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Background  Indigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in  Baphicacanthus cusia . Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown.    Results  One gene encoding EPSPS was isolated from  B. cusia . Quantitative real-time PCR analysis revealed that  BcEPSPS  was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that  BcEPSPS  is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed  BcEPSPS  protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of  BcEPSPS  in  Isatis indigotica  hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin.    Conclusions  The function of  BcEPSPS  in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of  BcEPSPS  on active compound biosynthesis in  B. cusia .

Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia