Reconstruction of erythromycin macrocyclic lactone synthesis pathway in Escherichia coli.
- Author:
Zhanghua HE
1
;
Yang WANG
;
Bingyu YE
;
Minglei SHI
;
Dong WANG
;
Qiusheng FAN
;
Fen HUANG
;
Zhihu ZHAO
Author Information
1. Beijing Institute of Biotechnology, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Anti-Bacterial Agents;
biosynthesis;
Cloning, Molecular;
Electrophoresis, Polyacrylamide Gel;
Erythromycin;
analogs & derivatives;
biosynthesis;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics
- From:
Chinese Journal of Biotechnology
2012;28(2):222-232
- CountryChina
- Language:Chinese
-
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 Xba I/Spe I 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.
