Constitutive Production Of Salvianic Acid A In Escherichia Coli And Separation

Salvianic acid A(SAA), an important natural phenolic acid, has wide applications in food and pharmaceutical industries. SAA is currently isolated from dried root of Chinese traditional herb salvia miltiorrhiza. The application of SAA is restricted because of its difficult extraction and the low amount in crude root. Now production of SAA by microorganism fermentation is becoming an important direction. In this paper, we engineered Escherichia coli to constitutively produce SAA and separated from fermentation broth.In this study, seven different strength promoters(lacUV5, trc, tac, gap, gaptb, p342 and p46) were chosen to regulate the expression of hpaBC and dldh. Through over-lap PCR, and restriction enzyme digestion and ligation, 13 different expression combinations of hpaBC and dldh were constructed. Then using ?-red homologous recombination technology, 13 expression combinations were integrated into chromosome of BKT5, and 13 different recombinant strains of BCL1 to BCL13 were obtained. The fermentation results in Erlenmeyer flasks showed that strain BCL13 produced the highest SAA with titer of 618mg/L, however strain BCL12 produced the lowest SAA with titer of 45mg/L. Changing the promoters of hpaBC and dldh led to SAA production of varied 13 folds.The optimum resin was screened from ten types of macroporous resins by static and dynamic absorption/desorption experiments and the major process parameters were also investigated. Adsorption kinetics curves and adsorption isotherms curves were used to describe adsorption behavior of SAA on macroporous resins. The best type macroporous resin was X5 resin and the adsorption capacity and desorption capacity of SAA were 10.66±0.54 mg/g dry resin and 9.13±0.47 mg/g dry resin, respectively. The purity and recovery yield of SAA in the final dry product were 90.2±1.5% and 81.5±23%, respectively.Constitutive production of SAA in engineered E. coli and efficient separation of SAA from fermentation broth would promote its applictions in food and pharmaceutical industries.