 10. In addition, most of these presented a greater similarity of sequence with enzymes from invertebrates, fungi, bacteria and other microalgae than with cellulases from plants; and the 3D modeling data obtained showed that both the main structures of the modeled proteins and the main amino acid residues implicated in catalysis and substrate binding are well conserved in Scenedesmus enzymes. We propose that these cellulase-producing phototrophic microorganisms could act as catalysts for the hydrolysis of cellulosic biomass fueled by sunlight. Keywords Cellulases, Scenedesmus, Endoglucanases, β-glucosidases, exocellulases 1. Introduction Generation of renewable energy resources and waste management are the major concern in twenty first century. Lignocellulosic agricultural and forest wastes are the promising feedstock for production of biofuel and value-added products due its high availability and low cost1 . Nevertheless, no commercial process has still been reported for the enzymatic hydrolysis of cellulose. The main reason is the high cost of the required enzymes, their low specific activity, their susceptibility to inactivation and the difficulty to recycle them2 . A group of naturally occurring cellulases are reported from heterotrophic microorganisms including bacteria and fungi3 . These organisms secrete cellulases to utilize cellulose as a carbon source. Bioconversion processes involve the hydrolysis of cellulose to produce reducing sugars; further fermentation of the sugars to ethanol and other bioproducts 4 . Cellulases hydrolyze the β-1,4 glycosidic bonds of the glucose polymer by two different ways, endoglucanases cut random positions along the cellulose chain, and exoglucanases progressively act on the terminal ends of the polymer, releasing either glucose molecules, or cellobiose3 . Finally, the cellobiose molecules produced are converted to glucose by intra- and extracellular β -glucosidases (EC 3.2.1.21), celludextrinases (EC 3.2.1.4), and cellodextrin phosphorylases (EC 2.4.1.49), depending upon the characteristic of each cellulolytic species 5 . Other than heterotrophs, cellulases belonging to glucoside hydrolase family (GH9) are also described from higher plants6 . However, it has been reported that plant cellulases participate in the biosynthesis and/or remodeling of cellulose rather than in its degradation 6,7 . Algae are phototrophs, ubiquitous with versatile metabolic pathways, which have been well exploited to obtained multiple products through algal refinery 8,9 . However, the presence of cellulases and cellulolytic activity was poorly described in algae. In 1966, Dvořáková-Hladká et al. reported the presence of β-glucosidase activity in S. obliquus , which allows it to grow using cellobiose as a substrate 10 . In 1970 Burczyk and col. reported the presence of extracellular cellulases in Scenedesmus obliquusbecause cell walls accumulated in the medium as a result of mother cells autospore release were deprived of the cellulose layer present in daughter cells. 11 . In 2012, Blifernez-Klassen and col. observed that the photoheterotrophic microalgae Chlamydomonas reinhardtii , was also capable of degrading and assimilating exogenous cellulose 3 . This interesting finding led us to investigate the presence of cellulases in S. quadricauda . This organism is a freshwater, non-mobile green algae which usually forms colonies of four cells. It belongs to the same class of green algae (Chlorophyceae) as the genus Chlamydomonas . S. quadricaudahas gained great importance due to its high capacity for effluent treatment, CO2 capture and biofuel production as we showed in a previous work 9 . The S. quadricaudaLWG002611 genome was sequenced and functional genes of different metabolic pathways were identified, such as those involved in the synthesis of triacyl glycerol (TAG) 9 . However, no 2 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. evidence on cellulase secretion and cellulose utilization in this alga was found or reported, as well as genes that encode proteins with glycoside hydrolase activity have not yet been described. In this work, we have identified different genes in the genome sequence of S. quadricauda LWG002611, belonging to GH1, GH5, GH9 and GH10 families. Furthermore, a comparative bioinformatic analysis was conducted in several available Scenedesmaceae algae genome (Scenedesmus obliquus EN0004 v1.0, Scenedesmus obliquusUTEX B 3031, Scenedesmus obliquus var. DOE0013 v1.0,Scenedesmus sp. NREL 46B-D3 v1.0, and S. quadricaudaLWG002611) to identify multiple homologs of endoglucanase, β-glucosidase and exocellulase genes. Additionally, a phylogenetic analysis and a 3D protein modeling were achieved. Our results showed that the 3D structures of all the modeled domains obtained and the main catalytic amino acid residues implicated in cellulolytic activity are well conserved in the Scenedesmus analyzed enzymes. These new findings open the opportunity to identify new cellulases from algae, as well as carry out their functional characterization to be used in biotechnological applications. 2. Methods 2.1. Sequence search, alignment and phylogenetic analysis Cellulase sequences of S. quadricauda were identified from genome sequence data of our previous study 9 using protein folding homology analysis by Phyre2 12 and Blast-N similarity study 13 with Monoraphidium neglectum taken as reference, and their details are included in table 1. Other analyzed sequences of Scenedesmus were taken from PhycoCosm 14 or NCBI {https://www.ncbi.nlm.nih.gov/} and their accession numbers are shown in table 2. Conserved domains, signal peptide, and GH-family assignment were identified with Prosite patterns 15 , DeepLoc 16 and PredAlgo17 . The sequences were aligned and processed with Clustal Omega 18 and visualized with ESPript 3.019 . To construct the phylogenetic trees, all the sequences were aligned with sequences from phylogenetically distant β-1,4-endoglucanases, β-glucosidases or exocellulases (respectively) from microalgae, fungi, plants, invertebrates and bacteria and processed with Gblock v0.91b before analyzing them in MEGA 6.0620,21 . Enzymes signal peptides were not included in the phylogenetic analysis. The phylogenetic trees were built by Maximum Likelihood method in MEGA 6.06 version with the model and the restrictions suggested by the program. Phylogenies were determined by Bootstrap Analysis of 100 replicates. 