Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan.
FEMS Microbiol Lett. 2012 Jun;331(1):81-8. doi: 10.1111/j.1574-6968.2012.02556.x. Epub 2012 Apr 17.
We identified a highly expressed protein (BUNA2) by two-dimensional gel electrophoresis from the hyper lignin-degrading fungus Phanerochaete sordida YK-624 under wood-rotting conditions. Partial amino acid sequences of BUNA2 were determined by LC-MS/MS analysis, and BUNA2 gene (bee2) and promoter region were PCR-cloned and sequenced. The bee2 promoter was used to drive the expression of the manganese peroxidase gene (mnp4) in P. sordida YK-624. Eighteen mnp4-expressing clones were obtained, with most showing higher ligninolytic activity and selectivity than wild-type YK-624. Examination of the ligninolytic properties of the most effective lignin-degrading transformant, BM-65, cultured on wood meal revealed that this strain exhibited higher lignin degradation and MnP activities than those of wild type. Transcriptional analysis confirmed the increased expression of recombinant mnp4 in the transformant. These results indicate that use of the bee2 promoter to drive the expression of ligninolytic enzymes may be an effective approach for improving the lignin-degrading properties of white-rot fungi.
我们通过在腐朽木材条件下，对高产木质素降解真菌栓菌 YK-624 进行二维凝胶电泳，鉴定到一种高表达蛋白（BUNA2）。通过 LC-MS/MS 分析，我们确定了 BUNA2 的部分氨基酸序列，并通过 PCR 克隆和测序获得了 BUNA2 基因（bee2）及其启动子区域。我们将 bee2 启动子用于驱动栓菌 YK-624 中的锰过氧化物酶基因（mnp4）表达。得到了 18 个 mnp4 表达克隆，其中大多数表现出比野生型 YK-624 更高的木质素降解活性和选择性。对在木屑上培养的最有效的木质素降解转化子 BM-65 的木质素降解特性进行研究，发现该菌株表现出比野生型更高的木质素降解和 MnP 活性。转录分析证实了重组 mnp4 在转化子中的表达增加。这些结果表明，利用 bee2 启动子驱动木质素降解酶的表达可能是提高白腐真菌木质素降解特性的有效方法。
