Reconstruction Of The Erythromycin Biosynthetic Pathway In E. Coli

Historically, the natural product-or natural product analogs-derived small molecule drugs are the most effective ways to combat human diseases. The discovery of the antibiotics of last century saves the life of hundreds of millions people. At the same time, the widely usage of antibiotics also causes the emergence of antibiotics resistance pathogens, especially the Vancomycin-resistant Enterococci, methicillin-resistant Staphylococcus aureus and other "superbugs". which seriously threat human health. It is imminent to develop new antibiotics by exploring and utilizing the natural small molecule resource more efficiently through the approach of combinatorial biosynthesis of natural small molecule pathways.Polyketide antibiotics, manily from actinomyces, are a large class of drugs with diverse structures and activities and currently widely used in clinic. Due to the features of their pathway genes clustering together, the strictly corresponding between the order of different enzyme active sites and the assemble steps, the final polyketide structure, the modular polyketide pathway particularly suits for the producting of "unnatural" natural small molecules by combinatorial biosynthesis. The combinatorial biosynthesis of polyketide is expected to play an important role in the fight against resistant bacterias. The erythromycin is a typical example of polyketide antibiotics. By the structure modification, it is most likely to produce new, second even third generation erythromycin and derivatives with different activities. To develope new erythromycin, the establishment of a stable and efficient combinatorial biosynthesis platform for erythromycin synthesis is particularly important.The molecular mechanisms of erythromycin biosynthesis have been clarified, most genes involved in the erythromycin synthesis have also been shown to be functionally expressed in E. coli. By the extensive study, the 6dEB (6-deoxyerythronolide B) and its derivatives were successfully synthesized in E. coli. A breakthrough of post-modification of 6dEB was also achieved.Based on our previous work, this study was mainly to reconstruct the erythromycin synthesis pathway and explore the possibility of combinatorial and directed engineering of erythromycin biosynthesis in E. coli. The biosynthesis of erythromycin include the synthesis of polyketide scaffold 6dEB and its glycosylation modification, which involved dozens of genes with different activities, some synthesise need to be posttranslationally modified. Firstly, we choose the E. coli BAP1 whose genome was integrated with SFP gene as the expression host to ACP protein. Secondly, facilitate the cloning and assembling of the diverse genes, over-lap PCR mediated point mutation was introduced into the common expression vector pET-22b. pET-28a and get the multi-genes co-expression vector pET-m22b, pET-m28a repetitively. Thirdly, the whole complex synthetic pathway was artifically divided into two parts:6dEB synthesis and its modification. While the synthesis of 6dEB involves the PKS gene eryAⅠ, eryAⅡ, eryAⅢfrom Saccharopolyspora erythraea, which will express polyketide synthase module and will catalyze the extension and cyclization of carbon chain, and the propionyl-CoA carboxylase genes accA1, pccB from Streptomyces coelicol, which are responsible for the supply of precursors to 6dEB synthesis, for the 6dEB post-modification, the glycosylation genes eryBⅡ, eryBⅣ, eryCⅡ, eryCⅢ, eryBⅥ, eryBⅤfrom Saccharopolyspora erythraea, tylAⅠ, tylCⅢ, tylCⅦfrom Streptomyces fradiae and desⅠ, desⅡ, desⅣ, desⅤ, desⅥfrom Streptavidin Venezuel need to be cloned. To promote the correct folding of polyketide synthase and other heterologys genes, the chaperone genes groEL and groES of E. coli need to be co-expressed. Basing on the above design, we first cloned all the 22 genes needed to construct the whole erythromycine synthetic pathway. Then using the isoschizomers XbaI/SpeI of multi-gene co-expressed vectors pET-m22b and pET-m28a, the related genes were assembled into a series multiple-genes recombinant plasmid pBJ144, pBJ130, pBJEM and pBJDE. After transformation, IPTG induction, SDS-PAGE analysis showed individual genes were expressed correctly. Most genes could co-express evidently, but minority was not obvious. To construct the 6dEB synthesis pathway, the recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 and got the recombinant BAP1(pBJ144/pBJ130). After inducing at low temperature, adding propionate as substrate, the crude product was validated by the mass spectrometry and the 6dEB yield was about 10 mg/L. The 6dEB post-modification recomibnant were obtained by the cotransformation of BL21 with pBJEM and pBJDE. After inducing at low temperature fermentation, adding 6dEB as a substrate, the crude product was analyzed by the mass spectrometry. Unfortunately, the corresponding product was not detected, which indicated the expression level of multi-genes involved in glycosylation modification of 6dEB might need to be adjusted appropriately.In summary, by the co-expression of multi-genes, the synthetic pathway of 6dEB was successfully assembled and reconstructed in E. 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.