2.2. Protein 3D modeling The protein 3D models were generated with RaptorX Contact Prediction Server 22 . Superposition between each model and the templates was done using the align command in PyMOL 2.3.1 version {The PyMOL Molecular Graphics System}. The regions implicated in substrate binding and activity were manually annotated using the pattern sequences or 3D structure of cellulase templates available in Prosite and RCSB Pdb database (rcsb.org 23 ). 3. Results and Discussion 3.1. Identification of genes encoding different cellulases in Scenedesmus LWG002611 In the present work, we performed an analysis of Scendesmus quadricauda LWG002611 draft genome sequence, and eleven endoglucanases, β-glucosidases and exoglucanases gene sequences have been detected by protein folding homology analysis by Phyre2 and similarity study withMonoraphidium neglectum, a closely related species which has been taken a reference sequence for draft genome analysis. These sequences have shown 82.26-98.38% similarity with the reference sequences of M. neglectum (Table 1). According to the amino acid sequence analysis by Pfam two GH9 (Scequ2611|3068 and Scequ2611|4665), one GH5 (Scequ2611|2009), three GH1 (Scequ2611|3544, Scequ2611|9833 and Scequ2611|10006), one GH10 (Scequ2611|547), and four undefined GH family short proteins (Scequ2611|8404, Scequ2611|9353, Scequ2611|13370 and Scequ2611|13657) were detected (Table 1, Supplementary file 1) 3 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. 3.2. In silico analysis of Scenedesmaceaecellulases We have used different bioinformatic tools to analyze the cellulases present in five different species of Scenedesmus:Scenedesmus obliquusEN0004 v1.0, Scenedesmus obliquus UTEX B 3031, Scenedesmusobliquus var. DOE0013 v1.0, Scenedesmus sp . NREL 46B-D3 v1.0, Scenedesmus sp PABB004 and Scenedesmus quadricaudaLWG 002611. The presence of conserved motifs, the arrangement of diverse domains (catalytic domain, carbohydrate-binding module (CBM) and linker regions) and the phylogenetic relationship between the proteins were determined. Their amino acid sequences were compared with enzymes previously characterized from others taxonomic groups. The cellulases sequences analyzed in this study were classified with KEGG ENZYME Database Entry by Phycocosm 14 , into three groups: (i) endo-β-glucanases (EC 3.2.1.4), (ii) β-glucosidases (EC 3.2.1.21) and (iii) cellobiohydrolases or exoglucanases (EC 3.2.1.91) (Table 2). In S. quadricauda LWG002611 sequences with a higher length than 300 amino acids and with higher similarity with previously studied cellulases are code by the genes: Scequ2611|2009, Scequ2611|9833 and Scequ2611|547 (percentage of identity: 98.32%, 93.93% and 82.26% respectively, table 1), which were chosen to be included in the posterior bioinformatic analysis 3.2.1. Ενδο-β-γλυςανασες Among the selected species we found thirty genes that encode different endoglucanases. Their catalytic domains belong to the GH5 and GH9 family of CAZymes (Carbohydrate-Active Enzymes) 24 and seven of these proteins present CBM from families 1 and 2. While CBM2 were found only in GH5 endoglucanases, CBM1 were associated with some GH9 endoglucanases (Figure 1 and Table 2). Our results showed that three GH9 endoglucanases, Sceob1|18668, SceobE4|579152, SceobDOE|757385, present Big 1 domains in their C-terminal. Big 1 is a bacterial immunoglobulin (Ig)-like domain, usually present in GH9 endoglucanases. The functions of these Ig-like modules are not clear; however, they are supposed to be involved in the catalytic efficiency or in the structural stability of GH9 endoglucanases 25,26 . On the other hand, KAF8065624.1 present a LPMO domain in its N-terminal region. LPMO is a lytic polysaccharide monooxygenase domain that usually act synergically with GH9 domains, increasing the cellulolytic enzyme activity27 . Most of the GH9 endoglucanases analyzed are secreted or anchored to the cell membrane (with the catalytic domain localized to the outer surface of the plasma membrane). On the other hand, only three identified proteins GH5 endonucleases are predicted to be secreted, while the other four identified proteins would be cytoplasmic enzymes (Table 2). The analysis performed with the Prosite Database showed three highly conserved regions present in GH9 endoglucanases which contains conserved residues important for their catalytic activity 5,28 (Figure 2). The first region comprises the DAGD motif, where the first Asp (Asp54, Nasta 1KS8 numbering) is an active site residue. The second region contains a conserved RPHHR sequence, where the first His (His359, Nasta 1KS8 numbering) is also part of the active site of GH9 endoglucanases. Finally, Region III contains two Asp and Glu residues (Asp399 and Glu412, Nasta 1KS8 numbering) that would be involved in catalysis. Thus, all the proteins identified and analyzed in this study contain four acidic residues, D, H, D and E, in the mentioned regions, which would be part of the active site of GH9 endoglucanases and would be involved in catalysis, with the exception of ScsoPA4|KAF8062061.1 and SceobDOE|1035052 proteins, that lack the catalytic D from Region I and the H residue from Region II, respectively (Fig 2.A.). S. quadricauda LWG002611 possess another two hypothetical GH9 endoglucanase proteins (Scequ2611|3068 and Scequ2611|4665) showing a high similarity with respect to the analyzed Scenedesmus endoglucanases (Scequ2611|3068 34-36% with KAF6265438.1 and KAF8066338.1, Scequ2611|4665 30% to KAF6264795.1) but they lack the essential Glu residue from Region III (Fig 2B). Further studies are needed to determine if Scequ2611|3068 and Scequ2611|4665 are catalytically inactive enzymes or if they have a different mechanism than traditional glucosyl hydrolases. 