Research On The Synthetic Metabolism And Regulation Mechanisms Of Indigo In Pseudomonas Putida B4

Blue is a vital class of edible pigment, and natural blue pigment is particularly scarce at present. The annual global demand of pigment is about 800000t, of which the demand of blue pigment is over 80000t. The edible blue pigment allowed for ues by GB7655.1-2005 and GB7655.2-2005 is Brilliant Blue and Brilliant Blue Aluminum Lake, which is called Indigotine (FD&C Blue No.2) in USA. All of them are chemically synthesized.Indigo is an important category of food pigment. Due to the high price of natural extracted indigo and the great toxicity and serious pollution of chemical synthesis indigo for using Aniline, Nitrobenzene and Phthalic Anhydride, the bio-production of indigo has gradually become the focous of researches on natural indigo.In previous work, we isolated an indigo-producing strain and indentified it as Psedudomonas putida B4. We demonstrated that Psedudomonas putida B4 expressed high activities of styrene monooxygenase in indigo fermentation. The styrene monooxygenase was considered to be close related to the ablity of producing bio-indigo. It was confirmed that there were styAB and styC genes in P. putida B4 by bioinformatics analysis.In this work, we constructed the styAB gene over-expressed strain B4-01, the styC gene over-expressed strain B4-02 and sty ABC gene over-expressed strain B4-03. Then, the styAB gene knock-out strain B4-04 and styC gene knock-out strain B4-05 were constructed respectively. According to the results of growth curve, gene expression level, activities of styrene monooxygenase and concentration of indigo in all recombiniant strains, the oxidation of indole was the key point and the styAB gene was the key gene in the whole bio-indigo synthesis. The styAB gene has significant regulating effect on the expression of styC gene. In the meanwhile, there was interaction effect between the styAB and styC genes, since the knock-out of styC gene could brought the decline of styAB expression level and styrene monooxygenase activity.The styAB and styABC gene were prokaryoric expressed in E. coli DH5a respectively to construct the genetically modified strains E. coli AB and E. coli ABC. The fermentation results showed that the styAB gene could endow the recombinant strains the ability of producing indigo as the key of indigo synthesis. By contrast, the expression of styC gene had no effect on the indigo production in the recombinant strains. Then the conditions of indigo fermentation were optimized through the response surface method. The optimized conditions wers substrate concentration of 0.95mg/mL, fermentation temperature of 29℃, initial pH of 6.9. The production of indigo was 67.65mg/mL under the optimized conditions, which was improved about 5.7 folds comparing to the wild type strain P. putida B4.There was a two-component regulating system located the upstream of sty cluster. The expression of regulating system styS and styR gene were both constitutive in P. putida B4, whereas expression of the styAB gene was inducible which was induced by styrene and its analogue indole. The styS was activated by the styrene and indole then promoted the expression of styR gene, then the transcription of styAB gene was motivated. The results revealed that the expression level was closely linked to the regulating effect of styS and styR genes.We obtained the regeneration enhanced system of Coenzyme factor NADH by constructing the maeB gene over-expressed strain in this work. For the enhanced system, the yield of bio-indigo increased about 20% comparing to the wild type strain B4, and the expression level of styAB gene and the activity of styrene monooxygenase was improved correspondingly. Conversely, the expression level of maeB gene and the activity of malic enzyme was enhanced partly when the styAB gene was over-expressed in B4-01. The results showed that there was coordinated regulation between NADH and styAB gene.In this research, we disccussed the biosynthesis mechanisms of indigo on the basis of the differences of indigo concentration, monooxygenase activity and gene expression level in all strains. The key points of bioindigo synthesis and transcription regulating model were confirmed through our research. Furthermore, the coordinated regulation between NADH and indigo biosynthesis was stated as well in this study. This work provided the theoretical basis and foundation of research for the construction of high yield bio-indigo system in future.