Effects Of Vgb Gene And BldD Gene Over-expression On Nosiheptide Production In Streptomyces Actuosus

Nosiheptide (Nos) is a sulfur-containing polypeptide antibiotic which is produced by Streptomyces actuous. It belongs to the class of thiopeptide antibiotics which include siomycin, micrococcin, thiocillin, thiostrepton and so on. It predominantly inhibits gram-positive bacteria, and the minimum inhibitory concentration (MIC) is only8ng/mL. For some gram-negative bacteria, it likewise has some certain degrees of inhibition. Due to the advantages of low toxicity, high antibacterial activity, no residue and so on, the nosiheptide has been developed for an efficient livestock feed additive which has extensive application in the livestock growth, disease prevention and controlling, but lower yield is the main factor which limites the application. This study mainly focuses on the exogenous genes expression to increase the nosiheptide yield.Hemoglobin is an oxygen binding protein, which widely exists in animals, plants and microorganisms. Vitreoscilla hemoglobin (VHb) is prokaryote class hemoglobin which can be induced to meet the life needs when the surrounding oxygen concentration is relatively low. With the continuous development of genetic engineering, it is found that the vgb gene which expresses the VHb is able to express stably in E. coli, fungi, Pseudomona, Streptomyce, and the VHb can significantly improve the production of a-amylase, actinorhodine, erythromycin.bid genes are global regulatory genes in Streptomyces, including bldA, bldB, bldC, bldD, bldH, bldK, ect, among which the bldD gene has more obvious regulatory effects on morphological differentiation and antibiotic production than others. Knocking out the bldD gene in Streptomyces coelicolor, the mutant shows the "bald" phenotype owning to lacking aerial hyphae. At the same time, the antibiotics output of actinorhodin, undecylprodigiosin, calcium-dependent antibiotic becomes low in different degrees. When the bldD gene is deleted in Saccharopolyspora erythraea, the yield of erythromycin reduces, and over-expression of bldD gene in S. erythraea can significantly improve the erythromycin output. In this study, the spores of S. actuosus were firstly diluted into plates, and then different clones were picked out for fermentation. Spectrophotometer was the main method to determine nosiheptide production. Besides, appling PEG-mediated protoplast transformation, the expression vector pZM, pZM-vgb, pZMW and pZMW-vgb were transformed into S.actuosus to construct the industrial strains including S.actuosus/pZM, S.actuosus/pZM-vgb, S.actuosus/pZMW and S.actuosus/pZMW-vgb. The results demonstrated the nosiheptide yield in industrial strain of S.actuosus/pZMW-vgb was increased by50%compared with the original strain. What’s more, we successfully transformed the bldD gene into S.actuosus to construct the industrial strain S.actuosus/pZMW-bldD. Applying the same methods to determine nosiheptide production, the results revealed the nosiheptide yield in S.actuosus/pZMW-bldD was increased by68%compared with the original strain.In this study, overexpression of vgb gene and bldD gene in S.actuosus were carried out to investigate the effects of these genes on nosiheptide production. This study not only provided a basis for researching the regulatory genes to improve the nosiheptide output, but also opened up the idea for exploring bldD gene to improve the production of other antibiotics.