为研究差向异构结构域基因的序列特征以及其在非核糖多肽生物合成中的立体异构化特性，以海洋链霉菌 Streptomyces sp. X66基因组DNA为模板，采用PCR技术克隆获得目的基因序列，经测序及生物信息学分析表明，该基因序列长 1 099 bp，Blast序列比对该基因与Streptomyces albidoflavus strain W68的E结构域基因序列相似度达99.45%，具有典型的差向异构结构域的功能区域，且包含差向异构结构域独有的保守基序HHxxxD，证明扩增的基因片段为差向异构结构域基因序列。理化分析显示：其拟编码366个氨基酸，理论等电点为5.69，原子组成为C1752H2738N510O523S3，不稳定指数为32.68，平均亲水系数为-0.15，编码产物为酸性亲水稳定蛋白，且不含信号肽和跨膜结构，二级结构以α-螺旋和无规卷曲为主，SDS-PAGE显示其分子量约为55 ku，与预期相符。通过对差向异构结构域基因的研究，以期为进一步解析差向异构结构域功能及新型非核糖体多肽的研发提供科学依据。

To study the sequence characteristics of epimerization domain genes and their stereo-isomerization in the biosynthesis of non-ribosomal peptides, an epimerization domain gene was cloned from the genomic DNA of Streptomyces sp. X66. Bioinformatics analysis showed that the gene sequence length was 1,099 bp. Blast sequence alignment showed that the similarity between the cloned gene and the epimerization domain gene of Streptomyces albidoflavus strain W68 is 99.45%, and that the former has a typical functional region of the epimerization domain. It contains a unique conserved motif (HHxxxD) of the epimerization domain, which proves that the amplified gene fragment is an epimerization domain gene sequence. Physical and chemical analysis showed that it can encode 366 amino acids, and that its theoretical isoelectric point is 5.69. Its atomic composition, instability coefficient, and total average hydrophilicity were found to be C1752H2738N510O523S3, 32.68, and -0.15, respectively. Its coding product is an acidic hydrophilic stable protein without a signal peptide and transmembrane structure. The secondary structure of the protein comprises an α helix and random coil. SDS-PAGE showed that its molecular weight is about 55 ku, consistent with the expected value. By studying such epimerization domain genes, we hope to provide a scientific basis for the follow-up study of epimerization domain functions and the development of new non-ribosomal peptides.

辽宁省自然科学基金项目（20180550858）