An Integrative Volatile Terpenoid Profiling and Transcriptomics Analysis for Gene Mining and Functional Characterization of AvBPPS and AvPS Involved in the Monoterpenoid Biosynthesis in Amomum villosum

Abstract

Amomum villosum, also known as Fructus Amomi, has been used to treat digestive diseases such as abdominal pain, vomiting, and dysentery. Volatile terpenoids are the active metabolites in the essential oil of Fructus Amomi. Nevertheless, downstream genes responsible for activating metabolites biosynthesis in A. villosum still remain unclear. Here, we report the use of an integrative volatile terpenoid profiling and transcriptomics analysis for mining the corresponding genes involved in volatile terpenoid biosynthesis. Ten terpene synthase (TPS) genes were discovered, and two of them were cloned and functionally characterized. AvTPS1 (AvPS: pinene synthase) catalyzed GPP to form α-pinene and β-pinene; AvTPS3 (AvBPPS: bornyl diphosphate synthase) produced bornyl diphosphate as major product and the other three monoterpenoids as minor products. Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggested that AvPS might play a role in biotic defense. On the other hand, the most active ingredient, bornyl acetate, was highly accumulated in seeds and was consistent with the high expression of AvBPPS, which further indicated that AvBPPS is responsible for the biosynthesis of bornyl acetate, the final metabolite of bornyl diphosphate in A. villosum. This study can be used to improve the quality of A. villosum through metabolic engineering, and for the sustainable production of bornyl acetate in heterologous hosts.

Bornyl Diphosphate Synthase From Cinnamomum burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast

“…In this study, we identified a (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii that catalyzed GPP to form (+)-borneol under the hydrolysis of CIAP. This is the first time an enzyme related to (+)-borneol synthesis was mined from C. burmannii , and it has the highest specificity for (+)-borneol production ( Wise et al, 1998 ; Despinasse et al, 2017 ; Hurd et al, 2017 ; Wang et al, 2018 ). The K m value of CbTPS1 (5.11 μM) for GPP is consistent with SBS (3.0 μM) ( Wise et al, 1998 ) and slightly lower than other reported monoterpene synthases (13.10–26.12 μM), which indicated CbTPS1 had a higher affinity for GPP.…”

“…It was classified into the TPS-b subfamily, which contains three motifs of typical terpene synthases, namely the RRX 8 W motif responsible for monoterpenoid cyclization ( Chen et al, 2011 ); and the DDXXD and NSE/DTE motifs in the C-terminal domain, which are responsible for metal-dependent ionization and substrate binding ( Chen et al, 2011 ). Homologous alignment analysis showed that CbTPS1 shared highest sequence identities with SBS (41.75%) from Salvia officinalis ( Wise et al, 1998 ; Figure 3 ), followed by AvBPPS (40.10%) from Amomum villosum ( Wang et al, 2018 ), LaBPPS (38.68%) from Lavandula angustifolia ( Despinasse et al, 2017 ), and LdBPPS (36.20%) from Lippia dulcis ( Hurd et al, 2017 ).…”

“…It catalyzes the universal precursor GPP to form (+)-bornyl diphosphate, and is then dephosphorylated to produce the target product (+)-borneol ( Figure 1 ). BPPSs have been identified from several plants, including Salvia officinalis (SBS), Lavandula angustifolia (LaBPPS), Lippia dulcis (LdBPPS), and Amomum villosum (AvBPPS) ( Wise et al, 1998 ; Despinasse et al, 2017 ; Hurd et al, 2017 ; Wang et al, 2018 ). However, all these enzymes produced multiple products, such as α-pinene, β-pinene, camphene, and limonene, with the largest amount of (+)-borneol produced by SBS, accounting for 57.8% of the total products.…”

Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes

“…BPP is then dephosphorylated to form borneol ( Fig. 1B) (Wise et al, 1998;Despinasse et al, 2017;Hurd, Kwon & Ro, 2017;Wang et al, 2018). The BPPS gene has been functionally characterized in four species, Salvia officinalis, Lavandula angustifolia, Lippia dulcis and Amomum villosum.…”

Peer Review #2 of "Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes (v0.1)"

“…BPP is then dephosphorylated to form borneol (Fig. 1b) (Croteau 1998;Despinasse et al 2017;Hurd et al 2017;Wang et al 2018). The BPPS gene has been functionally characterized in four species, Salvia officinalis, Lavandula angustifolia, Lippia dulcis and Amomum villosum.…”