Cloning and Homologous Expression of Novel Lignin Peroxidase Genes in the White-Rot Fungus Phanerochaete sordida YK-624
Cloning and Homologous Expression of Novel Lignin Peroxidase Genes in the White-Rot Fungus Phanerochaete sordida YK-624
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白腐菌Phanerochaete sordida YK-624新型木质素过氧化物酶基因的克隆及同源表达
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Hirofumi Hirai
中科院分区：
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作者：
Tatsuki Sugiura;K. Yamagishi;Toshiyuki Kimura;T. Nishida;H. Kawagishi;Hirofumi Hirai
从白腐菌sordida Phanerochaete YK-624中克隆了编码YK-LiP1和YK-LiP2的基因，并构建了该基因的同源表达体系。通过退化RT-PCR和RACE-PCR分离得到两个全长cdna （ylpA和ylpB）。天然YK-LiP1和YK-LiP2的n端氨基酸序列分析结果表明，ylpA和ylpB分别编码YK-LiP2和YK-LiP1。利用从P. sordida YK-624克隆的甘油醛-3-磷酸脱氢酶启动子（PsGPD）驱动ylpA的表达。将表达载体pGPD-g-ylpA转化为sordida YK-624尿嘧啶营养不良突变体UV-64。在含有过量氮源的培养基中生长4天后，转化子以活性形式分泌YlpA蛋白，而内源性YK-LiP1和YK-LiP2不产生。纯化的YlpA蛋白的物理和催化性能与YK-LiP2非常相似。这些结果表明重组YK-LiP2同源表达成功。
Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.