Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris.
A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae alpha-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for the construct encoding the alpha-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115.
Jönsson
L J
LJ
Applied Microbiology, Lund Institute of Technology/ Lund University, P. O. Box 124, S-22100 Lund, Sweden. leif.jonsson@tmb.lth.se
Saloheimo
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M
Penttilä
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D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D042845:Laccase; D000072235:Mating Factor; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D018076:DNA, Complementary; D004271:DNA, Fungal; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D010088:Oxidoreductases; D042845:Laccase; D000432:Methanol