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

Lithospermum erythrorhizon (red gromwell; zicao) is a medicinal and economically valuable plant belonging to the Boraginaceae family. Roots from L. erythrorhizon have been used for centuries based on the antiviral and wound-healing properties produced from the bioactive compound shikonin and its derivatives. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated from L. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly for L. erythrorhizon using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using the L. erythrorhizon genome, we identified several additional p-hydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkannin-producing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression of LePGT1 in L. erythrorhizon hairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway.

The origin and biosynthesis of the naphthalenoid moiety of juglone in black walnut.

Several members of the Juglandaceae family produce juglone, a specialized 1,4-naphthoquinone (1,4-NQ) natural product that is responsible for the notorious allelopathic effects of black walnut (Juglans nigra). Despite its documented ecological roles and potential for being developed as a novel natural product-based herbicide, none of the genes involved in synthesizing juglone have been identified. Based on classical labeling studies, we hypothesized that biosynthesis of juglone's naphthalenoid moiety is shared with biochemical steps of the phylloquinone pathway. Here, using comparative transcriptomics in combination with targeted metabolic profiling of 1,4-NQs in various black walnut organs, we provide evidence that phylloquinone pathway genes involved in 1,4-dihydroxynaphthoic acid (DHNA) formation are expressed in roots for synthesis of a compound other than phylloquinone. Feeding experiments using axenic black walnut root cultures revealed that stable isotopically labeled l-glutamate incorporates into juglone resulting in the same mass shift as that expected for labeling of the quinone ring in phylloquinone. Taken together, these results indicate that in planta, an intermediate from the phylloquinone pathway provides the naphthalenoid moiety of juglone. Moreover, this work shows that juglone can be de novo synthesized in roots without the contribution of immediate precursors translocated from aerial tissues. The present study illuminates all genes involved in synthesizing the juglone naphthoquinone ring and provides RNA-sequencing datasets that can be used with functional screening studies to elucidate the remaining juglone pathway genes. Translation of the generated knowledge is expected to inform future metabolic engineering strategies for harnessing juglone as a novel natural product-based herbicide.