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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。
The pathways for the synthesis of erythromycin macrolide (6-deoxy-erythronolide B, 6dEB) were reconstructed in E. coli. Firstly, the genes necessary for the synthesis of 6dEB were cloned into the multi-gene tandemly-cotransfected vector to obtain the single-gene recombinant plasmids, and then the tandem combination of related genes was achieved by using the characteristics of XbaⅠ / Recombinant plasmids pBJ130 and pBJ144. BAP1 (pBJ130 / pBJ144) containing 6dEB synthetic pathway was co-transformed into BAP1 by multi-gene recombinant plasmid. SDS-PAGE results showed that all the genes in the pathway were significantly expressed. After low temperature fermentation, the crude product was detected by mass spectrometry To 6dEB, its production is about 10 mg / L. The successful construction of the 6dEB synthetic pathway in Escherichia coli has provided a platform for the modification and modification of erythromycin macrolides, as well as the complete reconstruction of erythromycin synthesis pathway in E. coli and the development of polyketide antibiotics Combined biosynthesis provides a reference.