| Summary: | The BAHD acyltransferase family is a unique class of plant proteins that acylates plant metabolites and participates in plant secondary metabolic processes. However, the BAHD members in <i>Lithospermum erythrorhizon</i> remain unknown and uncharacterized. Although the heterologously expressed <i>L. erythrorhizon</i> BAHD family member LeSAT1 in <i>Escherichia coli</i> has been shown to catalyze the conversion of shikonin to acetylshikonin in vitro, its in vivo role remains unknown. In this study, the characterization, evolution, expression patterns, and gene function of <i>LeBAHDs</i> in <i>L. erythrorhizon</i> were explored by bioinformatics and transgenic analysis. We totally identified 73 <i>LeBAHDs</i> in the reference genome of <i>L. erythrorhizon</i>. All <i>LeBAHDs</i> were phylogenetically classified into five clades likely to perform different functions, and were mainly expanded by dispersed and WGD/segmental duplication. The in vivo functional investigation of the key member <i>LeBAHD1/LeSAT1</i> revealed that overexpression of <i>LeBAHD1</i> in hairy roots significantly increased the content of acetylshikonin as well as the conversion rate of shikonin to acetylshikonin, whereas the CRISPR/Cas9-based knockout of <i>LeBAHD1</i> in hairy roots displayed the opposite trend. Our results not only confirm the in vivo function of <i>LeBAHD1/LeSAT1</i> in the biosynthesis of acetylshikonin, but also provide new insights for the biosynthetic pathway of shikonin and its derivatives.
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