Cloning And Characterization Of The Zhongshengmycin Biosynthetic Gene Which Encodes β-Lysine Adenylation Enzyme From Streptomyces Lavendulae Var. Hainanensis

Streptomyces lavendulae var. hainanensis was isolated from the soil of Hainan Island in China, which could produce a kind of agricultural antibiotic—Zhongshengmycin that belongs to the Streptothricins-family antibiotics. It is a kind of multi-component antibiotic which contains a series of peptidyl nucleoside antibiotics that could effectively control some bacterial and fungal diseases of plants. This work bases on the related biosythetic genes which have been reported for the same Streptothricins (npsA and sttA). The β-lysine adenylation enzyme gene involved in Zhongshengmycin biosynthesis was colned from S. lavendulae var. hainanensis, and it's sequence analysis and function validation was carried out. First, we designed some degenerate primers according to the 4 conserved signature sequences A3,A5,A8,A10 of NpsA (AJ315729) and SttA (Y10293), and a 1.025kb DNA fragment was cloned from the chromosome DNA of S. lavendulae var. hainanensis UV-69 strain which encoded a peptide of 341 amino acids. An alignment of this peptide with the amino-acid residues of NpsA and SttA showed that their similarity is above 75%,which confirmed the previously proposed function of the peptide as a β-lysine adenylation enzyme.And it's function is similar with the function of A-domains of nonribosomal peptide synthetase as well as NpsA and SttA, so it was named Zhongshengmycin peptide synthetase. Then,recombinant plasmid pK2(pKC1139::611bp zpsA) was used for zpsA gene disruption in UV-69.The plasmid was inserted into the chromosome by homogenous recombination between partial zpsA in the plasmid and zpsA in the chromosome. Disruptants were confirmed by PCR.Shaking flask experiments showed that the assay titer of disruptants was far lower than that of UV-69.This revealed that zpsA gene was involved in Zhongshengmycin biosythesis, and encoded a key enzyme in the biosynthetic path of Zhongshengmycin. In order to effectively construct the disruptants, the factors that would affect the protoplast formation and regeneration were investigated.At last, the optimum examination conditions were determinated.The culture was shake-cultured at 28℃for 40 hours in improved liquid YEME media supplanted with 0.5% glycine. The mycelia was washed with 10.3% sucrose ,then incubated in P-buffer with 3mg/ml lysozyme at 35℃for 1.5 hour. Under the conditions, the amount of prepared protoplasts was up to 3.76×10~7/ml which was estimated by microscopic counts. HCM media was determined as regeneration medium, in which the regeneration frequency was 11.6%.The PGE1000 working concentration was 25% when plasmid pK2 was transformed to S. lavendulae var. hainanensis UV-69.