4 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Cellulases usually present linkers with length from 6–14 residues long up to >100 residues 5,29 . FromScenedesmus endoglucanases analyzed, most of the CBM1 containing enzymes showed putative P/Srich or poliQ linkers, mainly located between the GH9 and the CBM regions (Fig 2.A.). These proline or glutamine rich spacers constitute a rigid type of linkers which are thought to act as spacers to avoid non-native interactions between domains that may affect the correct folding of proteins30 . In addition, the linkers would allow cellulases to push forward on the exterior of the polysaccharide with a caterpillar-like movement 5,31 . In contrast to what was described in GH9 endoglucanases from other algae, such as Chlamydomonas , Volvox and Gonium 5 CBM1 domains are located either at the C- or N-terminus of some the studied Scenedesmus GH9 endoglucanases. Notably, both, the N- and the C-terminus CBM1 analyzed, are cysteine-rich domains as previously described in Chlamydomonas . A phylogenetic tree was also constructed (Fig. 3) using Gblock and the MEGA 6.06 software from the alignment of the amino acid sequences from Scenedesmus GH9 enzymes together with homologous sequences identified with Blast-P from invertebrates, fungi, plants and bacteria. The organization of the tree branches suggest that Scenedesmus GH9 endoglucanases are evolutionarily closer to termites, worms, sea urchins and bivalves GH9 cellulases (red branches of the tree) rather than the enzymes from higher plants, fungi and bacteria. Figure 4 shows the 3D model of the Sceobl1|32711 GH9 and CBM1 domains constructed with RaptorX Contact Prediction. The model created presents a similar fold to that described for previously characterized GH9 endoglucanases 5,32 with a (α/α)6 -barrel fold. Besides, the catalytic amino acid residues are positioned in a similar spatial location when Sceobl1|32711 model was superposed with 1ks8 template (an endocellulase from the termite Nasutitermes takasagoensis 40.24% identical to Sceobl1|32711 (79% cover). On the other hand, its N-terminal CBM1 showed a high sequence identity (99.6%, cover 35%) with the cellulose-binding domain of endoglucanase I fromTrichoderma reesei (PDB entry: 4BMF) and its model present a good spatial conservation when both models where superposed. Regarding GH5 endoglucanases, they present the consensus pattern [LIV]-[LIVMFYWGA](2)-[DNEQG][LIVMGST]-{SENR}-N-E-[PV]- [RHDNSTLIVFY] 15 . The C-terminal Glu is an active site residue. The predicted catalytic residues, Glu168 and Glu309 (Pyrho 3W6M numbering) are strictly conserved in all the GH5 endonucleases analyzed (Figure 5). The Figure 6 shows the GH5 endoglucanase phylogenetic tree. The tree branching organization suggest that most of the GH5 proteins analyzed are evolutionarily closer to those of other microalgae and higher plants. However, the group of enzymes containing a CBM2 are closer to fungal and bacterial endoglucanases, suggesting a microbial origin. The homology model of the Sceobl1|14060 GH5 domain present the (α/β)8 TIM barrel fold classical of GH5 family 33 (Figure 7). The superposition with Pyrho 3W6M PDB structure (an hyperthermophilic endocellulase from Pyrococcus horikoshii ) showed a conservation of the catalytic amino acid spatial location. Respects its N-terminal CBM2 domain, Sceobl1|14060 has a high sequence identity (97.3% identity, 68% cover) with 2RTT PDB structure (a chitin-binding domain of Chi18aC from Streptomyces coelicolor ); moreover, both models showed a high structural and spatial conservation. 3.2.2. β-γλυςοσιδασες In the the analyzed genomes we found twenty-nine putative β-glycosidases, all of them belonging to the GH1 family of CAZymes (Figure 8). The most common enzymatic activities reported for glycoside hydrolases of this family are β-glucosidases and β-galactosidases. It has been previously described that one of the highly conserved regions in GH1 sequences has a glutamic acid residue and is classified as GH1 1 34 . This region between positions 388-392 (Nanochloropsis β-glucosidase GH1 numbering, PDB code: 5YJ7,) presents a conserved sequence (V/I)TENG. The Glu residue would 5 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. participate in the cleavage of the glycosidic bond by acting as a nucleophile was first identified as Glu358 in a β-glucosidase from Agrobacterium35 . 34 . This catalytic nucleophile In our work, the conserved region (V/I)TENG was found in all the GH1 sequences analyzed: (Figure 9.A) GH1 1. The extended region defined as consensus is: [LIVMFSTC]-[LIVFYS] [LIV]-[LIVMST]-E-NG-[LIVMFAR]-[CSAGN]. All of the proteins chosen in this work possess in this region the Glu involved in catalysis followed by Asn and Gly, as can be seen in Figure 9. However, the four amino acids upstream Glu residue appear to differ in the proteins, with Ile-Trp-Ile-Thr being predominant. As a second signature pattern, the conserved region GH1 2 (Figure 8.A) was chosen for our analysis. This region, defined as: F-x-[FYWM]-[GSTA]-x-[GSTA]-x-[GSTA](2)-[FYNH]-[NQ]-x-E-x-[GSTA], is located at the N-terminal of GH1 β-glucosidases, however, it may not be present in some proteins of this family. The alignment of Sceobl|9031 and Sceobl|10236 sequences showed that these proteins do not contain that region (Figure 9.A). On the other hand, the protein SceoblEN4 |617109, possesses only nine of the fifteen amino acids established as consensus, which is equivalent to 60 %, and therefore would be considered that this domain is present but with some variations in the amino acid sequence. Similarly, Sceobl1|35463 and Scequ2611|9833 proteins have only 46 and 40 % of the consensus sequence, respectively. Scesp1|1644545 only possesses the last four amino acids of this consensus sequence, while the other eleven, do not correspond to the established consensus. The sequence of KAF8060308.1 BGLU11 shows some particular characteristics in this region. Although it possesses 86 % sequence identity respect to the established consensus sequence, it presents an insertion of several amino acids downstream the consensus (34 residues), before x-E-x-[GSTA]. This protein also contains an additional domain in its N-terminal region, a protein disulfide isomerase domain (cl36828: ER PDI fam Superfamily,36 ), previously involved in protein folding 37 . Interestingly, only five of the proteins analyzed contains the first Phe residue, which is characteristic of the GH1 2 pattern. It is interesting to note that none Scenedesmaceae β- glycosidases analyzed in this study presents CBM nor linkers. On the other hand, S. quadricauda LWG002611 have a hypothetical GH1 β-glucosidase protein, named Scequ2611|3544, with high similarity with other Scenedesmus endoglucanases (86% cover and 29.45 % identity with KAF8059426.1) but that lacks the region containing the catalytic Glu residue (Figure 9.B). The phylogenetic tree performed shows four β-glucosidase subgroups: (i) the plant GH1 subgroup, (ii) the GH1 from algae, (iii) the GH1 from fungi, and (iv) the GH1 from bacteria (Figure 10). The