Construction Of Genetically Engineered Streptomyces

Streptomyces is an important antibiotic producing bacteria, their metabolic processes of in-depth study can be explained from the genetic level in vivo metabolite changes and the impact on the target metabolite, so that industrial production strain optimization of the molecular orientation.Acetic acid is the end product of anaerobic metabolism in Streptomyces. Accumulation of acetic acid inhibits the growth of mycelium and the expression of exogenous protein. Acetyl-CoA is an important secondary metabolites of some precursors, in theory block the production of acetic acid acetyl-CoA pathway could promote cell growth, while increasing the output of the target product. Knockout of the key rate-limiting enzyme phosphofructokinase gene pfkA1 of glycolytic pathway could promote carbon metabolic flux shift from the glycolytic pathway to the pentose phosphate pathway, thus we could research the two metabolic pathways of glucose metabolism, also different metabolic ways in the middle of the secondary metabolites of the synthetic product of the role of metabolic transformation of primary guide for further study. The fermentation process of Streptomyces lydicus needs high demand for oxygen. The ability of Vitreoscilla hemoglobin (VHb) to capture and release oxygen can better meet the needs of oxygen in the fermentation of host cells. Therefore, the expression of Vitreoscilla hemoglobin in Streptomyces can improve the needs of high oxygen and promote the body metabolism, and improve the production of purpose productIn this paper, two steps are taken to knock out the key gene pta-ackA and pfkA1, according to RED recombination principle. By homology comparison, the key gene was amplified. By PCR, enzyme digestion, the connection will be cloned into cosmid vector construct intermediate plasmid. The plasmid was transformed into E. coli BW25113/pIJ790 body, the resistance marker apr fragments, which were amplificated by PCR, were electrotransfered to E. coli BW25113/pIJ790 to disrupt the taget genes. Then constructed a knockout vector. The knockout vector introduced into Streptomyces by polyethylene glycol-mediated transformation of protoplasts. Through relaxation training, screened lack of Streptomyces pta-ackA deletion mutant strain AS01 and pfkA1 deletion mutant strain AS02. According to Vitreoscilla hemoglobin gene vgb gene sequence, and codon preference of Streptomyces design PCR primers, the part of the vgb gene mutation in codon to be. Vgb gene fragment was then cloned into plasmid pIB139, the downstream of the strong promoter. Integration vector pIB139-vgb was constructed. And the integration vector was introduced into Streptomyces through protoplast transformation. Streptomyces AS03 with vgb expression was selected through antibiotic resistance.Construction of genetic engineering Streptomyces laid a very important change in the basis to study gene function, and metabolic networks in Streptomyces.