Biosynthesis Of Indigo By Cupriavidus Sp.IDO And Its Functional Genes

Indigo exhibits excellent fixation and lightfastness,thus it attracts the name‘the King of Dyes’.Compared to traditional chemical synthesis,microbial synthesis of indigo possesses the advantages of mild reaction condition,green and safety.Research on bacteria strain capable of indigo synthesis is limited,most of which have focused on Pseudomonas.The associated enzyme resource is mainly aromatic oxygenase,while few attempts have been made to explore the indole oxygenase,which exhibited better selectivity for indigo production.Herein,microbial synthesis of indigo was investigated from the aspects of the screening of indigo-producing bacteria,the optimization of synthesis conditions and the exogenous expression of functional genes.A bacteria strain named IDO was isolated from soil sample,and it was capable of growing and synthesizing indigo in the medium containing indole as the sole source of carbon.Results from 16S rRNA gene sequence analysis identified strain IDO as Cupriavidus sp.Based on HPLC/TOFMS analysis,indigo and indirub were found in the products of biotransformation of indole by strain IDO,then the pathway of biotransformation of indole to indigo was proposed.Strain IDO could produce 25 mg/L indigo from 100 mg/L indole under the optimal environmental conditions:temperature 30°C,rotary speed 150 r/min and pH 7.Most metal ions inhibited indigo production,while Fe³⁺enhanced its yield.Phenol has little influence in indigo production,while pyridine and quinolone deceased its yieald.The functional genes for biosynthesis of indigo by strain IDO were investigated using genome-wide sequencing technology combined with BLAST analysis.The oxygenase and reductase coded by ido1742-ido1743 in strain IDO showed more than 40%similarity with the corresponding components of reported indole oxygenases,and these gene clustes had the same structure.Thus this oxygenase composed by Ido1742 and Ido1743 might be indole oxygenase,and the two components were named IndA and IndB,selectively.Through phylogenetic tree analysis,we found that IndA belonged to a novel enzyme branch.Genes indAB were transferred into Escherichia coli BL21（DE3）（designated as IND_AB）and expressed in the recombinat E.coli.Blue product was formed in tryptophan medium.According to SDS-PAGE,protein IndAB was successfully expressed.The blue pigments synthesized by strain IND_AB using tryptophan as substrate was identified as indigo by HPLC/TOF/MS.The optimal conditions for indigo synthesis by strain IND_AB were:temperature 30°C and induction at time zero with 1 mmol/L IPTG.The optimal medium composition,determined by response surface methodology,contained 9.12 g/L yeast extract,12.20 g/L NaCl,0.54 g/L tryptophan.Under this condition,the yields of indigo reached the peak as 133 mg/L after 16 h.In addition,strain IND_AB were able to produce indigo-like pigments from various indole derivatives such as methylindoles,nitroindoles,chloroindoles.According to the analysis of UV-vis spectra and LC-MS,the major products derived from indoles should be indigo with different substituent groups.