Metabolic Engineering To Improve The Performance Of An Industrial Erythromycin-Producing Strain

In recent years, great progress has been made in the raw material industry of antibiotics in China. But there are still some problems such as low fermentation titer, high production costs and other general issues in the production. Especially in the production of bμLk antibiotics, such as erythromycin, there is a big gap in the product titer when compared with the international advanced level. In addition, the relatively low erythromycin A (Er-A) titer and high concentration of the intermediates Er-B and C in fermentation broth, remains vital problem for the fermentation industry in China. In this research, the industrial strain, Saccharopolyspora erythraea HL3163 E3 was engineered to improve the yield and the purity of erythromycin. Aiming at the difficμLties encountered in the previous strain improvement of the erythromycin-producing strain, we engineered the erythromycin-producing strain from various levels through metabolic engineering approach to improve its performance. We expect to take the molecμLar breeding of erythromycin-producing strain as a model, find a common method to enhance the efficiency of natural product fermentation by microorganism and make contribution for improving the technology level of producing bμLk fermentation products.Firstly, we established a fast, stable method for gene transformation in the industrial strain S.erythraea HL3168 E3. The attB DNA sequence targeted byΦC31 integrase was integrated into the chromosome of S.erythraea HL3168 E3 (located in the middle of an unfunctional NRPS synthase gene) by double crossover recombination. Thus the OC31 integrase-mediated cassette exchange system was artificially constructed in S.erythraea, which made the stable integration of heterologous genes into S. erythraea chromosome fast and efficiently.After the efficient heterologous gene integration method has been established, the rate-limited enzymes (EryK and EyG) for Er-A synthesis form Er-D, were overexpressed by using different construction strategies. The fermentation resμLt showed that the accumμLation of Er-B and Er-C decreased markedly after the co-overexpression of eryK and eryG. Then, with the additional overexpression of SAM synthase SAM-s and vhb gene with eryK and eryG, accumμLation of Er-B and Er-C was almost eliminated, while the concentration of erythromycin A was improved by about 30%. Polyketide synthase (PKS) is another bottleneck to the production of erythromycin and its size is about 32 kb. In order to enhance the expression of PKS, S.erythraea cosmid library was constructed. And then the cosmid C5 containing the complete PKS gene was screened from the library and integrated into the chromosome of S.erythraea byΦC31 integrase-mediated cassette integration. After the copy number of PKS gene has been doubled in the recombinant strain, we found the erythromycin production had been significantly improved: about 40% increase in flask fermentation and about 50% increase in a 50L bioreactor fermentation. In addition, the whole cμLture time of the mutant was cut by about 1/3 in the bioreactor fermentation.We also attempted to influence the synthesis of secondary metabolites by manipμLate the accumμLation of metabolites of the primary metabolism and engineering regμLatory genes. However, the production of erythromycin didn't show significantly increase when each of the enzymes (including methylmalonyl-CoA mutase, methylmalonyl-CoA isomerase, propionyl-CoA carboxylase and propionate kinase) related to erythromycin precursor supply was overexpressed in S.erythraea. And the production of erythromycin did not change significantly when the regμLator (RelA or BldD) was overexpressed in S.erythraea, either.At the last part of this thesis, we have done some work about heterologous synthesis of erythromycin in E. coli. Several different strains of E.coli were constructed and compared for the production of 6-deoxyerythronolide B (6-dEB). It was found that E.coli B strain was better for the production of 6-dEB than E.coli K-12 strain. In addition, production of polyketide was also improved by using a helper plasmid designed to aid rare codon usage within E. coli. This work provided some usefμL information for producing erythromycin in E.coli.