Plant Research International, Wageningen, the Netherlands; Platform Green Synthetic Biology, Delft University of Technology, Delft, the Netherlands.
Plant Biotechnol J. 2014 Feb;12(2):174-82. doi: 10.1111/pbi.12124. Epub 2013 Sep 25.
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.
贝壳杉烯是北美红杉心材中的主要萜烯之一。它是一种氧化的倍半萜烯，据推测它是由柠檬烯衍生而来。柠檬烯和贝壳杉烯都被用于调味柑橘类饮料，被认为是商业规模上使用的最有价值的萜烯之一。通过对北美红杉木材进行大规模 EST 测序获得的假定萜烯合酶基因进行功能评估，发现了一种柠檬烯合酶基因（CnVS）。该基因在树的不同组织中的表达与贝壳杉烯含量密切相关，表明 CnVS 代表了 C. nootkatensis 中贝壳杉烯生物合成途径中的第一个专用基因。该基因属于萜烯合酶的 gymnosperm-specific TPS-d 亚家族，其蛋白序列与已知的柑橘柠檬烯合酶相似度较低。在体外，CnVS 在不同 pH 值和温度条件下表现出较高的稳健性，这是在不同宿主和生理条件下应用的潜在有益特性。生物技术生产倍半萜烯已被证明是可行的，但表达柑橘柠檬烯合酶的微生物菌株的生产力较低，表明需要优化柠檬烯合酶的活性。事实上，CnVS 在酿酒酵母中的表达表明有更高产量的潜力。在优化的 Rhodobacter sphaeroides 菌株中，CnVS 的表达将柠檬烯的产量提高了 14 倍，达到 352mg/L，使产量达到具有工业潜力的水平。
