Isolation And Identification Of High Pigment-producing Gluconobacter Oxydans FBFS 97 And Analysis Of The Pigment Synthesis Pathways Based On Its Genome

Acetic acid bacteria(AAB)are a large class of important bacteria,which have unique incomplete oxidization characteristics,can incompletely oxidize alcohols,sugars and sugar alcohols to yield the relative products,which are used in the fields of medicine and food.For example,AAB can produce the key intermediate of the antidiabetic drug Miglitol(a glucosidase inhibitor),6-deoxy-6-amino(N-hydroxyethyl)-?-1-furanasorbose,the precursor of vitamin C,2-keto-L-gulonic acid,as well as dihydroxyacetone,acetic acid,gluconic acid and sorbitol.Many AAB strains can also produce pigments,but their composition and biosynthesis pathways are not clear.In this study,firstly a high yield pigment strain of Gluconobacter oxydans FBFS 97 was isolated and identified.Then its genome was sequenced and analyzed,and the genes related to melanin,riboflavin and porphyrins were found.Finally,the physicochemical properties of melanin and the medium composition to produce riboflavin were investigated.1.Isolation and identification of a high-yield-pigment strain G.oxydans FBFS 97Using GYC(Glucose,yeast extract and CaCO₃)medium,four strains of AAB were selected as high-yield pigment strains of AAB.Among them,two AAB strains were screened from AAB strains preserved in our laboratory and named FBFS 97 and FBFS 82,respectively,both can produce brown pigment.The other two strains were isolated from flowers and fruits samples and named FBFS SE and FBFS S18 respectively,both can produce pink pigment.According to their morphological,biochemical and physiological characteristics,they belong to the genera of Gluconobacter and Acetobacter,respectively.Subsequently,based on the sequence analysis of 16S rRNA,FBFS 97 and FBFS 82 were identified as G.oxydans,while FBFS SE and FBFS S18 were identified as A.tropicalis and A.indonesiensis,respectively.FBFS 97 can produce a huge amount of extracellular brown pigment,so in the following experiments,the strain will be further investigated.2.Genome sequencing of FBFS 97 and analysis of genes related to pigment productionThe results of genome sequencing analysis have shown that the genome size of FBFS97 is about 4.0Mb and a G+C content in the genome is 66.6%.The genome encodes 3500genes of which 2752 genes with function prediction and 748 genes with unknown functions.Further analysis has revealed that the genome of FBFS 97 contains the genes related to the biosynthesis pathways of melanin,riboflavin and porphyrins.The results of UHPLC-MS/MS determination prove that the three above mentioned kinds of pigments exactly existed in the fermentation broth of FBFS 97.Due to the high contents of melanin and riboflavin in the broth,these two pigments were further researched.3.Characterization of physicochemical properties of melanin produced by FBFS 97After 14 days of culture at 28?in the GY(Glucose and yeast extract)liquid-state medium,FBFS97 could produce the maximum production of melanin,up to about 12-15mg/L.The extracted melanin can not be dissolved in water and most organic solvents,but be dissolved in 1 M Na OH or 1 M KOH.It is acid-resistant and can be decolorized by oxidants.The extracted melanin was confirmed to be exact melanin by ultraviolet-visible spectrophotometry,Fourier-transformed infrared spectroscopy,thin layer chromatography,element analysis,differential scanning calorimetry and scanning electron micrograph.The IC₅₀ value of the extracted melanin for scavenging 50%DPPH radical was 36.94?g/m L,and the IC₅₀ value of antioxidant activity for ABTS was 4.06?g/m L.4.Optimization of the medium composition for riboflavin production by FBFS 97FBFS 97 was cultured in GY liquid-state medium at 28?and riboflavin production was monitored from 0 to 72h.The results showed that riboflavin began to produce at about24 h.The riboflavin production increased significantly at about 32 h.At 64 h,riboflavin production was the highest,which was 0.33 mg/L,after that,riboflavin production began to decline.In order to improve the production of riboflavin,Plackett Burman design and central composite design were used to optimize the medium composition for riboflavin production by FBFS 97.Finally,the optimal medium for riboflavin production by FBFS 97 was obtained as follows:fructose 25 g/L,tryptone 12.5 g/L,K₂HPO₄ 9 g/L,CaCl₂ 0.06 g/L.In this medium,the riboflavin produced by FBFS 97 was as high as 23.24 mg/L.