Metabolic Engineering Of The Tailing Enzymes To Obtain Overproduction Of Erythromycin A,B,C, Or D In The Industrial Erythromycin-producing Strain

Erythromycins are a group of 14-members macrolide antibiotics produced by Saccharopolyspora erythraea. Erythromycin A (Er-A) is the main product in the broth of S. erythraea, additionally, a few members in the erythromycin family, including B, C, and D, were also present in the culture broth of S. erythraea. Er-A is widely used as the material for the synthesis of the new generation erythromycin. Er-D is the first intermediate to show antibacterial activity, Er-B and C show less activity than Er-A. However, Er-B shares the relative acid stability of the second generation Er-A derivatives, and Er-C can be converted to megalomicin and its analogues. They may be used as the lead compounds for generation of new derivatives through semi-synthesis and bioconversion. In order to improve the production of Er-A and acquire high accumulation of Er-B, C and D, the biosynthesis pathway from Er-D to Er-A in the industrial strain S. erythraea HL3168 E3 was altered by genetic manipulation of the two tailoring enzymes in the biosynthesis of Er-A:EryK (erythromycin C-12 hydroxylase, a P-450 monooxygenase responsible for erythromycin C-12 hydroxylation) and EryG (erythromycin O-methyltransferase, an S-adenosylmethionine-dependent O-methy ltr ansferase).First of all, the expression cassette PermE-eryK-eryG-eryK was integrate into the chromosome of the industry strain HL3168 E3 using theφC31 integrase-mediated cassette exchange system, generating the recombinant strain S. erythraea-KGK. In the fermentation broth of recombinant strain KGK, the proportion of Er-A was improved to 91.1%, and titer of Er-A also increased by 18.6%. This result showed thatφC31 integrase-mediated cassette exchange system is feasible for optimization of erythromycin production in industry strain.In order to overproduce only Er-C, Er-B, or Er-D, we inactivated eryG gene, eryK gene, or both of them in industrial strain S. erythraea HL3168 E3, generating mutant strain QL-G, QL-K, or QL-KG. In these mutant strains, Er-A was eliminated in the culture broth, while Er-C, Er-B and Er-D was accumulated as the major component to the concentration of 2.48, 1.70 and 2.15 mg/ml, respectively. Moreover, it was found that the industrial strain HL3168 E3 is amenable to genetic engineering of the Er-A pathway without a decrease of the Er titer. And analysis of the erythromycin congeners produced by recombinant strains throughout the fermentation process indicated that the EryG-catalyzed methylation of erythromycin D to B is more efficient than the EryK-catalyzed hydroxylation of erythromycin D to C. So, the results in our report showed that overproduction of Er-A, B, C and D could be obtained in our four mutant strains, and the information obtained in this study may contribute to the further investigations of improving erythromycin production.