Scesp1|1509300, SceobDOE|17466, SceoblEN4|575894, and SceobDOE|32074 proteins are grouped in a branch close to the bacteria enzyme, that proposes the possible acquisition of these genes by horizontal transfer. On the other hand, the Scequ2611|9833 protein was found within a large group of GH1 enzymes from algae. This result suggests the correct inference of its sequence and the possibility that orthologous genes are those that code for proteins found on a nearby branch. There is at least one representative of each species corresponding to a genus in each branch of the group of proteins from algae. This result suggests that the different β-glucosidases present in the different species could fulfill the same function and that it would not be redundant within the genus. The homology model of the Sceob1|9434 β-glucosidase constructed with RaptorX Contact Prediction is shown in Figure 11. The superposition with Nannochloropsis oceanica BGLN1 β-glucosidase crystal structure (PDB code: 5YJ7) shows a catalytic amino acid positional conservation in the central region. Also, it presents an overall structure of TIM barrel, and the ENG residues conserved, which suggest a reliable protein function assignment of this new protein in Scenedemus quadricauda. 3.2.3. Exocellulases Exoglucanases or cellobiohydrolases (CBH) (EC 3.2.1.74; 1,4-β-D-glucan-glucanhydrolase) catalyze the successive hydrolysis of residues from the reducing and non-reducing ends of the cellulose polysaccharide, re- 6 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. leasing cellobiose molecules as main product of the reaction 38 . These enzymes account for 40 to 70% of the total component of the cellulase system, and are able to hydrolyze crystalline cellulose. An excellent candidate for use as a bait to explore algal genomes is the GH10 from Cellulomonas fimi (CexP07986 Uniprot). High resolution crystal structures are available and there is a large literature on the kinetic characterization of the enzyme and the identification of amino acid residues important to the mechanism of catalysis 39 . Among all the Scenedesmaceae genomes studied we found at least eleven enzymes, all of them putative GH10 bifunctional cellulase/xylanase proteins (Figure 12A). These enzymes are monomeric proteins with a molecular mass ranging from 50 to 65 kDa, although there are smaller variants (41.5 kDa) in some fungi, such as Sclerotium rolfsii 40 . The calculated molecular mass of five of the eleven algal proteins studied were higher (between 70 and 100kDa), while in the other six enzymes is within the expected range (table 2). In general, most of the proteins containing GH10 domains show a structure that matches an eightfold α/ β-barrel with a profound channel in the center 41 . However, it has been proposed that the exocellulases from the GH10 family form a transient tunnel by the extension of some loop regions upon substrate binding42 . It has been reported that GH10 enzymes present a double-displacement ’retaining’ hydrolysis mechanism, where one catalytic residue acts a nucleophile and the other acts as a general acid/base43 . The catalytic nucleophile in Cex is Glu274 and the putative acid/base catalyst is Glu168 44 . As shown in figure 12B, both residues are full conserved in the algal protein sequences. However, they were replaced by an Asp and Ile residues in the Scequ2611|547 protein. We also performed a phylogenetic tree for the GH10 exoglucanase (Figure 13) from the alignment of the amino acid sequences fromScenedesmus GH10 enzymes together with homologous sequences from invertebrates, fungi, plants and bacteria. The branches distribution suggests the GH10 analyzed proteins are evolutionarily closer to those of other microalgae and higher plants. In addition, we built a sequence homology model of the Sceob1|4623 exocellulase using RaptorX Contact Prediction is shown in Figure 14. The superposition with Cellulomonas fimi exocellulase crystal structure (PBD code: 1EXG) shows a spatial location conservation of the algal protein residues Glu236 and Glu338 with respect to catalytic residues from the bacterial protein, also suggesting the conservation of the catalytic site. Conclusion The discovery of new microbial sources of cellulases is a crucial strategy to reduce costs of various industrial processes using such enzymes. Cellulases are produced by various microorganisms including bacteria, fungi and actinomycetes. Recently was reported that they are also produced by some animals like termites and crayfish without certainties about his role in vivo 45 . The search, isolation and identification of new cellulose degrading microorganisms from different environments are of crucial importance to get new cellulases with unique and distinctive characteristics. Microalgae are considered a valuable source of new enzymes with biotechnological potential. However, the presence of cellulolytic enzymes is meagre studied form this photosynthetic microorganisms. Different works published during the last decade report cellulolytic activity (either by experimental evidence or by bioinformatic analysis) in C. reinhardtii , V. carteri , G. pectorale andA. protothecoides but the genus Scenedesmus had not been analysed3,5,46 . This is the first bioinformatic analysis of Scenedesmaceae cellulases reported. It comprises GH5 and GH9 β-1,4-endoglucanases, GH1 β-glucosidases and GH10 exoglucanases. Our results shows that GH9 endoglucanases analyzed are phylogenetically closer to invertebrates, termites and bivalve rather than higher plant, bacteria or fungi. On the other hand, most of GH1 β-glucosidases analyzed are evolutionarily closer to enzymes of other microalgae, however, four of them are grouped in a branch close to the bacteria enzymes, 7 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. result that suggests the probable gaining of their genes by horizontal transfer. In contrast, GH5 and GH10 studied enzymes are evolutionarily closer to enzymes of other microalgae and higher plants. Most of the analyzed enzymes present signal peptides for membrane anchoring or extracellular secretion. This result suggests the presence of extracellular cellulolytic machinery in Scenedesmaceae. Only some of the analyzed enzymes were found to have additional modules and linkers besides its GH domains, and particularly a few endoglucanases have CBM modules, from CBM1 and CBM2 families. The combination of GH catalytic domains together with CBMs and, in some cases linkers, propose that these cellulases would present an enhanced cellulolytic activity. The presence of this battery of enzymes in the photoheterotrophic algaeScenedesmus suggest that these organisms are perfectly prepared for use of cellulose as carbon source. This strategy would represent an advantage that would have allowed Scenedesmaceae to occupy many environments in nature. The findings reported in this work explores just one family within the Chlorophyta taxon, but it increases the evidence in favor of the presence of conserved cellulolytic machinery in photoheterotrophic organisms and encourages to continue with the search for cellulases in other species of microalgae. References 1 Saini, J. K., Saini, R. & Tewari, L. Lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments. 