Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway

Abstract

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon

“…10% of shikonin is produced as ester derivatives, which are secreted by cells, and plant masses can be easily enlarged in liquid suspension cultures 6 . Our multiple omics study identified 10 genes as being strongly involved in shikonin biosynthesis and 16 as being strongly involved in shikonin secretion 9 . The VIGS methodology described in this study will likely be utilized for functional analysis of these genes to determine the entire shikonin biosynthetic pathway and to understand mechanisms underlying its secretion.…”

“…Shikonin is a meroterpene synthesized via two independent biosynthetic pathways, a common phenylpropanoid pathway and an isopreniod pathway 5 . Although several enzymes and genes involved in the initial steps of shikonin biosynthesis have been identified to date [6][7][8][9][10] , genes and proteins involved in later steps have not yet been identified, especially those involved in the crucial steps required to form the naphthalene ring.…”

“…To identify candidate genes involved in these later steps of shikonin biosynthesis, we performed large-scale transcriptome and comparative proteome analyses, based on results showing that shikonin production can be strictly regulated by many chemical and physical factors 9 . For example, shikonin production is strongly inhibited by ammonium ion and illumination, but is markedly enhanced in M9 medium.…”

“…Shikonin production is also tissue specific, being produced solely in the epidermis, but not in other root tissues, such as inside the central cylinder 6 . These characteristics enabled the identification of more than 10 putative candidate genes involved in shikonin biosynthesis 9 and more than 16 putative candidate genes involved in shikonin secretion from cells 11 .…”

Hybrid de novo genome assembly of red gromwell (Lithospermum erythrorhizon) reveals evolutionary insight into shikonin biosynthesis

“…Current efforts have extended to include synthetic chemistry for producing shikonin, alkannin, and derivatives with higher specificity and potency 12,13 . Moreover, the use of comparative transcriptomics (e.g., [14][15][16] ) and proteomics 17 approaches for elucidating the shikonin/alkannin pathway has uncovered several gene candidates and led to the identification of the geranylhydroquinone hydroxylase (GHQH; CYP76B74) 18 . Comparatively less attention has focused on the evolutionary origin of shikonin/alkannin pathway genes 16,19 and on the ecological significance of producing the compounds 3 .…”