红霉素大环内酯合成通路在大肠杆菌中的重建

Reconstruction of erythromycin macrocyclic lactone synthesis pathway in Escherichia coli

摘要:

研究在大肠杆菌中重建了红霉素大环内酯 (6-脱氧-红霉内酯B，6dEB) 合成通路。先将参与6dEB合成所必需的基因分别克隆于多基因串联共表达载体中，获得单基因重组质粒；再利用载体中XbaⅠ/SpeⅠ互为同尾酶的特性实现相关基因的串联组合，获得多基因重组质粒pBJ130和pBJ144。将多基因重组质粒共转化BAP1，获得含6dEB合成通路的工程菌株BAP1 (pBJ130/pBJ144)，SDS-PAGE检测结果显示通路中各基因均有明显的表达；进行低温发酵，产物粗提后质谱检测到6dEB，其产量约10 mg/L。表明成功实现了6dEB合成通路在大肠杆菌中的重建，为红霉素大环内酯的改造和修饰提供了平台，也为红霉素合成通路在大肠杆菌中的完整重建以及聚酮类抗生素的组合性生物合成提供了参考。

Abstract:

We reconstructed the erythromycin macrocyclic lactone (6-deoxyerythronolide B, 6dEB) synthesis pathway in Escherichia coli. We first cloned all the genes needed to synthesize the 6dEB into multi-gene co-expressed vectors. Then using the recognition sequences of isoschizomers XbaⅠ/SpeⅠ of vectors, we assembled the related genes into a series multiple-genes recombinant plasmids pBJ144, pBJ130. The recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 to get the recombinant BAP1(pBJ144/pBJ130). SDS-PAGE analysis showed that individual genes were expressed correctly. After inducing at low temperature, adding propionate as substrate, we validated the crude product by mass spectrometry and the 6dEB yield was about 10 mg/L. These results indicated that the synthetic pathway of 6dEB was successfully assembled and reconstructed in Escherichia coli, which will greatly facilitate the reconstruction of whole erythromycin synthesis pathway and finally help to establish a stable research platform for developing of new derivatives of erythromycin and combinatorial biosynthesis of polyketide-type antibiotics.