Molecular cloning and functional characterization of the shikimate kinase gene from Baphicacanthus cusia

<i>Baphicacanthus cusia</i> (Nee) Bremek, a perennial herbaceous plant with medicinal properties, has limited genomic insights regarding the genes involved in its indole alkaloid biosynthesis pathway. In this study, the <i>BcSK</i> gene was isolated and cloned from the transcriptome data of <i>B. cusia</i>. The full-length cDNA of <i>BcSK</i> is 1,657 bp, comprising a 265 bp 5' UTR, a 507 bp 3' UTR, and an 885 bp ORF encoding 295 amino acids. The exon-intron structure of <i>BcSK</i> consists of four exons and three introns. Bioinformatics and phylogenetic analyses revealed a high degree of homology between <i>BcSK</i> and its counterparts in various plant species. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that <i>BcSK</i> expression was significantly altered under abiotic stress conditions, including methyl jasmonate (MeJA), abscisic acid (ABA), and ultraviolet (UV) radiation. The gene was predominantly expressed in flowers compared to roots, stems, and leaves. Subcellular localization analysis indicated that <i>BcSK</i> is primarily expressed in chloroplasts, confirming that the conversion of shikimic acid to shikimate-3-phosphate occurs in this organelle. Prokaryotic expression and enzyme activity assays demonstrated that the heterologously expressed <i>BcSK</i> protein catalyzed the conversion of shikimic acid to shikimate-3-phosphate. Furthermore, the ectopic overexpression of <i>BcSK</i> in <i>Isatis indigotica</i> significantly enhanced the biosynthetic flux toward indole alkaloids, including indole, indigo, and indirubin. In conclusion, this study identifies and characterizes a novel <i>BcSK</i> gene, providing new insights and potential applications for the metabolic engineering of <i>B. cusia</i>.