3 Biotech5 , 337-353, doi:10.1007/s13205-014-0246-5 (2015). 2 Lynd, L. R., Weimer, P. J., van Zyl, W. H. & Pretorius, I. S. Microbial cellulose utilization: fundamentals and biotechnology.Microbiology and molecular biology reviews : MMBR 66 , 506-577, table of contents (2002). 3 Blifernez-Klassen, O. et al. Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii. Nature communications 3 , 1214, doi:10.1038/ncomms2210 (2012). 4 Menon, V. & Rao, M. 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Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs). Biochimie 95 , 1865-1870, doi:10.1016/j.biochi.2013.06.009 (2013). 41 Gao, F., Jiang, Y., Zhou, G. H. & Han, Z. K. The effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets. Br Poult Sci 48 , 480-488, doi:10.1080/00071660701477320 (2007). 42 Schubot, F. D. et al. Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum.Biochemistry 43 , 1163-1170, doi:10.1021/bi030202i (2004). 43 Gilkes, N. R. et al. Structural and functional relationships in two families of beta-1,4-glycanases. European journal of biochemistry / FEBS 202 , 367-377, doi:10.1111/j.1432-1033.1991.tb16384.x (1991). 44 MacLeod, A. M., Lindhorst, T., Withers, S. G. & Warren, R. A. The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants.Biochemistry 33 , 6371-6376, doi:10.1021/bi00186a042 (1994). 45 Watanabe, H. & Tokuda, G. Animal cellulases. Cellular and molecular life sciences : CMLS 58 , 11671178, doi:10.1007/PL00000931 (2001). 46 Vogler, B. W. et al. Characterization of plant carbon substrate utilization by Auxenochlorella protothecoides. Algal Research 34 , 37-48, doi:https://doi.org/10.1016/j.algal.2018.07.001 (2018). Table 1: Predicted cellulases# of S. quadricauda LWG 002611 S. No. Genes Genes Predicted mRNAs Predicted proteins Predicted proteins Predicted ID Length (bp) Length (bp) Length (aa) Size (KD) GH Fami 10 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. S. No. Genes Genes Predicted mRNAs Predicted proteins Predicted proteins Predicted 1. 2. 3. 4. 5. 6. 8. 9. 10. 11. 12. 2009 3068 8404 9353 4665 3544 9833 10006 13370 13657 547 2176 3243 1472 635 3365 4540 5359 1578 1613 523 4033 963 1539 303 225 1332 1272 1173 540 291 363 2165 321 513 101 75 443 424 390 181 96 121 734 34.45 54.83 11.19 7.69 48.97 48.28 43.37 10.78 10.78 11.91 80.85 GH5 GH9 inc inc GH9 GH1 GH1 GH1 inc inc GH10 # Sequences are separately provided in Supplementary file; inc: Inconclusive result due to short sequence Table 2 Organism ID/PBD ENDOGLUCANASES Scenedesmus 365111 obliquus EN0004 v1.0 Scenedesmus 579152 obliquus EN0004 v1.0 Scenedesmus 610731 obliquus EN0004 v1.0 199 Scenedesmus obliquus UTEX B 3031 Scenedesmus 8271 obliquus UTEX B 3031 Scenedesmus 14060 obliquus UTEX B 3031 Scenedesmus 18668 obliquus UTEX B 3031 25222 Scenedesmus obliquus UTEX B 3031 Scenedesmus 35062 obliquus UTEX B 3031 Scenedesmus 19147 obliquus UTEX B 3031 Conserved domains Cell Location Size (aa), mol.wt Taxonomic group CBM2, GH5 Cytoplasm 555, 59.86 kDa Bacteria GH9 Cytoplasm 716, 75.57 kDa Green algae, Insect 5TM, GH9 Extracellular 891,91.43 kDa Termites 1TM, GH9 Cell Membrane/ Extracelular 665, 70.87 kDa Earthworms GH9 Extracellular 489, 53.02 kDa CBM2, GH5 Cytoplasm 551, 59.37 kDa Sea urchin (animal). Insect Bacteria CBM1, GH9 Extracellular 710, 74.88 kDa Green algae, Insect GH9 Extracellular 861, 88.71 kDa Green algae, Sea urchin GH9 Extracellular 487, 53.70 kDa Termites, insects CBM1, GH9 Extracellular 730, 77.78 kDa Thermophilic bacterium 11 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 32711 1TM, GH9 Extracellular 629, 67.22 kDa Microalgae, Sea urchin, Insect 1035052 GH9 Extracellular 439, 47.68 kDa Earthworms, Chordates 739074 GH9 Extracellular 479, 51.60 kDa Green algae, Sea urchin 757385 GH9 Extracellular 733, 77.30 kDa Green algae, Termites 776386 1TM, GH9 Cell Membrane (GH9 outside) 521, 57.08 kDa Termites 826696 1TM, GH9 Cell Membrane (GH9 outside) 611, 65.76 kDa Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus obliquus var. DOE0013 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus PABB0004 Scenedesmus PABB0004 1002809 GH5 Extracellular 419, 45.87 kDa 1008808 CBM2 GH5 Cytoplasm 518, 55.90 kDa Earthworms, Termites Green algae (una sola coccomyxa) Fungi, cellulolityc bacteria Bacteria 761682 CBM1, GH9 Extracellular 682, 72.02 kDa Green algae, Worm 956336 GH9 Cytoplasm 511, 55.45 kDa Worm, Termite 1003724 GH5 Cytoplasm 424, 46.48 kDa Archaea, Bacteria 1274183 GH9 Extracellular 473, 50.60 kDa Sea urchin Termites 1508728 GH5 Extracellular 451, 49.56 kDa Worms, termite 1655835 GH9 Extracellular 360, 38.96 kDa Amoeba, Bacteria, worms 1693221 GH9 Extracellular 614, 66.93 kDa Green algae, Termite KAF8061121.1 (celF) KAF8066338.1 (celD) CBM1, GH9 Cytoplasm 769, 80.61 kDa LPMO, GH9 Cell membrane 672, 69.77 kDa Green algae, sea urchin Green algae, Worm Anemones 12 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Scenedesmus PABB0004 KAF8065624.1 (celF) GH9 Extracellular 1166, 119.94 kDa Scenedesmus KAF8062061.1 PABB0004 (celZ) Scenedesmus 2009 quadricauda LGW0026011 βΓΛΥ῝ΟΣΙΔΑΣΕΣ GH9 Cell membrane 766, 77.74 kDa GH5 Cell Membrane (GH5 outside) 321, 34.33 kDa Organism Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 Scenedesmus sp. NREL 46B-D3 v1.0 [Scenedesmus sp. PABB004] [Scenedesmus sp. PABB004] [Scenedesmus sp. PABB004] [Scenedesmus sp. PABB004] S.quadricauda LWG 002611 Scenedesmus obliquus var. DOE0013 Scenedesmus obliquus var. DOE0013 Scenedesmus obliquus var. DOE0013 Scenedesmus obliquus var. DOE0013 Scenedesmus obliquus var. DOE0013 Scenedesmus obliquus var. DOE0013 EXOCELLULASES Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus EN0004 v1.0 Scenedesmus obliquus UTEX B 3031 Scenedesmus obliquus UTEX B 3031 v1.0 v1.0 v1.0 v1.0 v1.0 v1.0 Green algae, Sea anemone Crustaceans, Green algae, Termite Bivalve. Green algae, Bacteria ID/PBD 574933 575894 610267 617109 2100 3494 8342 9031 10236 17466 23136 26740 32074 32663 35463 88126 1297936 1298554 1298717 1507547 1644545 1509300 KAF8062692.1 KAF8062654.1 KAF8060308.1 KAF8059426.1 9833 67487 177517 746725 797715 882370 1019541 Conserved domains GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 / PDI GH1 GH1 GH1 GH1 GH1 GH1 GH1 GH1 Cell Location Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Extracellular Membrane Extracellular Extracellular Extracellular Membrane Membrane Extracellular Extracellular Extracellular Chloroplastic Membrane Extracellular Extracellular Extracellular Mitochondrion Cytoplasm Extracellular Cytoplasm Cell membrane Extracellular Cell membrane Size (a 565, 62. 