Cloning And Functional Research Of Amt Gene From Saccharopolyspora Spinosa

Nitrogen is a very important constituent of cellular components such as proteins, nucleic acid and a number of cofactors contain nitrogen as one of the elements. The nitrogen metabolism plays a critical role in all living organisms. Ammonia (ammonium ion) is one of the most convenient form of nitrogen absorbed by the organism, it is also one of the main transport form of nitrogen. Ammonium ion being in the center of nitrogen metabolism is the precursor of amino acids, nucleic acids and cofactors. The uptake of NH4+from environment and the transportation of NH4+between organizations is mediated by the cell plasma membrane transporter protein (Ammonium carrier) through a active transport process. Therefore, we overexpressed ammonium transporter proteins (ammonium transporter) in Streptomyces coelicolor M145and Streptomyces lividans TK24in order to improve their nitrogen environment, and to further explore the impact of ammonium transporter protein Amt on M145and TK24nitrogen metabolism.In this study, the ammonium transporter gene was amplified using genomic DNA of Saccharopolyspora spinosa as a template. The PCR product was inserted into pMD18-T vector through traditional molecular technical and sequenced. The fragment containing the amt gene was obtained by digested the pMD18T-amt by NdeⅠ and BglⅡ. This fragment was then inserted into the E. coli-Streptomyces shuttle vector pMF yielding pMF-amt. pMF-amt was then introduced into Streptomyces coelicolor M145and Streptomyces lividans TK24by conjugation. Amt protein was successfully expressed under the bidirectional promoter PactⅠ/Ⅲ. The phenotypes between wild-type and the transformants were analyzed in detail.The wild type and the transconjugants were cultivated on solid plate culture and liquid shake flask.The productions of the secondary metabolites actinorhodin were calculated and cell morphology were observed under microscope. Results showed that Transformants M145/pMF-amt can produce2.85times more Actinorhodin than the parental strain M145in shake flask, while the actinorhodin production of transformants TK24/pMF-amt were improved30.02times in comparison to the wide type. Meanwhile, the difference between the production of M145and M145/pMF, TK24and TK24/pMF, which contained the empty vector had no statistically difference. Furthermore, the cultivation on R4C agar plate demonstrated that the tranformants can synthesized pigments earlier than the wile type and can produce more spores than the parental strain.In conclusion, the coding gene of the ammonium transporter protein was first cloned from Saccharopolyspora spinosa, and then heterologous expressed in mode strain Streptomyces coelicolor M145and Streptomyces lividans TK24. This work established certain foundation for the following research.