Study On The Strain Improvement By Protoplast Fusion And Its Molecular Biology From Micromonospora Inyoensis

Sisomicin, an important aminoglycosides in clinic, belongs to 4,6-disubstituted deoxystreptamine antibiotics, and is produced by Micromonospora inyoensis. In this research work, a high sisomicin-producing strain S96 was obtained from M. inyoensis by means of cell-fusion and molecular biology methods, and a sisomicin resistance gene sisR was cloned from S96.Strain S96 was isolated from protoplast fusion of M.inyoensis strain T125 which produces sisomicin. , It was found that this strain was different from original strain both in pigment production and mycelian growth based on its mycelia morphology, slant culture characteristics, carbohydrate utilization and submerged culture and had many advantages over original strain.The immersed culture characteristics of strain S96 were studied. The formulation of fermentation medium was optimized by orthogonal design. Fermentation process was also improved depending on the dissolved oxygen curve and the typical metabolic characteristic curves of strain S96. The titer of sisomicin by strain S96 could be reached to 1600ug/ml.when the optimized conditions were adopted in a pilot-plant fermenter, which was close to the level of that in flask.A sisomicin resistance gene(sisR) was tried to be cloned by PCR amplification from genomic DNA isolated from M. inyoensis, in which primer pairs were designed based on grmA gene sequence from a gentamicin-producing strain of M. purpurea. A series of different DNA fragments were obtained from PCR amplification, and were sub-cloned into vector pUC19 for further identification. Five specific transformants containing target DNA fragments which could resist high concentrations of sisomicin (>1000|ag/ml) were obtained. One of them was designated as LY102 and its sequence was determined. The alignment among LY102 and other related genes showed that sisR gene obtained from this work was a new sisomicin resistance gene found in M. inyoensis that differs from any known genes.LY102 fragment was cloned into pUC19/18 vectors and sisR gene was expressed in Escherichia coli DH5a under the control of its own promoter sequence and ribosome-binding site(GGAGG) instead of lacZ promoter. It was found that two open reading frames (orfl and orf2) in the complete nucleotide sequence of 849 base pairs were coordinately responsible for the expression of sisomicin resistance. A comparison of the predicted amino acid sequence of sisR with the deduced amino acid sequences of three 16S rRNA methylases grmB grmA and sgm showed an extensive similarity to 92 %, 87 % and 85%, respectively.