Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Abstract

Summary Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large‐scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm‐specific TPS‐d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14‐fold to 352 mg/L, bringing production to levels with industrial potential.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

“…Farnesyl pyrophosphate (FPP) was synthesized by the FPP synthase IspA from E. coli. FPP, in turn, is converted to (ϩ)-valencene via the (ϩ)-valencene synthase CnVS from Callitropsis nootkatensis (35). In the newly constructed (ϩ)-valencene producer strain VLC4, the CnVS-encoding gene was expressed after its adaptation to the codon usage of C. glutamicum.…”

“…These values are in the same range as those obtained with other bacteria and higher than those obtained with eukaryotic microorganisms. To the best of our knowledge, the highest titers and volumetric (ϩ)-valencene productivities so far were described for Rhodobacter sphaeroides expressing the mevalonate operon from Paracoccus zeaxanthinifaciens and the codon-optimized (ϩ)-valencene synthase CnVS, which reached a titer of 352 mg · liter Ϫ1 and a productivity of 4.88 mg · liter Ϫ1 · h Ϫ1 (35). The yeasts Saccharomyces cerevisiae (titer, 1.36 mg · liter Ϫ1 ; productivity, 18 g · liter Ϫ1 · h Ϫ1 ), Schizophyllum commune (titer, 16.6 mg · liter Ϫ1 ; productivity, 115 g · liter Ϫ1 · h Ϫ1 ), and Pichia pastoris (titer, 51 mg · liter Ϫ1 ; productivity, 1 mg · liter Ϫ1 · h Ϫ1 ), however, proved suitable for the in situ conversion of (ϩ)-valencene to more valuable products, such as (ϩ)-nootkatone (35,54,55).…”

“…In a recent study, for instance, the IPTGinduced production of (ϩ)-valencene, a natural constituent of the essential oils of citrus fruits, faced serious growth impairment in C. glutamicum (34). Here, only moderate production could be obtained of this bicyclic sesquiterpenoid, which is used as an additive by the food and beverage industry, due to its pleasant orange-like odor (35,36).…”

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

“…Applications for terpenoids include medicine (Gershenzon and Dudareva, 2007;Nosten and White, 2007), natural flavouring (Beekwilder et al, 2014), platform chemicals and perfumes (Alonso-Gutierrez et al, 2013), next generation biofuel compounds (Peralta-Yahya et al, 2011;Wang et al, 2011), cosmetics (McVean and Liebler, 1999), animal feed (Johnson et al, 1980), as well as food colorants (Baker and Günther, 2004).…”

A process economic assessment of hydrocarbon biofuels production using chemoautotrophic organisms

“…The current bulk price of squalene is $15-20/ kg, which translates to about $2027-2702/bbl-fuel. Other isoprenoids, such as lycopene, valencene, beta-carotene, etc., are also high value products whose production are demonstrated in nonnative hosts (Alper et al, 2005;Beekwilder et al, 2014;Verwaal et al, 2007). Wax ester (a health product commonly sold as Jojoba oil) is a fatty ester for which we have also demonstrated production in Rba.…”