Expression, Purification And Functional Identification Of Sansanmycin Biosynthesis Gene SsaX

Sansanmycins, produced by Streptomyces sp. SS isolated from Guizhou province of China, are members of a uridyl peptide antibiotic family. Sansanmycins were reported to exert antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa, and more interesting, Mycobacterium tuberculosis H37Rv and multidrug-resistant M. tuberculosis strains. The uridyl peptide antibiotic family also includes closely related pacidamycins, napsamycins and mureidomycins which contain a non-proteinogenic meta-tyrosine (m-Tyr) in their chemical structures. The biosynthetic gene clusters for pacidamycin, napsamycin and sansanmycin have been cloned, and most of the pacidamycin biosynthetic genes have been characterized. The PacX of the biosynthetic gene cluster for pacidamycins was recently biochemically characterized as a phenylalanine hydroxylase to catalyze the formation of m-Tyr from phenylalanine (Phe). Bioinformatics analysis showed that ssaX in sansanmycin gene cluster is highly homologous to pacX, thus SsaX might be responsible for the synthesis of m-Tyr in the peptide backbone of sansanmycins.Here, ssaX was amplified from the genomic DNA of Streptomyces sp. SS by PCR and cloned into an expression vector pET16b. The fusion protein was expressed in Escherichia coli BL21(DE3) as an N-terminal His10-tagged protein and identified by Western blotting. In order to increase the yield of soluble His10-SsaX in E. coli, the fermentation conditions were optimized and the strain was cultured at 37 ℃ until the OD6oo=0.6, and then induced with 0.02 mM IPTG at 16 ℃ for 24 h. The His10-SsaX in the cellular lysate supernatant was subjected to nickel affinity chromatography and eluted with the optimized elution buffer containing 200 mM imidazole. His10-SsaX was purified from E. coli with a yield of 9.9 mg/L and the purity of the crude protein is about 61.7%. The purified His10-SsaX was mixed with L-Phe and (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride (6R-BH4) in sodium phosphate buffer, and the mixture was incubated at room temperature and then subjected to HPLC analysis. The results showed that the His10-SsaX could catalyze the synthesis of m-Tyr from Phe. There was also small amounts of para-Tyr (p-Tyr) formed from the reaction mixture, and the production of p-Tyr compared to m-Tyr is about 1:8.In conclusion, in this work, an N-terminal His10-tagged SsaX protein was cloned, expressed, and purified from E. coli. The reaction results confirmed that SsaX is a phenylalanine 3-hydroxylase that catalyzes the synthesis of m-Tyr in the peptide backbone of sansanmycins from Phe.This work not only provides a new method for the synthesis of m-Tyr, but also provides a basis for sansanmycin genetic manipulation to get more antibacterial derivatives with higher activity.