795, 87. 596, 64. 484, 54. 490, 54. 589, 64. 707, 78. 389, 43. 361, 40. 814, 89. 558, 61. 560, 61. 917, 100 573, 64. 477, 54. 539, 59. 512, 57. 489, 53. 488, 53. 600, 66. 461, 52. 825, 91. 1210, 13 744, 75. 2136, 22 1038, 10 391, 43. 62.11 kD 579, 61. 513, 57. 563, 62. 496, 54. 255, 24. 352887 587573 4623 16336 GH10 GH10 GH10 GH10 Cytoplasm, Soluble Cytoplasm, Soluble Extracellular Cytoplasm, Soluble 635, 849, 498; 440; 13 70. 93. 55. 49. Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Scenedesmus Scenedesmus Scenedesmus Scenedesmus Scenedesmus Scenedesmus Scenedesmus obliquus UTEX B 3031 obliquus var. DOE0013 v1.0 obliquus var. DOE0013 v1.0 obliquus var. DOE0013 v1.0 obliquus var. DOE0013 v1.0 PABB0004 quadricauda LGW0026011 10793 977091 243254 752374 750376 KAF8058849.1 547 GH10 GH10 GH10 GH10 GH10 GH10 GH10 Extracellular Cytoplasm, Soluble Extracellular Cytoplasm, Soluble Cytoplasm, Soluble Mitochondrion, Membrane Extracellular. Fig 1 Fig.1 Domain architecture of the putative endoglucanases enzymes in the proteome of Scenedemaceae. The figure shows all predicted proteins containing domains annotated as glycosyl hydrolases in families GH9 (A ), or GH5 (B ), in the analyzed strains. Red and yellow squares represent signal peptides and transmembrane domains, respectively. CBM: carbohydrate binding domain. LPMO: Lytic polysaccharide monooxygenases Fig 2A 2B Fig.2 A GH9 family endoglucanase alignment. Alignment between Scenedesmus GH9 endoglucanases, and Nasta|1KS8 A and Trire|4BMF A amino acid sequences used as the template for 3D modeling. Red asterisk above the alignment indicates catalytic residues, characterized in Nasta|1KS8. Cyan asterisk indicate cellulose binding residues and purple asterisk indicate conserved cysteines. Other conserved positions are shown in red and boxed in blue. CBM domain is framed in grey. Abbreviations: ScespPA4: Scenedesmus sp. PABB004, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, Sceob1:Scenedesmus obliquus UTEX B 3031, SceobDOE: Scenedesmus obliquus var. DOE0013 v1.0, SceobE4: Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Nasta:Nasutitermes takasagoensis and Trire: Trichoderma Reesei . B Scequ2611|3068 and Scequ2611|4665 amino acid sequences alignment. Alignment between Scenedesmus GH9 endoglucanases, and two enzymes from close species. Red asterisk above the alignment indicates the catalytic residues. Other conserved positions are shown in red and boxed in blue. Abbreviations: ScespPA4: Scenedesmus sp.PABB004, Scesp1: Scenedesmus sp . NREL 46B-D3 v1.0, Scequ2611:Scenedesmus quadricauda LWG 002611, Monne: Monoraphidium neglectum and Rapsu: Raphidocelis subcapitata Fig. 3 14 443; 393; 495; 839; 410; 829; 735; 49, 44. 55. 92. 46. 90. 79. Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Fig. 3 GH9 family endoglucanase Phylogenetic tree. The tree is based on the alignment of the regions obtained with Gblock and it was built by Maximum Likelihood method in MEGA 6.06 version, under the LG + G model, suggested by MEGA. Phylogenies were determined by Bootstrap Analysis of 100 replicates. Branch lengths are proportional to distances. Bootstrap values are shown above branches. The dark green branches contain GH9 from plants, the lighter green ones correspond to GH9 from algae, the orange ones contain GH9 from fungi, the red ones correspond to invertebrates and those colored purple contain GH9 from bacteria. Abbreviations: Gonpe: Gonium pectorale, Volca:Volvox carteri, Chlre: Chlamydomonas reinhardtii, Sceob1:Scenedesmus obliquus UTEX B 3031, SceobDOE: Scenedesmus obliquus var. DOE0013 v1.0, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, ScespPA4: Scenedesmus sp. PABB004, SceobE4:Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Tetob: Tetradesmus obliquus , Rapsu:Raphidocelis subcapitata, Retfl: Reticulitermes flavipes , Copfo: Coptotermes formosanus , Mesnu: Mesocentrotus nudus , Methi: Metaphire hilgendorfi , Rhiso: Rhizoctonia solani , Citun: Citrus unshiu , Mucpr: Mucuna pruriens, Macco:Macleaya cordata , Artan: Artemisia annua , Anaco:Ananas communis , Jatcu: Jatropha curcas, Eucgr:Eucalyptus grandis , Maldo: Malus domestica , Spiol:Spinacia oleracea , Actch: Actinidia chinensis var. chinensis , Jugre: Juglans regia , Leepe: Leersia perrieri,Oryba: Oryza barthii, Basme: Basidiobolus meristosporus,Neoca: Neocallimastix californiae, Pirfi: Piromyces finnis , Anaro: Anaeromyces robustus, Pirsp: Piromyces sp.E2, Paele: Paenibacillus lentus, Flasp: Flammeovirga sp. SJP92, Hahch:Hahella chejuensis KCTC 2396, Marba: marine bacteriumAO1-C, Niasp: Niastella sp . CF465, Niavi: Niastella vici,Chisp: Chitinophaga sp. YR573, Chisa: Chitinophaga sancti , Allop: Allonocardiopsis opalescens, Celsp: Cellulomonas sp ., Celsh: Cellulomonas shaoxiangyii , Celce: Cellulomonas cellasea DSM 20118, Strsp: Streptomyces sp . Ru87, Plasp:Plantactinospora sp. KBS50, Micro: Microbispora rosea,Miccr: Micromonospora craniellae, Micya: Micromonospora yangpuensis , Micen: Micromonospora endolithica, Micsp:Micromonospora sp. CNZ295 15 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Fig. 4 Fig. 4 3D modeling of GH9 endoglucanase 32711 from Scenedesmus obliquus UTEX B 3031. A. Proposed model of Sceobl1|32711 (pink) superimposed to Nasta|1KS8 A GH9 and Trire| 4BMF A CBM1 templates (cyan); putative catalytic and binding residues are shown. B. Inset of catalytic residues area. Abbreviations: Nasta: Nasutitermes takasagoensis and Trire: Trichoderma Reesei Fig. 5 Fig. 5.GH5 family endoglucanase alignment. Multiple alignment between Scenedesmus GH5 endoglucanases, and Pyrho|3W6M A used as the template for 3D modeling. The multiple sequence alignment was performed using Clustal Omega program and ESPript 3.0. Red asterisk above the alignment indicates catalytic residues. Cyan asterisk indicate cellulose binding residues. Other conserved positions are shown in red and boxed in blue. CBM domain is framed in grey. Abbreviations: ScespPA:Scenedesmus sp. PABB004, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, Sceob1: Scenedesmus obliquus UTEX B 3031, SceobDOE:Scenedesmus obliquus var. DOE0013 v1.0, SceobE4:Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Pyrho: Pyrococcus horikoshii Fig. 6 Hosted file image11.