Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression

• Published in: Applied microbiology and biotechnology 2006-07, Vol.71 (4), p.493-501
• Main Authors: Xiao, Y. Z, Hong, Y. Z, Li, J. F, Hang, J, Tong, P. G, Fang, W, Zhou, C. Z
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Aromatic compounds; Base Sequence; Basidiomycetes; Basidiomycota - enzymology; Basidiomycota - genetics; Basidiomycota - metabolism; Biological and medical sciences; Biotechnology; Cloning; Cloning, Molecular; copper; Copper - metabolism; DNA, Fungal - chemistry; DNA, Fungal - genetics; Enzymatic activity; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; gene expression regulation; Gene Expression Regulation, Enzymologic; genes; Isoenzymes; isozymes; laccase; Laccase - biosynthesis; Laccase - genetics; Laccase - metabolism; messenger RNA; Promoter Regions, Genetic; Resorcinols - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Sequence Alignment; Sequence Analysis, DNA; Sequence Analysis, Protein; TATA box; Toluidines - metabolism; Trametes
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-005-0188-2

Three novel laccase isozyme genes, lacA, lacB, and lacC, have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE, and CreA consensus sequences were identified in the lacA, lacB, and lacC promoter regions. Different from the TATA boxes of lacA and lacB at about −100, the TATA box of lacC is located at −172. For all the isozymes, copper ion is essential for laccase synthesis in Trametes sp. AH28-2. More interestingly, different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of laccase A (LacA) while 3,5-dihydroxytoluene mainly stimulating the production of laccase B (LacB). Quantitative reverse transcriptase–polymerase chain reaction showed that the accumulation of laccase messenger RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures. The glucose-repression effect on laccase expression in Trametes sp. AH28-2 was also observed. Furthermore, lower Cu²⁺ concentration (lower than 0.5 mM) can induce LacA and a novel laccase (LacC), and the latter will disappear when Cu²⁺ concentration is increased up to 1−2 mM. Upon induction by 3,5-dihydroxytoluene, the ratio of LacA to LacB decreased in the later phase of induction.