Construction Of Natamycin High-yield Strains

Natamycin or Pimaricin, is a26-member polyene macrolide antibiotic, which has the ability to inhibit the fungal and yeast. Pimaricin is a safe product for people, because its low toxicity to mammalian cells, pimaricin is widely used in food industry for preventing fresh food from mold contamination and in treatment fungal disease. However, because of the high cost and low yield of natamycin limited its application. Therefore, it’s important to improve the natamycin production by gene engineering.Use gene technology to increase natamycin production by increasing the copies of gene pimM, adpA, metk and vgb in this paper.Cloning16S rRNA of NTF4-245then constructing the evolutionary tree to determine NTF4-245and S. natalensis has the high homology, NTF4-245is one of S. natalensis. Cloning the gene pimM and pimMp, then ligated with vector pl168and pSET152constructing the expression vector pxql and pxq2, respectively. The ermE*p-pimM fragment was an enzymatic digestion from pxq2, then inserted into vector pSET152constructing plasmid pxq3.The three plasmids and other four plasmids which contain adpA, metk and vgb are trandform into NTF4-245through conjugation, spectively. Though selecting by antibiotic and PCR verified, obtain seven genetic engineered strains FNQ1～FNQ7. RT-PCR determined the gene expression of engineering strains.Compared the natamycin inhibition zone and the natamycin production in the fermentation medium of engineering strains with NTF4-245, finally confirm the highest-yield strains of natamycin are FNQ3.3and FNQ6.3. The natamycin production improve28.79%and28.99%compare with NTF4-245, respectively. Analyzing the relationship of gene and the production of natamycin show that pimM act as an activator in NTF5-245, adpA, metk, vgb can increase the natamycin yield of NTF4-245through connection in series. Though the growth curve of FNQ3.3, FNQ6.3and NTF4-245demonstrate that FNQ3.3grows well, the genes in FNQ6.3may influence the cells growing speed.