Cloning And Expression Of Vitreoscilla Hemoglobin Gene In Streptomyces Fradiae

Streptomyces fradiae is a kind of strict areobacterium that can produce Tylosin; Its fermentation broth is so mucous that it cannot dissolve too much oxygen, as a result, DO becomes one of the resticted factors that affects the increase of Tylosin yield. This study was carried out to improve the transmission of oxygen by expression of Vitreoscilla hemoglobin(v/z6) gene in Streptomyces fradiae in order to promote synthesis of Tylosin and cell growth.An expression vector, pIJ8600, contains the Streptomyces phage C31 attachment site for chromosomal intergration and a strong promoter(PtipA) which is inducible by thiostrepton. vhb gene expression vector, pWQ2005, was constructed by cloning vhb gene under control of PtipA, and pWQ2005 was introduced into Streptomyces fradiae from E. coli ET12567( pUZ8002) by conjugal transfer. PCR analysis indicated that vhb gene has been successfully intergrated into the chromosome, CO binding difference spectrum analysis revealed the vhb gene in pWQ2005 induced by thiostrepton has expressed Vitreoscilla hemoglobin with bio-activation. Shask flask fermentation experiments preliminarily showed that expression of vhb gene could dramatically promote synthesis of Tylosin and improve cell growth. However, different inflences were remarkablely produced by the thiostrepton induced in different time.The plasmids, pSET152 and pIJ8600, are both the same kind of vectors, but the former doesn't contain promoter for expression of exotic gene. An expression vector, pWQ1116. was constructed by cloning vhb gene with native promoter into pSET152. However, vhb gene couldn't be expressed in Stretomyces fradiae under oxygen-resticted conditions, the probable reason for that is the RNA polymerase of Stretomyces fradiae couldn't recognize the native promoter of vhb gene.Promoter of Erythromycin resistance gene (PermE) can be constitutively expressed in Streptomyces, vhb gene without native-promoter was placed under control of PermE via SOE-PCR(Splicing of Overlap Extension by PCR), so vhb gene relied on PermE to gain expression. Site-directed mutagenesis was performed to enable vhb gene to express better in Streptomyces fradiae, and two site-mutagenesised vhb gene expression vectors. pWQ2004 and pWQ1023, were constructed. At the same time, the original vhb gene expression vector, pWQ1118, was consructed as control by using the similar way. According to CO binding difference spectrum analysis results, all of the three vectors have expressed VHb with bio-activation in Streptomyces fradiae, but comparaed with the original gene, the site-mutagenesised vhb gene has expressed a little more VHb.Candidate conjugants(Streptomyces fradiae/pWQ2004) had been screened, and the subsequent fermentation results showed that exprssion of vhb gene in Streptomyces fradiae could promote cell growth and Tylosin yield, and the promotion displayed more eminently under more hypoxic conditions.