Supplementary Data

Cloning and heterologous expression of a novel ligninolytic peroxidase gene from the poroid brown-rot fungus Antrodia cinnamomea, by S. T. Huang, S. S. Tzean, B. Y. Tsai and H. J. Hsieh

Microbiology vol. 155, part 2, pp. 424-433

Fig. S1. Map of expression vector pQE31-ACLnP, containing a T5 promoter and an inserted 6xHis-tag.

Fig. S2. Comparison of amino acid sequences of ACLnP with extracellular peroxidases of other wood-rot basidiomycetes: Phanerochaete chrysosporium PCLIG1 (Q01775), PCLIG2 (AAA33735.1), PCLIGA (CAA38177.1), PCLIGH8 (P06181.1), PCLIGH2 (P11542.2), PCLIGJ (AAD46494.1), PCLIGH5 (AAA33734.1), PCLIG3 (CAA35939.1), PCMnP (AAA33743.1), PCMnP1 (AAA33742.1), PCMnP2 (AAB30859.1), PCMnP3 (AAA62243.1); Trametes versicolor TVLPGIV (CAA83228.1), TVLPG1 (AAA34049.1), TVLPG3 (CAA83146.1), TVLPG7 (CAA83147.1), TVLPGV (CAA54398.1), TVMNP2 (CAA83148.1); Pleurotus eryngii PEVPS1 (Q9UVP6.1), PEVP (AAZ04666.1), PEVPL1 (Q9UR19.1), PEVPL2 (O94753.1); Coprinellus disseminatus CdP (AAZ14938.1); Coprinus macrorhizus CmP (S78602); Coprinopsis cinerea CcP1 (CAA49216.1); Coprinus cinereus CcP2 (1H3J); Arthromyces ramosus P (BAA09861.1); Agaricus bisporus AbMnP (CAG27835.1); Ganoderma applanatum GaaMnP (BAA88392.1); Ganoderma australe GauMnP (ABB77244.1); Ganoderma formosanum GfMnP (ABB77243.1); Trametes cervina TCLIG (BAE46585.1); Armoracia rusticana ArHP (CAA00083.1); Magnaporthe grisea PP (EAA53513.1).

Fig. S3. Nucleotide and translated amino acid sequences of ACLnP. Introns are numbered I-XII. Inverted triangle, start of transcript; asterisk, stop codon; green dotted underline, transmembrane protein helix structure; purple arrow, signal peptide cleavage site; star, proximal haem ligand and distal histidines; diamond, three residues adjacent to the proximal histidines; green letters, possible glycosylation sites. The four TATA boxes and three CAAT elements are shown in blue; putative regulatory elements are indicated by underlining (two SP1 and HSF, one AP1, AP2, RFX2, C/EBP and GATA binding factor: SP1, stimulating protein 1; HSF, heat-shock factor; AP1, factor involved in response to oxidative stress/oxygen detoxification and metal resistance; AP2, activator protein 2; RFX2, enhancer factor; C/EBP, enhancer binding protein). The 5′ upstream sequences of ACLnP were predicted by using MatInspector (www.genomatix.de) and AliBaba 2.1 (www.gene-regulation.com) software.

Fig. S4. Comparison of the number, position and size of the introns (orange blocks) of ACLnP with genes of LiPs, MnPs and VPs from other white-rot basidiomycetes. The distance between vertical lines represents 500 nt. LiPs: ACLnP is from A. cinnamomea ACT1; PCLiG1 (M24082.1), PCLiG2 (M92644.1), PCLiGA (X54257.1), PCLiGH2 (X15599.1), PCLiGJ (AF140062.1) and PCLiGH5 (X55343.1) are from Phanerochaete chrysosporium; TVLiG1 (M64993.1), TVLiG3 (Z30666.1), TVLiG5 (D86493.1) and TVLiG7 (Z30667.1) are from Coriolus versicolor. MnPs: PCMNP1 (M77513.1), PCMNP2 (S69963.1) and PCMNP3 (U70998.1) are from Phanerochaete chrysosporium; STVMNP2 (Z30668.1) is from Coriolus versicolor. VPs: PEVP (DQ056374.1), PEVPL1 (AF007223.1), PEVPL2 (AF007224.1) and PEVPS1 (AF175710.1) are from Pleurotus eryngii.

Fig. S5. Analysis of LiP, BG, EBG and 18S RNA from the mycelium (blue bars) or fruiting body (orange bars) of A. cinnamomea by Q-PCR.

Fig. S6. Alignment and comparison of ACLnP from five fosmid clones [b16 (ACLnP), g20, k16, k17, p11]. With the exception of clone g20, with 8 nucleotide substitutions, the sequences in the other four fosmid clones are identical.

Fig. S7. (a) Molecular model of ACLiP, consisting of 13 α-helices, 3 β-sheets, 2 Ca²⁺ binding sites, an internal haem functional block, and N and C termini, predicted based on the available crystallographic structures of LiP, MnP, VP and peroxidase from other white-rot basidiomycetes. (b) Stereo view of the partial molecular structure of ACLnP. The internal haem functional block is surrounded by conserved amino acids. The possible short- and long-range electron transfer routes between amino acids, or between amino acids and haem (dotted line) were predicted using PyMOL software (http://pymol.sourceforge.net).

Fig. S8. Superimposition of 3-D molecular structures of ACLnP (ACLiP, orange) with Phanerochaete chrysosporium LiP (PCLiP, green) and MnP (PCMnP, purple), Pleurotus eryngiiVP (PEVP, blue) and Arthromyces ramosus peroxidase (ArP, pale blue) retrieved from the PDB database (www.rcsb.org/pdb/home/home.do). Calcium (Ca²⁺), pale green; manganese (Mn²⁺), pale grey.

Fig. S9. The ACLnP gene containing 12 introns. The 770 bp at the 3′ end without EcoRV, PstI and EcoRI restriction sites was used as the gene-specific probe for Southern blot analyses.

Fig. S10. Alignment of ACLnP (ACLiP, clone g20) with the VP precursor VPL1 sequence from the NCBI database.

Table S1. Primers used for PCR and Q-PCR

Table S2. Properties of ACLnP and the prediction of regulatory elements in the promoter region

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