bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Two pectate lyases from Caldicellulosiruptor bescii with the same CALG domain had 2 distinct properties on plant biomass degradation 3 Hamed I. Hamoudaa,b,c, Nasir Alia, Hang Sua,b, Jie Fenga, Ming Lua,†and Fu-Li Li a,† 4 a Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuel, 5 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 6 Qingdao 266101, China 7 b University of Chinese Academy of Sciences, Beijing 100039, China. 8 c Egyptian Petroleum Research Institute, Nasr City 11727, Cairo, Egypt. 9 †Corresponding authors: Dr. Ming Lu (E-mail: [email protected]) and Dr. Fu-Li Li 10 (E-mail: [email protected]), Qingdao Institute of Bioenergy and Bioprocess Technology, 11 Chinese Academy of Sciences, Qingdao 266101, China 12 13 Keywords: Caldicellulosiruptor, Pectin, Pectate lyase, Polysaccharide lyase, Concanavalin 14 A-like lectin/glucanase (CALG) 15 16 17 18 19 20 21 22 23 24 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Abstract 27 Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using 28 selected microorganisms that carry pectinolytic enzymes. Pectate lyases that cleave 29 α-1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making 30 and fruit softening. However, reports on pectate lyases with high thermostability are few. Two 31 pectate lyases (CbPL3 and CbPL9) from a thermophilic bacterium Caldicellulosiruptor bescii 32 were investigated. Although these two enzymes belonged to different families of 33 polysaccharide lyase, both were Ca2+-dependent. Similar biochemical properties were shown 34 under optimized conditions 80 °C ‒85 °C and pH 8‒9. However, the degradation products on 35 pectin and polygalacturonic acids (pGA) were different, revealing the distinct mode of action. 36 A concanavalin A-like lectin/glucanase (CALG) domain, located in the N-terminus of two 37 CbPLs, shares 100% amino acid identity. CALG-truncated mutant of CbPL9 showed lower 38 activities than the wild-type, whereas the CbPL3 with CALG knock-out portion was reported 39 with enhanced activities, thereby revealing the different roles of CALG in two CbPLs. 40 I-TASSER predicted that the CALG in two CbPLs is structurally close to the family 66 41 carbohydrate binding module (CBM66). Furthermore, substrate-binding assay indicated that 42 the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but 43 CALG showed weak interaction with a number of lignocellulosic carbohydrates, except 44 sodium carboxymethyl cellulose and sodium alginate. Finally, scanning electron microscope 45 analysis and total reducing sugar assay showed that the two enzymes could improve the 46 saccharification of switchgrass. The two CbPLs are impressive sources for degradation of 47 plant biomass. 48 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 Importance 50 Thermophilic proteins could be implemented in diverse industrial applications. We sought to 51 characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium 52 Caldicellulosiruptor bescii. The two enzymes had high optimum temperature, low optimum 53 pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding 54 module (CBM66) was identified in two CbPLs with sharing 100% amino acid identity. 55 Deletion of CBM66 obviously decreased the activity of CbPL9, but increase the activity and 56 thermostability of CbPL3, suggesting the different roles of CBM66 in two enzymes. 57 Moreover, the degradation products by two CbPLs were different. These results revealed 58 these enzymes could represent a potential pectate lyase for applications in paper and textile 59 industries. 60 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 1. Introduction 62 Pectin is an intercellular cement that gives plants structural rigidity; it was discovered at 63 concentrations of 15% to 30% in fruit fibers, vegetables, legumes, and nuts (1). In terms of 64 chemistry, it is an intricate polysaccharide with a molecular weight in the range 20–400 kDa 65 (2). The simplest structure of pectin is a linear polymer of galacturonic acids with 66 α-1,4-galacturonosidic linkages, which is named as homogalacturonan (HG). More 67 complicated forms of pectin are linked by D-galacturonate and L-rhamnose residues, which 68 are designated as types I and II rhamnogalacturonan (RGI and RGII, respectively). The side 69 chains of these RGI and RGII are mostly modified by neutral sugars, such as D-galactose, 70 L-arabinose, D-xylose, and L-fucose (3). 