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Fig. 6 GH5 family endoglucanase Phylogenetic tree. The tree is based on the alignment of the regions obtained with Gblock and it was built by Maximum Likelihood method in MEGA 6.06 version, under the WAG + G model suggested by MEGA. Phylogenies were determined by Bootstrap Analysis of 100 replicates. Branch lengths are proportional to distances. Bootstrap values are shown above branches. The dark green branches contain GH5 from plants, the lighter green ones correspond to GH5 from algae, the orange ones contain GH5 from fungi, and those colored purple contain GH5 from bacteria. Abbreviatures: Aciva:Acidovorax valerianellae, Xanal: Xanthomonas albilineans , Xanca: Xanthomonas campestris pv. Campestris , Xanor:Xanthomonas oryzae pv. Oryzae, Acici: Acidovorax citrulli , Xantr: Xanthomonas translucens pv. Translucens, Xylfa: Xylella fastidiosa, Acian: Acidovorax anthurii , Acisp: Acidovorax sp. MR-S7 , Canma: Candidatus Magnetobacterium bavaricum , Thiba: Thiotrichales bacterium, Ulisp:Uliginosibacterium sp., Halsp: Halothiobacillus sp.,Massp: Massilia sp., Albte: Albitalea terrae, Crepo: Crenothrix polyspora , Metis: Methylomagnum ishizawai , Hydsp:Hydrogenophaga sp. A37 , Polbr: Polyangium brachysporum,Abypr: Abyssibacter profundi , Rapsu: Raphidocelis subcapitata , Monne: Monoraphidium neglectum, Sceob1:Scenedesmus obliquus UTEX B 3031, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, SceobDOE: Scenedesmus obliquus var. DOE0013 v1.0, Tetob: Tetradesmus obliquus , SceobE4: Scenedesmus obliquus EN0004 v1.0, Kleni: Klebsormidium nitens, Neoca: Neocallimastix californiae, Pirfi: Piromyces finnis,Pirsp: Piromyces sp., Anaro: Anaeromyces robustus, Cocsu:Coccomyxa subellipsoidea, Chleu: Chlamydomonas eustigma,Monsp: Monosporascus sp ., Micco: Micromonas commode,Glyso: Glycine soja, Vigra: Vigna radiata var. radiata,Phaan: Phaseolus angularis, Arath: Arabidopsis thaliana,Braca: Brassica campestris, Macco: Macleaya cordata Fig.7 Fig. 7 3D modeling of GH5 endoglucanase 14060 from Scenedesmus obliquus UTEX B 3031. A. Proposed model of Sceobl1|14060 (pink) superimposed to Pyrho|3W6M A GH5 and Strco|2RTT A CBM2 templates (cyan); putative catalytic and binding residues are shown. B. Inset of substrate binding residues area. C. Inset of catalytic residues area. Abbreviations: Strco:Streptomyces coelicolor ; Pyrho: Pyrococcus horikoshii. Fig.8 16 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Fig. 8 Domain architecture of the putative β-glucosidases enzymes in the proteome of Scenedemaceae. The figure shows all predicted proteins containing domains annotated as glycosyl hydrolases in families GH1, in the analyzed strains. Red, yellow, magenta and cyan boxes represent signal peptides, transmembrane domains, RAMA domain, and ER-PDI superfamily domain, respectively Fig. 9A Hosted file image14.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Hosted file image15.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus 9B Φιγ. 9.Α ΓΗ1 φαμιλψ β-γλυςοσιδασε αλιγνμεντ. (ὃλορ ονλινε, δουβλε ςολυμν). Multiple alignment between Scenedesmus GH1 endoglucanases, and Nanoc|5YJ7 A, sequence used as the template for 3D modeling. The multiple sequence alignment was performed using Clustal Omega program and ESPript 3.0. Red asterisk above the alignment indicates the catalytic residues. Abbreviations: ScespPA4:Scenedesmus sp . PABB004, Scesp1: Scenedesmus sp . NREL 46B-D3 v1.0, Sceob1: Scenedesmus obliquus UTEX B 3031, SceobDOE:Scenedesmus obliquus var. DOE0013 v1.0, SceobE4:Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Nanoc: Nannochloropsis oceanica .B. Scequ2611|3544 amino acid sequences alignment. Alignment between Scenedesmus 3544 putative endoglucanase, and enzymes from close species. The multiple sequence alignment was performed using Clustal Omega program and ESPript 3.0. Red asterisk above the alignment indicates catalytic residues. Abbreviations: ScespPA4: Scenedesmus sp. PABB004, Scequ2611: Scenedesmus quadricauda LWG 002611, Monne: Monoraphidium neglectum and Rapsu: Raphidocelis subcapitata Fig. 10 Hosted file image17.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Fig. 10 GH1 family endoglucanase Phylogenetic tree.The tree is based on the alignment of the regions obtained with Gblock and it was built by Maximum Likelihood method in MEGA 6.06 version, under the LG + G model, suggested by MEGA. Phylogenies were determined by Bootstrap Analysis of 100 replicates. Branch lengths are proportional to distances. Bootstrap values are shown above branches. The dark green branches contain GH1 from plants, the lighter green ones correspond to GH1 from algae, the orange ones contain GH1 from fungi, and those colored purple contain GH1 from bacteria. Abbreviations: Sceob1:Scenedesmus obliquus UTEX B 3031, SceobDOE: Scenedesmus obliquus var. DOE0013 v1.0, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, ScespPA4: Scenedesmus sp. PABB004, SceobE4:Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Botbo: Botryobasidium botryosum , Aspud:Aspergillus udagawae , Rasem: Rasamsonia emersonii , Exoaq:Exophiala aquamarine , Aurna: Aureobasidium namibiae , Aurme: Aureobasidium melanogenum , Aurpu: Aureobasidium pullulans , Baupa: Baudoinia panamericana , Aciri:Acidomyces richmondensis , Horwe: Hortaea werneckii , Dotse: Dothistroma septosporum (strain NZE10 / CBS 128990) (Mycosphaerella pini), Zymtr: Zymoseptoria tritici , Cerze: Cercospora Zeina , Cerbe: Cercospora berteroae , Ceret: Cercospora beticola , Calfi: Caldanaerobius fijiensis , Actre:Actinoplanes regularis , Vulte: Vulcaniibacterium tengchongense , Vicva: Victivallis vadensis , Rubsp:Rubrivirga sp. , Lewag: