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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/molecularbiosystems Page 1 of 30 Molecular BioSystems 1 Depiction of Carbohydrate-Active Enzymes Diversity in Caldicellulosiruptor sp. F32 at 2 Genome Level Reveals Insights into Distinct Polysaccharides Degradation Feature 3 4 Dong-Dong Meng1, Yu Ying1,2, Kun-Di Zhang1, Ming Lu1, and Fu-Li Li1* Manuscript 5 6 1Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao 7 Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 8 266101, China 2 9 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Accepted 10 266071, China 11 12 *Corresponding author: Fu-Li Li, Qingdao Institute of Bioenergy and Bioprocess Technology, 13 Chinese Academy of Sciences, Qingdao, 266101, China. Tel.: 86-532-80662655. Fax: 14 86-532-80662778. E-mail: [email protected] 15 BioSystems 1 Molecular Molecular BioSystems Page 2 of 30 16 Abstract 17 Thermophilic bacterium Caldicellulosiruptor sp. F32 can utilize cellulose-, 18 hemicellulose-containing biomass, including unpretreated wheat straw. We have conducted a 19 bioinformatics analysis of the carbohydrate-active enzyme (CAZyme) in the genome of 20 Caldicellulosiruptor sp. F32, which reveals a broad substrate range of the strain. Among 2285 Manuscript 21 predicted open reading frames (ORFs), 73 (3.2%) CAZyme encoding genes, including 44 22 glycoside hydrolases (GHs) distributing in 22 GH families, 6 carbohydrate esterases (CEs), 3 23 polysaccharide lyases (PLs), 21 glycosyl transferases (GTs), and 25 carbohydrate-binding 24 modules (CBMs) were found. An in-depth bioinformatics analysis of CAZyme families that 25 target cellulose, hemicellulose, chitin, pectin, starch, and β-1,3-1,4-glucan degradation were Accepted 26 performed to highlight specialized polysaccharides degrading abilities of strain F32. A great 27 number of orthologous multimodular CAZymes of Caldicellulosiruptor sp. F32 were found in 28 other strains of genus Caldicellulosiruptor. While, a portion of the CAZymes of 29 Caldicellulosiruptor sp. F32 showed sequence identity with proteins from strains of genus 30 Clostridium. A thermostable β-glucosidase BlgA synergistically facilitated enzymatic 31 degradation of Avicel by endo-1,4-β-glucanase CelB, which indicated that synchronous action of BioSystems 32 synergism between CAZymes enhanced the lignocellulose degradation of Caldicellulosiruptor sp. 33 F32. 34 35 Keywords: CAZymes; Glycoside hydrolase; Lignocellulose; Synergism; Caldicellulosiruptor 36 2 Molecular Page 3 of 30 Molecular BioSystems 37 Introduction 38 Thermophilic bacteria of genus Caldicellulosiruptor (Topt 65–78 °C) are robust and efficient 39 carbohydrate degrading microorganisms, which secrete a series of carbohydrate active enzymes 40 (CAZymes) to deconstruct lignocellulosic biomass1. Gram-positive Caldicellulosiruptor spp. 41 have been isolated from a wide variety of ecological niches. Most of the species can metabolize Manuscript 42 an extensive monosaccharides, oligosaccharides, and polysaccharides, including crystalline 43 cellulose, xylan, and switchgrass2. Furthermore, since no carbon catabolite repression (CCR) 44 system exists in C. saccharolyticus, this strain has been shown to be able to ferment C5 and C6 45 sugars simultaneously, which was a highly desirable feature for lignocellulose conversion3, 4. All 46 isolates are given a diverse set of cell wall degrading enzymes (CWDEs) as an excellent Accepted 47 CAZymes reservoir for carbohydrate degradation, which are potential consolidated 48 bioprocessing (CBP) platforms for biomass-to-biofuel conversion. Cellulolytic enzymes systems 49 can be divided into two main categories, namely cellulosomal and noncellulosomal. Genus 50 Caldicellulosiruptor adopts an intermediate strategy, secreting many free multi-modular 51 cellulases containing multiple binding and catalytic domains5. 