71 Pectin depolymerization is an initiating step for the bioconversion of plant biomass. 72 Therefore, pectate lyases, a group of enzymes that break down pectin in nature, are used as an 73 additive in generating commercial cellulase cocktails and consolidated-bioprocessing (CBP) 74 microorganisms (4). The mechanism of action of pectate lyase is to cleave the 75 α-1,4-galacturonosidic linkage of polygalacturonic acid (pGA) by β-elimination reaction, 76 thereby forming a double bond between positions C4 and C5 in oligo-galacturonates (1, 4). 77 Through the carbohydrate-active enzyme (CAZy, www.cazy.org) database (5), pectate lyases 78 are classified into families-1, -2, -3, -9, and -10 of polysaccharide lyases. Most of these lyases 79 are identified from microorganisms, such as those from the genera of Bacillus, Clostridium, 80 Erwinia, Fusarium, Pseudomonas, Streptomyces, and Thermoanaerobacter, which are mostly 81 alkaline (pH 8–11) and Ca2+-dependent (4). 82 Pectate lyases are widely applied in industries, such as paper making, fruit softening, juice 83 and wine clarification, plant fiber processing, and oil extraction (6). Biotechnological usage is 84 highly dependent on the biochemical properties of these enzymes, such as temperature, pH, 85 and salt concentration (1). Commercial pectate lyases were mostly isolated from the 86 mesophilic bacterium Bacillus, including B. subtilis, B. licheniformes, B. cereus, B. circulens, 87 B. pasteurii, B. amyloliquefaciens, and B. pumulis (1, 4). Among them, the recombinant 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 88 BacPelA from B. clausii had high cleavage activity on methylated pectin. Its maximum 89 activities are observed at pH 10.5 and 70 °C and showed the highest degumming efficiency 90 reported to date (7). A pectate lyase pelS6 from B. amyloliquefaciens S6 strain displayed 91 highly thermostability and survival in a wide range of pH, which could be suitable for juice 92 clarity (8). To identify thermostable and highly active enzymes, the number of studies 93 investigating thermophilic microorganisms, such as those from the genera of Thermotoga and 94 Caldicellulosiruptor, has been growing recently (9-14). 95 A previous study indicated that a pectin-deconstruction gene cluster is vital for the growth 96 of thermophilic C. bescii on plant biomass (15). Fig. 1A shows three adjacent genes, 97 Cbes_1853, Cbes_1854, and Cbes_1855 that encode a rhammogalacturonan lyase CbPL11, a 98 pectate lyase CbPL3, and a pectate disaccharide lyase CbPL9, respectively (16). Alahuhta et 99 al. presented a crystal structure of the catalytic domain of CbPL3 and described CbPL3 as a 100 unique pectate lyase with a low optimum pH (11, 17). However, the detailed properties of 101 these pectate lyases remained unclear. Meanwhile, CbPL3 and CbPL9 share the same 102 N-terminal domain, which is predicted as a concanavalin A-like lectin/glucanase (CALG) 103 domain with 100% protein sequence similarity. The function of this CALG is still unknown. 104 In this study, the pectate lyases CbPL3 and CbPL9 and their truncated mutants were 105 heterologously expressed in E. coli. The properties of the different domains in the two 106 enzymes were biochemically and functionally analyzed. 107 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 108 2. Materials and methods 109 2.1. Chemicals and Strains 110 Mono-galacturonic acid, di-galacturonic acid, tri-galacturonic acid, polygalacturonic acid, 111 citrus pectin, apple pectin, rhamnogalacturonic acid, CMC-Na, Avicel, beechwood xylan, 112 alginate, and cellobiose were purchased from Solarbio Science & Technology (Beijing, 113 China). The vector pEAZY blunt-E2, competent cells E. coli BL21 (DE3) pLysS, and Plasmid 114 mini Prep Kit were purchased from TransGen Biotech (Beijing, China). Isopropyl 115 β-D-1-thiogalactopyranoside (IPTG) and ampicillin were purchased from Sigma-Aldrich (St. 116 Louis, MO, USA). 117 2.2. Gene cloning, protein expression, and purification 118 The genomic DNA was isolated from C. bescii (18). Two pectate lyases CbPL3 and CbPL9 119 were submitted to the NCBI database with GenBank Accession Nos. ACM60942 and 120 ACM60943, respectively (19).