Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis)

Abstract

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…This indicates that geranylgeraniol can be produced in the absence of the nerolidol synthase, but that the nerolidol synthase also has geranylgeraniol synthase activity. This is surprising considering that exactly the same protein sequence was used as in the previous study, where linalool and geranyllinalool, but not geranylgeraniol, were reported (Green et al, 2012). We presume that differences in in vitro conditions used previously, compared to our in vivo experiment, are responsible for this discrepancy.…”

“…Plasmid construction processes are listed in Supplementary Materials: Table S2. The genes encoding trans-nerolidol synthase (AcNES1; GenBank-KX064240) from Actinidia chinensis (Green et al, 2012), and 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) synthase (EfmvaS; GenBank-KX064238) and bi-functional acetoacetyl-CoA thiolase /HMG-CoA reductase (EfmvaE; GenBank-KX064239) from E. faecalis (Wilding et al, 2000) were synthesized by GenScript (Piscataway, NJ, USA) with codon optimization according to the codon bias of S. cerevisiae (Supplementary Materials: Table S3). .…”

“…S1). AcNES1 is known to have additional linalool/geranyllinalool synthase activities (Green et al, 2012); however, geraniol, linalool, farnesol, and geranyllinalool, were all below the limit of dectection in strain N6 (Supplementary Materials: Fig. S1).…”

Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple

“…Details of the primers are given in Supplemental Table S8. Recombinant TPS enzymes were expressed under autoinducible conditions and purified by immobilized affinity metal chromatography according to previous methods (Green et al, 2007(Green et al, , 2012b. Solvent extraction assays for terpene identification and enantiomeric determinations were carried out as described previously (Green et al, 2012b).…”

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering

“…To date, numerous flower-specific terpene synthases have been characterized and isolated, as they are accountable for the development of monoterpene linalool (A. thaliana, Anthirrhinum majus, H. coronarium and Clarkia breweri) (Nagegowda et al 2008;Ginglinger et al 2013;Yue et al 2014), 1,8-cineole (Citrus unshiu, H. coronarium and Nicotiana suaveolens) (Roeder et al 2007;Li and Fan 2007), myrcene (Alstroemeria peruviana and A. majus) (Aros et al 2012), E-(β)-ocimene (A. majus and H. coronarium) (Shimada et al 2005;Fan et al 2003Fan et al , 2007 and the sesquiterpenes α-farnesene (Actinidia deliciosa and H. coronarium) (Nieuwenhuizen et al 2009;Yue et al 2015), nerolidol (A. chinensis and A. majus) (Green et al 2011), valencene (Vitis vinifera) (Lücker et al 2004), germacrene D (V. vinifera, Rosa hybrid and A. deliciosa) (Lücker et al 2004;Nieuwenhuizen et al 2009) β-ylangene, β-copaene, β-cubebene, α-bergamotene (Cananga odorata var. fruticosa) (Jin et al 2015) and β-caryophyllene (Ocimum kilimandscharicum, Daucus carota) (Yahyaa et al 2015;Jayaramaiah et al 2016).…”