1 2 3 4 5 Journal name: Applied Microbiology and Biotechnology Manuscript Title: Identification of a thermostable fungal lytic polysaccharide monooxygenase and evaluation of its effect on lignocellulosic degradation The names of the authors: Ruiqin Zhang a, Yucui Liu a, Yi Zhang b, Dan Feng a, Shaoli Hou c, Wei Guo a, Kangle Niu a, Yi Jiang a, Lijuan Han a, Lara sindhu a, Xu Fang a,* 6 7 8 9 10 11 12 13 14 15 The affiliations and addresses of the authors: a State Key Laboratory of Microbial Technology, Shandong University No. 72 Binhai Road, Qingdao 266237, China b Department of Plastic and Reconstructive Surgery, Jinan Central Hospital Affiliated to Shandong University No. 105 Jiefang Road, Jinan 250013, China c Shandong Henglu Biotech. Co., Ltd. Room 101, Building 1, New technology demonstration garden, 1277 Xinyuan Avenue, Ji'nan 250000, China 16 17 18 19 20 21 *Correspondence author s Address: State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao 266237, China E-mail address: fangxu@sdu.edu.cn Tel: +86-532-58631507; Fax: +86-532-58631507 Electronic Supplementary Information (ESI) available: [Figure S1, Table S1-S3]. 22 23 24 25 26 27 28
29 30 31 Fig. S1 SDS-PAGE of purified TaAA9A E. coli and TcAA9A E. coli. All bands were identified using MALDI-TOF MS. Molecular mass marker in kda 32 33
34 Table S1 Plasmids and strains used in this study Plasmids Properties Source/Referen ce pet21a Escherichia coli protein expression vector Kim et al. 2015 TaAA9A pet-21a (+)-T7p-TaAA9A-T7t TcAA9A pet-21a (+)-T7p-TcAA9A-T7t pug6ptra E. coli cloning vector Jiang et al. 2016 pug6ptra-pxyn1-taaa9a-t trpc pug6ptra-pxyn1-tcaa9a-t trpc pug6-ptef1-pxyn1-taaa9a-ttrpc-pptra-ptra-tptr A-Ttef1 pug6-ptef1-pxyn1-tcaa9a-ttrpc-pptra-ptra-tptr A-Ttef1 Strains Description Source/Referen ce E. coli TaAA9A An AA9 LPMO from Thermoascus aurantiacus (GenBank Accession No. MK359139) was heterologously expressed in E. coli BL21. E. coli TcAA9A An AA9 LPMO from Talaromyces cellulolyticus (GenBank Accession No. MK359134) was heterologously expressed in E. coli BL21. Trichoderma reesei T1 (Control) T. reesei T1 (CCTCC M2015804) was used as the parent strain for the AA9 LPMOs heterologous expression experiment. The strain was derived from QM6a (ATCC 13631) by random mutagenesis. Compared with that of the original strain QM6a, the cellulase production by T1 was significantly improved. Therefore, T1 has been widely used in the production of cellulases. Tang et al. 2013; Wang et al. 2016; Zhang et al. 2009 T. reesei TX A β-glucosidase from Aspergillus niger was heterologously expressed in T. reesei T1. Wang et al. 2015b Tr TaAA9A An AA9 LPMO from T. aurantiacus (GenBank Accession No. MK359138) was heterologously expressed in T. reesei T1. Tr TcAA9A An AA9 LPMO from T. cellulolyticus (GenBank Accession No. MK359137) was heterologously expressed in T. reesei T1.
