Using The EryK Gene And EryG Gene To Improve The Yield Of Erythromycin And Identification Of SACE?0012 Gene Function In Saccharopolyspora Erythraea

The secondary metabolism of actinomycetes can produce various bioactive substances.According to the function,these materials can be divided into antibiotics,inmunoinhibitors,antiviral drugs,antitumor drugs.The erythromycin A(ErA)is produced by Saccharopolyspora erythraea,which can effectively cure the respiratory infections.Erythromycin is a kind of broad-spectrum antibiotics and resist to gram-positive bacterial infection.Thus there is important economic value to improve the erythromycin production.Microbial secondary metabolites are influenced by various physical and chemical factors,such as precursor supply,dissolved oxygen concentration,temperature and pH value.Traditional methods to enhance secondary metabolites are random mutating and screening the high-yielding strain.With the maturity of molecular biology technology,using of metabolic engineering method to modify the biosynthetic pathway can improve the secondary metabolites yield.In this study,The erythromycin biosynthetic gene eryK gene and eryG gene copies were increased,and led to improve the expression of C12 hydroxylase(EryK)and 3"-O-methyltransferase(EryG).When eryK gene and eryG gene copies were increased,erythromycin D(ErD)could maximum transform into erythromycin A(ErA),and eliminated the accumulation of middle product erythromycin B(ErB)and erythromycin C(ErC).This result would reduce the isolation and purification process of erythromycin and the environmental pollution.The genome of Sac.erythraea A226 was extracted,then we obtained the eryK and eryG gene fragments by using the PCR technology.The intermediate vector pUCKKG plasmid which included eryK gene and eryG gene fragments was successfully constructed,then The integrating expression plasmid pZMWKKG was successfully constructed.The pZMWKKG plasmid was transformed into Sac.erythraea ZMD by PEG mediated protoplast transformation,yielding ZMD/pZMWKKG strain.The eryK gene and eryG gene copies were appropriate increased in ZMD/pZMWKKG strain,therefore,we get the high yield of erythromycin strain by many times the screening of the clones and fermentation product for analysis.The secondary metabolism makes the erythromycin in Sac.erythraea,which is important medical value,meanwhile,Sac.erythraea will undergo complex morphological differentiation during the development of life cycle.The life cycle begins with the germination of the spores,then it will form the vegetative mycelium.In certain under the environmental pressure,filamentous substrate mycelium can form the aerial hyphae and spores.Another content of this research that the TetR family member SACE₀012 gene affects morphological differentiation in Sac.erythraea,and research the SACE₀012 gene and the SACE 7040 gene to a certain degree of functional linkages.The ?SACE₀012,?bldD/?SACE₀012,?SACE₇040/?SACE₀012 mutants was successfully constructed by the linearized fragment homologous recombination.These mutants,Sac.erythraea A226,?bldD and ?SACE₇040 are inoculated R3M incline to incubate at 30?,then we observed the growth of spores.The result showed that the ?SACE₀012 mutant formed the spores in advance,so we speculated the biological function of the SACE₀012 gene may be related to spore formation.The laboratory had reported the SACE₇040 gene was negative regulate to morphological differentiation in Sac.erythraea.In order to research the relationship of the SACE₀012 gene and the SACE₇040 gene,we disrupted the SACE₀012 gene in the ?SACE₇040 mutant,then we found spores growth in the?SACE₇040/?SACE₀012 mutant was the same as that in the ?SACE₀012 mutant and ?SACE₇040 mutant.We speculated the acting targets of the SACE₀012 gene and the SACE₇040 gene maybe upstream and downstream relationship,and not parallel relationship.The targets of SACE₇040 gene was located in the downstream of the bldD gene.Deletion of the SACE₀012 gene in the ?bldD mutant can not restore the growth of spores.The result implied that the target of the SACE₀012 gene maybe located in the upstream of the bldD gene.Thus we can conclude that the target of the SACE₀012 gene maybe located in the upstream of the SACE₇040 gene in the regulatory network.However,the detailed regulatory mechanism of the SACE₀012 gene remains to be further studied.This research lays the theoretical and experimental basis for improve the production of erythromycin and understand the regulate mechanism of morphological differentiation in Sac.erythraea.With the deepening of research to structure genes and regulatory genes,we can more accurately identify the function of certain genes and improve the yield of purpose production.We can also understand the regulate mechanism of morphological differentiation in Sac.erythraea.