Lewinella agarilytica , Ulvma:Ulvibacterium marinum , Arexa: Arenicella xanthan , Verba:Verrucomicrobiae bacterium, Sacde: Saccharophagus degradans, Lenar: Lentisphaera araneosa, Halhy:Haliscomenobacter hydrossis, Mansp: Mangrovimonas sp.,Urecr: Urechidicola croceus, Rosmi: 17 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. Roseivirga misakiensis, Polsp: Polaribacter sp., Flaba: Flaviramulus basaltis, Aquag: Aquimarina aggregate, Confl:Confluentibacter flavum, Helan: Helianthus annuus, Orypu:Oryza punctate, Dauca: Daucus carota subsp. Sativus,Solly: Solanum lycopersicum, Artan: Artemisia annua,Oryba: Oryza barthii, Braol: Brassica oleracea var. Oleracea, Leepe: Leersia perrieri, Cinmi: Cinnamomum micranthum f. kanehirae, Anaco: Ananas comosus, Orypu:Oryza punctate, Arahy: Arachis hypogaea, Vitvi:Vitis vinifera, Cajca: Cajanus cajan. Fig. 11 Φιγ. 11 ΓΗ1 β-γλυςοσιδασε 3Δ μοδελ 3D modeling of GH1 β-glucosidase 3494 from Scenedesmus obliquus UTEX B 3031. A.Proposed model of Sceobl1|3494 (pink) superimposed to Nanoc|5YJ7 A GH1 template (cyan); putative catalytic residues are shown. B. Inset of catalytic residues area. Abbreviations: Nanoc: Nannochloropsis oceanica Fig. 12 A B Hosted file image20.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Hosted file image21.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Fig. 12A Domain architecture of the putative exoglucanases enzymes in the proteome of Scenedemaceae The figure shows all predicted proteins containing domains annotated as glycosyl hydrolases in families GH10, in the analyzed strains. Red boxes represent signal peptides. Yellow squares represent transmembrane domains B GH10 family exoglucanase alignment. Multiple alignment between Scenedesmus GH10 exoglucanases, and Celfi|P07986, sequence used as the template for 3D modeling. The multiple sequence alignment was performed using Clustal Omega program and ESPript 3.0. Red asterisk above the alignment indicates catalytic residues. Abbreviations: ScespPA4: Scenedesmus sp. PABB004, Scesp1: Scenedesmus sp. NREL 46B-D3 v1.0, Sceob1:Scenedesmus obliquus UTEX B 3031, SceobDOE: Scenedesmus obliquus var. DOE0013 v1.0, SceobE4: Scenedesmus obliquus EN0004 v1.0, Scequ2611: Scenedesmus quadricauda LWG 002611, Celfi:Cellulomonas fimi . Fig. 13 Hosted file image22.emf available at https://authorea.com/users/500895/articles/581523-molecular-insightinto-cellulose-degradation-by-the-phototrophic-green-alga-scenedesmus Fig. 13 GH10 family exoglucanase Phylogenetic tree . The tree is based on the alignment of the regions obtained with Gblock and it was built by Maximum Likelihood method in MEGA 6.06 version, under the WAG + G model, suggested by MEGA. Phylogenies were determined by Bootstrap Analysis of 100 replicates. Branch lengths are proportional to distances. Bootstrap values are shown above branches. The dark green branches contain GH10 from plants, the lighter green ones correspond to GH10 from algae, the orange ones contain GH10 from fungi, and those colored purple contain GH10 from bacteria. Abbreviations: Isodo:Isoptericola dokdonensis, Isosp: Isoptericola sp. Jonde:Jonesia denitrificans , Celbo: Cellulomonas bogoriensis , Actfe: Actinotalea fermentans, Celgi: Cellulomonas gilvus , Celsp: Cellulomonas sp ., Celbi: Cellulomonas biazotea , Celal: Cellulomonas algicola, Celfi: Cellulomonas fimi 18 Posted on Authorea 1 Sep 2022 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.166030463.37247755/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. , Micha: Micromonospora haikouensis Micsp: Micromonospora sp ., Micec: Micromonospora echinaurantiaca, Micco:Micromonospora coxensis, Celsp: Cellulomonas sp. , Celfl:Cellulomonas flavigena , Ktera: Ktedonobacter racemifer , Actsp: Actinomadura sp . GC306, Actda: Actinomadura darangshiensis , Stral: Streptomyces alni, Thebi:Thermobispora bispora, Phach: Phanerodontia chrysosporium , Neopa: Neocallimastix patriciarum, Arath: Arabidopsis thaliana, Nagal: Naganishia albida, Gibze: Gibberella zeae , Fusox: Fusarium oxysporum f. sp. lycopersici ,Aurpu: Aureobasidium pullulans, Humgr: Humicola grisea var. Thermoidea, Maggr: Magnaporthe grisea, Magor: Magnaporthe oryzae, Talfu: Talaromyces funiculosus, Agabi: Agaricus bisporus , Phach: Phanerodontia chrysosporium, Hypje:Hypocrea jecorina , Ustma: Ustilago maydis ,Clapu: Claviceps purpurea, Penca: Penicillium canescens,Pensi: Penicillium simplicissimum, Talpu: Talaromyces purpureogenus, Aspac: Aspergillus aculeatus, Aspni:Aspergillus niger, Aspka: Aspergillus kawachii , Rhior:Rhizopus oryzae, Pench: Penicillium chrysogenum, Theau:Thermoascus aurantiacus, Aspte: Aspergillus terreus,Aspor: Aspergillus oryzae , Neofu: Neosartorya fumigata ,Neofi: Neosartorya fischeri, Aspcl: Aspergillus clavatus, Aspfl: Aspergillus flavus , Emeni: Emericella nidulans , Aspor: Aspergillus oryzae , Aurpu:Aureobasidium pullulans, Nagal: Naganishia albida. Fig. 14 Fig. 14 GH10 exoglucanase 3D model. 3D modeling of GH10 exoglucanase 4623 from Scenedesmus obliquus UTEX B 3031.A. Proposed model of Sceobl1|4623 (pink) superimposed to Celfi|P07986 GH10 template (cyan); putative catalytic residues are shown. B. Inset of catalytic residues area. Abbreviations: Celfi: Cellulomonas fimi Supporting information: Supplementary Figure 1: In a separate file Data Availability Statement Not applicable Conflict of interests The authors declare no potential financial or other interests that could be perceived to influence the outcomes of the research. No conflicts, informed consent, human or animal rights applicable. All authors declare agreement to authorship and submission of the manuscript for peer review. Acknowledgements This work was supported by Grants from Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET, PIP2015-0476), Agencia Nacional de Promoción Cientı́fica y Tecnológica (ANPCyT, PICT2016-0350), and Agencia Santafecina de Ciencia Tecnologı́a e Innovación (ASaCTeI IO2018-00098). JB, MVB and DGC are research members from CONICET. CND is assistant professor from Amity University Uttar Pradesh, Lucknow, India. MBV is a doctoral fellow from CONICET. Authorship. JB, MBV, DGC, MVB and CND designed the conception and delineation of the study; and prepared the manuscript and reviewed it before submission. MBV performed the in silico characterization. JB and MBV performed the acquisition of the data or analyzed such information. All authors read and approved the final manuscript. 19