Study On The Preparation Of Heterologous Expressed Blue Pigment-Indigoidine By Escherichia Coli

Indigoidine is a nontoxic microbial blue pigment,which has the ability of scavenging free radicals.It can be used as pigment and dye in food and printing and dyeing industry,to some extent,it can alleviate the problem of the scarcity of natural blue pigment in the market.Indigoidine was produced by microbial fermentation.Indigoidine with glutamine as precursor,which is a high content of amino acid in the cell,so there is no need to add high cost precursor.Based on E.coli-Staphylococci shuttle plasmid pBT2-ET-5R-EGFP,a recombinant E.coli engineering strain E-30s was constructed,which contained the indigoidine synthetase gene(idgS)from Streptomyces lavendulae and the 4 '-phosphopanthiylthioglycosyl transferase gene(sfp)from Bacillus subtilis.The fermentation medium and culture conditions of indigoidine were optimized.Under the same conditions,E.coli heterologous expression of indigoidine and commercial indigo were tested for pigment stability,and then indigoidine pigment was compared and evaluated.The specific research contents are as follows:(1)Removing the Tat signal peptide of plasmid pBT2-ET-5R-EGFP to construct the plasmid pBT2-5R-EGFP.And then use gene idgS-sfp replace 5R-EGFP of pBT2-5R-EGFP to construct the recombinant expression plasmid p30s and recombinant E.coli engineering strain E-30s.(2)LB medium was used as seed medium and initial fermentation medium.According to single factor experiment and orthogonal experiment,the optimum fermentation medium was decided.Indigoidine fermentation medium(g/L):peptone 5,yeast extract 10,glycerin 12.28,Na2HPO4·12H2O 9.55,KH2PO4 1.81,NaCl 4,HCOOK 5,MSG 10,MgSO4·7H2O 0.74.(3)Under the original variable temperature culture conditions,the final fermentation conditions were optimized by single factor experiment and orthogonal experiment,it adopts two-stage temperature and speed control mode,the final fermentation conditions were as follow:initial pH 7.5,inoculation volume 75mL,inoculability quantity 1%,the temperature of bacteria growth was 34? culture 4h change to 25?,the shaking speed of bacteria growth was 180 r/min culture 4h change to 240 r/min.Compared with the initial fermentation conditions.(4)Using N,N-dimethylformamide as solvent,the effects of light,UV,pH,temperature,oxidant,reductant and metal ion on the stability of indigoidine and indigo.Indigoidine exhibits much better overall stability than indigo,suggesting a broader prospect of development and application.