35 Table S2 Primers used in this study Name of Nucleotide sequence (5-3 ) primer TaAA9A-F TaAA9A-R TcAA9A-F TcAA9A-R TaAA9A -F TaAA9A -R TcAA9A-F CTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAATGGTGATGAT GATGATGAACCGGTATACAGCGGCGGACCCGGA AAGAAGGAGATATACATATGGCTAGCATGACTGGTGGACAG CAAATGGGTCGCGGATCCGAATTCCATGGTTTTG CTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAATGGTGATGAT GATGATGACAGCACGGTGGTGGTAATCACGGTG AAGAAGGAGATATACATATGGCTAGCATGACTGGTGGACAG CAAATGGGTCGCGGATCCGAATTCATGCCGAGCA CAAGGAAAACACGCACAAATAATCATCATGAGCTTCAGCAA GATCATCGCCACCGCC TGGATCGATCCGGTCGGCATCTACTTTAGCCGGTGTAGAGAG GAGGGCCAGGAAT GCAAGCTCAACTGCATAGTATCGACTTCAAGGAAAACACGC ACAAATAATCATCATGCCTTCTACTAAAGTCGCTGCCC TcAA9A-R GCTGTTTGATGATTTCAGTAACGTTAAGTGGATCGATCCGG TCGGCATCTACTTTAAAGGACAGTAGTGGTGATGACGG Pxyn1-F TGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGA TTCGTTACCCACAGCATATTTCGTTGGCTGGCA Pxyn1-R TtrpC-F TtrpC-R GATGATTATTTGTGCGTGTTTTCC AGTAGATGCCGACCGGATCGATCC ATGGGATCCCGTAATCAATTGCCCAGTTGGAACCTCTTACG TGCCGATCACGGACGGTCTTTTCCTCTTTTTTTC 36 37 38 39 40
41 Table S3 (a) Identification of affinity chromatography-purified AA9 LPMOs in E. coli AA9 Protein/module Protein NCBI MW a pi a Score b Sequence family Reference (kda) coverage b, % ID TaAA9A AA9 AGO68294.1 24.6 4.71 278.97 28.95 TcAA9A AA9 GAM42970.1 29.4 4.62 364.92 37.59 42 (b) Identification of SDS-PAGE bands of secreted proteins in Tr AA9 culture grown for 8 days Protein/module Protein NCBI MWa pi a Score b Sequence family Reference (kda) coverage b,% ID 43 44 TaAA9A AA9 AGO68294.1 26.5 5.45 12.45 8.43 TcAA9A AA9 GAM42970.1 31.3 4.96 6.65 8.71 a Hypothetical molecular weight and pi of the proteins b The score and sequence coverage (%) are based on the genome sequence information 45 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 File S1 Amino acid sequences of AA9s in this study > AGO68294.1 (TaAA9A) MSFSKIIATAGVLASASLVAGHGFVQNIVIDGKKYVIARRNQYPYMSNPPEVIAWSTTATDLGFVD GTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQL EFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYP QCINLQVTGGGSDNPAGTLGTALYHDTDPGTLINIYQKLSSYIIPGPPLYTG >GAM42970.1 (TcAA9A) MPSTKVAALSAVLALASTVAGHGFVQNIVIDGKSYSGYLVNQFPYESNPPAVIGWATTATDLGFVA PSEYTNADIICHKNATPGALSAPVAAGGTVELQWTTWPDSHHGPVISYLANCNGNCSTVDKTKL NFVKIDQGGLIDDTTPPGTWASDKLIAANNSWTVTIPSTIAPGNYVLRHEIIALHSAGNADGAQNY PQCINLEITGSGTAAPSGTAGEKLYTSTDPGILVNIYQSFGAANGAVATGSATAVATTAAASATATPT TLVTSVAPASSTSATAVVTTVAPAVTDVVTVTDVVTVTTVITTTVL The codon-optimized AA9 genes for expression in T. reesei in this study >MK359138 (TaAA9A) ATGAGCTTCAGCAAGATCATCGCCACCGCCGGCGTCCTCGCCAGCGCCAGCCTCGTCGCCGG CCACGGCTTCGTCCAGAACATCGTCATCGACGGCAAGAAGTACGTCATTGCCCGACGAAACC AGTACCCCTACATGAGCAACCCCCCTGAGGTCATTGCCTGGTCTACCACCGCCACCGACCTCG GCTTTGTCGACGGCACCGGCTACCAGACCCCTGACATTATTTGCCACCGAGGCGCCAAGCCTG GCGCCCTCACCGCCCCTGTCTCTCCTGGCGGCACCGTCGAGCTCCAGTGGACCCCTTGGCCT GACTCTCACCACGGCCCTGTCATTAACTACCTCGCCCCTTGCAACGGCGACTGCTCTACCGTC GACAAGACCCAGCTCGAGTTTTTTAAGATTGCCGAGTCTGGCCTCATTAACGACGACAACCCT CCTGGCATTTGGGCCTCTGACAACCTCATTGCCGCCAACAACTCTTGGACCGTCACCATTCCT