Wang Z M, Bleakley B H, Crawford D L, Hertel G, Rafii F
Department of Bacteriology and Biochemistry, University of Idaho, Moscow.
J Biotechnol. 1990 Feb;13(2-3):131-44. doi: 10.1016/0168-1656(90)90099-w.
A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.
将来自绿产色链霉菌T7A的木质素过氧化物酶基因克隆到质粒pIJ702中，并导入变铅青链霉菌TK64。用BglII消化的绿产色链霉菌基因组DNA（4 - 10 kb）经鸟枪法克隆到变铅青链霉菌中，该DNA片段先插入到pIJ702的黑色素（mel +）基因中。根据再生培养基上硫链丝菌素抗性（tsrr）/mel - 菌落的出现情况，筛选出携带插入DNA的pIJ702转化子。通过在tsr - 聚B - 411染料琼脂培养基上筛选转化子，从该群体中分离出表达木质素过氧化物酶的克隆。在变铅青链霉菌分泌的H2O2存在下，表达木质素过氧化物酶的克隆菌落使染料脱色。在筛选的1000个转化子中，发现了2个染料脱色克隆。其中一个，pIJ702/TK64.1（TK64.1），被进一步鉴定。TK64.1表达出显著的细胞外2,4 - 二氯苯酚（2,4 - DCP）过氧化物酶活性（= 绿产色链霉菌木质素过氧化物酶的检测方法）。在所采用的培养条件下，无质粒的变铅青链霉菌TK64的2,4 - DCP氧化活性背景水平较低。如在非变性聚丙烯酰胺凝胶上的活性染色分析所示，TK64.1分泌一种在变铅青链霉菌TK64中未观察到，但与绿产色链霉菌木质素过氧化物酶ALip - P3相似的细胞外过氧化物酶。该基因位于绿产色链霉菌基因组DNA的一个4 kb片段上。当编码过氧化物酶的质粒pIJ702.LP被纯化并用于转化三种不同的变铅青链霉菌菌株（TK64、TK23、TK24）时，所有测试的转化子都能使聚B - 411脱色。当在固态发酵过程中在木质纤维素上生长时，基因工程改造的变铅青链霉菌TK64.1对木质纤维素的降解能力略优于变铅青链霉菌TK64。这是关于克隆编码木质素降解酶的细菌基因的首次报道。
