Integrative analysis of the shikonin metabolic network identifies new gene connections and reveals evolutionary insight into shikonin biosynthesis

Abstract

SummaryPlant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shiknoin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.

Transcriptional dynamics of Chitinophaga sp. strain R-73072-mediated alkannin/shikonin biosynthesis in Lithospermum officinale

“…Similarly, at the pathway level, genes involved in “phenylpropanoid biosynthesis, and ubiquinone” and “other terpenoid-quinone biosynthesis” were enriched among DEG ( Figure 3B ). Overrepresentation of genes associated with the aforementioned KEGG or GO terms is noteworthy because they encompass genes that are directly connected to A/S production in L. officinale and other Boraginaceae ( Suttiyut et al, 2022 ).…”

“…Alkannin, its enantiomer shikonin, and its plethora of derivatives (A/S) are pharmaceutically bioactive SMs mainly produced in the root periderm of Boraginaceae ( Yazaki, 2017 ), and the A/S biosynthesis pathway has been already extensively studied in different Boraginaceae species including Lithospermum officinale ( Rai et al, 2018 ; Ahmad et al, 2022 ), L. erythrorhizon ( Yazaki, 2017 ; Takanashi et al, 2019 ; Auber et al, 2020 ; Song et al, 2020 , 2021 ; Suttiyut et al, 2022 ), Echium plantagineum ( Tang et al, 2020 ), and Arnebia euchroma ( Wang et al, 2019 ). Because of their interesting pharmaceutical properties and significant commercial value ( Papageorgiou et al, 1999 , 2008 ), there has been increasing interest in enhancing their production, e.g., through boosting its biosynthetic pathway, also by using beneficial microbes ( Rat et al, 2021 ).…”