52 Blumer-Schuette et al. have examined the CAZymes of eight numbers of this genus. Each BioSystems 53 strain contains a significant number of glycoside hydrolases (GHs, 37~77), carbohydrate binding 54 modules (CBMs, 15~28), polysaccharide lyases (PLs, 1~4), carbohydrate esterases (CEs, 4~9), 55 and glycosyl transferases (GTs, 27~35)2, which comprise 1%~3% of the genome. Comparison 56 analysis within the genus from genome level indicates 106 GHs distributing in 43 GH families 57 constitute the Caldicellulosiruptor pangenome, while 26 GHs from 17 families are conserved in 3 Molecular Molecular BioSystems Page 4 of 30 58 the core genome2. Though more than half of the lignocellulose degradation related proteins are 59 shared between all Caldicellulosiruptor species, genomic and proteomic studies in one individual 60 species are needed to distinguish the difference of carbohydrate utilization within the genus6. As 61 an efficient lignocellulolytic bacterium toward unpretreated plant biomass, analysis of CAZyme 62 families and its architecture composition at genome level is helpful to elucidate the ability of Manuscript 63 Caldicellulosiruptor species for deconstruction of plant polysaccharides. 64 Caldicellulosiruptor sp. F32 is an extremely thermophilic anaerobic bacterium, which was 65 isolated from biocompost with optimal growth temperature at 75 °C7. Compared with the type 66 stain C. saccharolyticus DSM 8903, Caldicellulosiruptor sp. F32 grew better on unpretreated 67 wheat straw, exhibited higher endoglucanase and xylanase activities in the secretome8. Three Accepted 68 CAZy enzymes, GH11 xylanase XynA (GenBank accession number JX030400), GH10 xylanase 69 XynB (GenBank accession number JX030401)9, and GH5 lichenase F32EG5 (GenBank 70 accession number KC958563, unpublished) have been characterized in detail. Further elucidation 71 of the abundant CAZymes is thus essential to understand the complexity process of the 72 lignocellulose deconstruction. This report focused on the CAZymes identification of strain F32 73 and provided a wealth of information to generate a comprehensive analysis of CWDEs in the BioSystems 74 host. Sequence and module annotation have been applied to predict functions of each CAZyme 75 proteins. Moreover, synergism between two cellulose-degrading enzymes was investigated. 76 Briefly, this will provide a better understanding of the plant-biomass degradation mechanisms of 77 the genus Caldicellulosiruptor. 78 4 Molecular Page 5 of 30 Molecular BioSystems 79 Materials and method 80 Genomic DNA isolation and genome sequencing 81 Caldicellulosiruptor sp. F32 (CGMCC 1.5183, China General Microbiological Collection 82 Center, Beijing, China) was cultured at 75 °C in an anaerobic jar containing modified GS-2 83 medium7. Genomic DNA was isolated10 from Caldicellulosiruptor sp. F32 and was sequenced by Manuscript 84 a standard shotgun strategy using GS FLX-454 pyrosequencing technology (Roche, Basel, 85 Switzerland). The numerous reads were assembled by using the 454 Newbler assembler software 86 (Roche) into hundreds of contigs, which were re-sorted subsequently to predict gene functions 87 using Glimmer11, GeneMark12, and Zcurve13. The genes were annotated through the National 88 Center for Biotechnology Information (NCBI) nr database14 and KEGG database15. Accepted 89 CAZymes annotation 90 All protein-encoding open reading frames (ORFs) from the Caldicellulosiruptor sp. F32 91 genome generated through nr and KEGG databases annotation, were further refined and screened 92 for carbohydrate-active modules using Carbohydrate-active enzyme ANnotatio (dbCAN, 93 http://csbl.bmb.uga.edu/dbCAN/index.php)16. To eliminate proteins identified by dbCAN but 94 those were not really CAZymes, all proteins were individually examined using NCBI’s BioSystems 95 Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)17 96 and Pfam (http://pfam.sanger.ac.uk/)18. These data sources were combined to assert the function 97 of each predicted proteins. When a gene appeared to be truncated and the missing sequence data 98 couldn’t be found within all the supercontig, the orthologous coding region in other 99 Caldicellulosiruptor isolates were used to modify the gene sequence from Caldicellulosiruptor 5 Molecular Molecular BioSystems Page 6 of 30 100 sp. F32 genome. Further annotation of selected proteins included protein similarities and 101 identities determination19, molecular size and isoelectric point prediction20, and signal peptide 102 prediction (SignalP v4.1)21. 103 Cloning, expression, and purification of Caldicellulosiruptor