ACCACCATTGCCCCTGGCAACTACGTCCTCCGACACGAGATTATTGCCCTCCACTCTGCCCAG AACCAGGACGGCGCCCAGAACTACCCTCAGTGCATTAACCTCCAGGTCACCGGCGGCGGCTC TGACAACCCTGCCGGCACCCTCGGCACCGCCCTCTACCACGACACCGACCCTGGCACCCTCA TTAACATTTACCAGAAGCTCTCTTCTTACATTATTCCTGGCCCTCCTCTCTACACCGGCTAA >MK359137 (TcAA9A) ATGCCTTCTACTAAAGTCGCTGCCCTTTCTGCTGTTCTAGCTTTGGCCTCCACGGTTGCTGGC CATGGTTTTGTGCAAAACATCGTTATCGACGGTAAATCTTACTCTGGATACCTTGTGAATCAG TTCCCCTACGAGTCCAACCCACCAGCTGTTATTGGGTGGGCAACAACTGCAACCGACCTGGG ATTCGTCGCTCCCAGTGAGTACACCAATGCAGACATTATCTGCCACAAGAACGCCACACCTG GCGCGCTTTCTGCTCCAGTTGCTGCAGGGGGCACTGTCGAGCTCCAGTGGACTACATGGCCC GATAGTCATCACGGTCCTGTCATCAGCTACCTCGCCAACTGCAATGGCAATTGTTCTACCGT GGATAAGACTAAGCTAAACTTTGTCAAGATTGACCAAGGTGGTTTGATCGACGATACTACCC CCCCGGGTACATGGGCTTCCGACAAACTTATCGCTGCCAACAACAGCTGGACTGTAACTATC CCCTCCACCATCGCGCCTGGAAACTACGTTTTGCGCCACGAAATCATTGCTCTTCATTCCGCT GGAAACGCAGACGGTGCCCAAAACTACCCTCAATGCATCAACTTGGAGATCACCGGCAGCG
86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 GAACCGCCGCTCCCTCTGGTACCGCTGGCGAAAAGCTCTACACCTCTACTGACCCCGGTATC TTGGTCAATATCTACCAATCCTTCGGTGCTGCCAATGGCGCTGTTGCCACTGGTTCTGCTACT GCGGTTGCTACGACTGCCGCTGCTTCTGCGACCGCTACTCCTACCACACTTGTTACCTCTGTC GCTCCAGCTTCATCTACCTCTGCCACTGCTGTTGTGACCACTGTCGCTCCTGCAGTAACTGAT GTCGTGACTGTCACCGATGTAGTTACCGTGACCACCGTCATCACCACTACTGTCCTTTAA The codon-optimized AA9 genes for expression in E. coli in this study >MK359139 (TaAA9A) ATGCATGGTTTTGTGCAGAATATTGTGATTGATGGCAAAAAATACGTGATTGCACGTCGTAATC AGTATCCGTATATGAGTAATCCGCCGGAAGTTATTGCATGGAGTACCACCGCAACCGATCTGGG TTTTGTGGATGGCACCGGTTATCAGACCCCGGATATTATTTGTCATCGTGGTGCAAAACCGGGC GCCCTGACCGCCCCTGTGAGTCCTGGTGGTACCGTGGAACTGCAGTGGACCCCGTGGCCGGA TAGTCATCATGGTCCGGTTATTAATTATCTGGCCCCGTGCAATGGTGACTGTAGCACCGTTGAT AAAACCCAGCTGGAATTTTTCAAAATTGCAGAAAGTGGTCTGATTAATGATGATAATCCGCCG GGCATTTGGGCCAGTGATAATCTGATTGCAGCAAATAATAGCTGGACCGTGACCATTCCGACC ACCATTGCCCCGGGCAATTATGTGCTGCGCCATGAAATTATTGCACTGCATAGCGCCCAGAATC AGGATGGTGCACAGAATTATCCGCAGTGTATTAATCTGCAGGTGACCGGCGGTGGTAGCGATA ATCCGGCAGGTACCCTGGGTACCGCACTGTATCATGATACCGATCCGGGCACCCTGATTAATAT CTATCAGAAACTGAGCAGCTATATTATTCCGGGTCCGCCGCTGTATACCGGTCATCATCATCATC ACCATTAA >MK359134 (TcAA9A) ATGCATGGTTTTGTGCAGAACATTGTGATTGACGGCAAAAGCTATAGCGGCTATCTGGTGAAC CAGTTTCCGTATGAAAGCAACCCGCCGGCGGTTATTGGTTGGGCAACCACCGCGACCGATTTA GGCTTTGTTGCGCCGAGCGAATATACCAACGCGGACATCATTTGCCATAAAAACGCGACCCCG GGTGCATTATCAGCACCTGTTGCGGCAGGTGGTACCGTTGAATTACAGTGGACCACCTGGCCG GATAGCCATCATGGCCCGGTGATTAGCTATCTGGCGAACTGCAACGGCAATTGCAGCACCGTG GATAAAACCAAACTGAACTTCGTGAAAATTGATCAGGGCGGCCTGATTGATGATACCACCCCT CCTGGTACCTGGGCGAGCGATAAACTGATTGCGGCGAACAACAGCTGGACCGTGACCATTCC GTCAACGATTGCGCCGGGCAATTATGTGCTGCGCCATGAAATTATTGCGCTGCATAGCGCGGG CAACGCAGATGGTGCGCAGAATTATCCTCAATGCATCAACCTGGAAATTACCGGCTCAGGTAC CGCAGCACCTAGCGGTACCGCGGGTGAAAAACTGTATACCAGCACCGATCCGGGCATTCTGG TGAACATTTATCAGAGCTTTGGCGCAGCAAATGGCGCGGTTGCGACCGGTTCAGCAACTGCA GTTGCAACTACCGCAGCAGCAAGCGCAACTGCAACCCCTACCACCTTAGTTACCAGCGTTGC GCCTGCATCATCAACCAGCGCGACTGCGGTTGTTACCACTGTTGCGCCTGCGGTTACCGATGT TGTGACCGTGACCGATGTTGTGACCGTGACCACCGTGATTACCACCACCGTGCTGCATCATCA TCATCATCACTGA