Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing

Xing Yana‡ · Alei Genga‡ · Jun Zhanga · Yongjun Weia · Lei Zhanga · Changli Qiana ·Qianfu Wanga ·Shengyue Wangb · Zhihua Zhoua*

aKey Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China,

bChinese NationalHumanGenomeCenter at Shanghai, Shanghai, 201203, China

‡These authors contributed equally to this work.

* Corresponding author. Mailing address: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences, Fenglin Rd 300, Shanghai 200032, China. Phone: (86) 21-54924050. Fax: (86) 21-54924049. E-mail:

Table S1 Primers, vectors and strains used in this study

Names / Characteristics / Experimental purposes / Sources
Primers (from 5' to 3')
en1f / TATCCATGGTGAAGAACATAATGGAC / Cloning of gene en1into pET 22b / This study
en1r / TATCTCGAGTTTCTCGCTTACAATCCT
xy2f / GGGGCATATGTTGTTGAAGAGAACATTT / Cloning of gene xyl2 into pET 22b / This study
xy2r / GGGGGTCGACAAATTATTGGCCTAGATA
en3f / GAAGCTGGAAAAACTGGAAC / Cloning of gene en3into pET 22r / This study
en3r / TATCTCGAGTTTACCAGCTGAAGCTG
en4f / TATGATATCATGATGAAGCTTAACAGAG / Cloning of gene en4 into pET 22r / This study
en4r / TATCTCGAGTTTTCCTGTCCATAAGG
en5f / ATACCATGGTGCAAACTTATGTAGAAG / Cloning of gene en5 into pET 22b / This study
en5r / TATCTCGAGTTACTTTTTCACAGTTCT
en6f / TATCCATGGTGAGACTTAATAGAGGC / Cloning of gene en6 into pET 22b / This study
en6r / TATCTCGAGTTTAATCCAGAGTGAGAG
bgl1f / TATGATATCATGGTTGAGCGGGTAAC / Cloning of bgl1 into pET 22r / This study
bgl1r / TATGTCGACTAACCTGAGGTCAAGCC
bgl2f / TATGATATCATGATGAATACTGTGGC / Cloning of bgl2 into pET 22r / This study
bgl2r / TATGTCGACGTTATTGATTACGCC
xyl1f / GGGGCATATGCCAGTTAAGTTTTAAGAAA / Cloning of xyl1into pET 22b / This study
xyl1r / GACGGTCGACGGGTACTGAATTATACAT
T7f / TAATACGACTCACTATAGGG / PCR verification / T7 promoter
T7r / TGCTAGTTATTGCTCAGCGG
Vectors
pET 22b / Ampr / Gene expression vector / Novagen
pET 22r / Kanar / Gene expression vector / Geng et al.(2012)
Strains
E.coli
BL21 / DE3 / Gene expression host / Novagen

Note: Restriction sites are underlined; Nonunderlined pimers indicated the corresponding genes wereligated to the EcoRV site of the vector.

Table S2Summary of 454 sequencing of pools of fosmids

BatchNoa. / Total No. of fosmids / Total No. of bases (kb) / Total assembled bases (kb) / Percentage of assembled reads (%) / Coverage / Status / Glycosyl hydrolase genes obtainedd
1 / 4 / 2,293 / 149 / n.d. / 14.3 / All finished / 5
2 / 10 / 14,356 / 377 / 95.11 / 35.9 / 8 finished / 17
3 / 16b / 27,641 / 258 / 98.45 / 43 / 1 finishedc / 5

aIn batch 4, a fosmid clone (T02C7) was sequenced alone using the sanger’s method; and In batch 5, the sequence of xyl2 was obtained usingtransposon insertion inactivation from a fosmid clone (XYL01C8). Thus, they were not presented in this table.

b One methanogenesis related fosmid clone and one hydrogenesis related fosmid clone were included.

c Only one methanogenesis related fosmid clone was finished.

dAnother 2 glycosyl hydrolase genes (bgl3 and xyl2) obtained from batch 4 and 5 was not included in this table.

n.d., not determined.

Table S3Fully assembled clones and their GH genes

Batch / Clone ID / Clone activities / GH genes / Putative activitiesa / Real activitiesb
1 / EXO02C10 / Exo-β-1,4-glucanase / en5 / Endo-β-1,4-glucanase / Endo-β-1,3-glucanase
1 / CMC03A5 / Endo-β-1,4-glucanase / en1 / Endo-β-1,4-glucanase / Endo-β-1,4-glucanase
1 / BGL04B5 / β-Glucosidase / bgl4 / β-Glucosidase / n.d.
1 / XYL07A11 / Endo-β-1,4-xylanase / xyl1 / Endo-β-1,4-xylanase / Endo-β-1,4-xylanase
2 / CMC3B6 / Endo-β-1,4-glucanase / en2 / Endo-β-1,4-glucanase / Unknown
enx3 / Endo-β-1,4-glucanase / Endo-β-1,4-glucanase
xyl3 / Exooligoxylanase
Endo-β-1,4-glucanase / n.d.
man1 / β-1,4-Mannanase / n.d.
2 / CMC6A4 / Endo-β-1,4-glucanase / en3 / Endo-β-1,4-glucanase / Endo-β-1,4-glucanase
2 / BGL02B4 / β-Glucosidase / bgl1 / β-Glucosidase / β-Glucosidase
2 / BGL4A6 / β-Glucosidase / bgl2 / β-Glucosidase / β-Glucosidase
2 / XYL1G3 / Endo-β-1,4-xylanase / xyl1 / Endo-β-1,4-xylanase / Endo-β-1,4-xylanase
2 / XYL3H9 / Endo-β-1,4-xylanase / xyl4 / Endo-β-1,4-xylanase / n.d.
bxy1 / β-1,4- xylosidase
α-N-arabinofuranosidase / n.d.
2 / XYL4C3 / Endo-β-1,4-xylanase / xyl5 / Endo-β-1,4-xylanase / n.d.
xyl6 / Endo-β-1,4-xylanase / n.d.
2 / XYL6G6 / Endo-β-1,4-xylanase / bgl2 / β-Glucosidase / β-Glucosidase
4c / T02C7 / β-Glucosidase / bgl3 / β-Glucosidase / n.d.

aPutative activities of the GHs were inferred by the activities of their nearest neighbors

b Real activities of the GHs were assayedbased on substrates that they could hydrolyze , see Table 3

cIn batch 4, the fosmid T02C7was sequenced alone using the sanger’s method

n.d., not determined

Fig. S1 Function-based screening for cellulase or hemicellulase activities. Hydrolysis halos on Congo Red screening plates indicated(A) endo-β-1,4-glucanase and (B) endo-β-1,4-xylanase activities. (C) A black halo indicated β-glucosidase activity

Fig.S2 Protein purification of the endo-β-1,4-glucanase,En1. (A) His-tag affinity chromatography of En1. Lane 1, 300 mM imidazole washout; lane 2, 20 mM imidazole washout; lane 3, elution buffer; lane 4, crude protein extract of the supernatant. (B) Furtherion exchange chromatography of En1. Lanes 1 to 9, washouts of different NaCl gradients from 0 to 400 mM

Fig. S3 RFLP patterns of 14 fosmidclones using BamHI. Lane 1 to lane 14 resulted from different fosmidclones. Lane M, λ HindIII-digested DNA marker. The resulting vector fragment is about 8 kb. Note that lanes 1,2,3 are similar, especially the bands in the red box, and that lane 7 and lane 12 are similar, especially the bands in the red box

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