Jönsson L J, Saloheimo M, Penttilä M
Applied Microbiology, Lund Institute of Technology/ Lund University, P. O. Box 124, S-22100 Lund, Sweden.
Curr Genet. 1997 Dec;32(6):425-30. doi: 10.1007/s002940050298.
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.
通过RNA-PCR从木质素分解真菌云芝中分离出编码漆酶的cDNA。该cDNA对应于基因lcc1，其编码一种由498个氨基酸残基组成的漆酶同工酶，前面有一个22个残基的信号肽。将lcc1 cDNA克隆到载体pHIL-D2中，以便在AOX1启动子的控制下在毕赤酵母中表达。发现用甲醇诱导后，转化体分泌活性重组酶。发现使用缓冲至pH 6.0的生长培养基并在培养过程中控制pH对于在液体培养物中获得活性很重要，甚至是必要的。通过将编码成熟酶的部分克隆到载体pPIC9中，研究了将天然分泌信号替换为酿酒酵母α因子前体-前导分泌信号的效果。发现编码天然漆酶信号序列的构建体所获得的活性比编码α因子分泌信号的构建体高七倍。发现利用毕赤酵母pep4突变株SMD1168与毕赤酵母GS115相比可提供两倍高的活性水平。
