Celedon J M, Bohlmann J
Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
Methods Enzymol. 2016;576:47-67. doi: 10.1016/bs.mie.2016.03.008. Epub 2016 Apr 13.
Terpenoid fragrances are powerful mediators of ecological interactions in nature and have a long history of traditional and modern industrial applications. Plants produce a great diversity of fragrant terpenoid metabolites, which make them a superb source of biosynthetic genes and enzymes. Advances in fragrance gene discovery have enabled new approaches in synthetic biology of high-value speciality molecules toward applications in the fragrance and flavor, food and beverage, cosmetics, and other industries. Rapid developments in transcriptome and genome sequencing of nonmodel plant species have accelerated the discovery of fragrance biosynthetic pathways. In parallel, advances in metabolic engineering of microbial and plant systems have established platforms for synthetic biology applications of some of the thousands of plant genes that underlie fragrance diversity. While many fragrance molecules (eg, simple monoterpenes) are abundant in readily renewable plant materials, some highly valuable fragrant terpenoids (eg, santalols, ambroxides) are rare in nature and interesting targets for synthetic biology. As a representative example for genomics/transcriptomics enabled gene and enzyme discovery, we describe a strategy used successfully for elucidation of a complete fragrance biosynthetic pathway in sandalwood (Santalum album) and its reconstruction in yeast (Saccharomyces cerevisiae). We address questions related to the discovery of specific genes within large gene families and recovery of rare gene transcripts that are selectively expressed in recalcitrant tissues. To substantiate the validity of the approaches, we describe the combination of methods used in the gene and enzyme discovery of a cytochrome P450 in the fragrant heartwood of tropical sandalwood, responsible for the fragrance defining, final step in the biosynthesis of (Z)-santalols.
萜类香料是自然界生态相互作用的强大介质，在传统和现代工业应用中有着悠久的历史。植物产生种类繁多的芳香萜类代谢产物，这使它们成为生物合成基因和酶的绝佳来源。香料基因发现的进展为高价值特殊分子的合成生物学开辟了新途径，可应用于香料香精、食品饮料、化妆品及其他行业。非模式植物物种转录组和基因组测序的快速发展加速了香料生物合成途径的发现。与此同时，微生物和植物系统代谢工程的进展为基于数千种构成香料多样性基础的植物基因的合成生物学应用建立了平台。虽然许多香料分子（如简单单萜）在易于再生的植物材料中含量丰富，但一些高价值的芳香萜类化合物（如檀香醇、龙涎醚）在自然界中却很罕见，是合成生物学感兴趣的目标。作为基因组学/转录组学助力基因和酶发现的一个代表性例子，我们描述了一种成功用于阐明檀香（檀香属檀香木）完整香料生物合成途径并在酵母（酿酒酵母）中进行重建的策略。我们探讨了与在大基因家族中发现特定基因以及回收在顽固组织中选择性表达的稀有基因转录本相关的问题。为了证实这些方法的有效性，我们描述了在热带檀香木芳香心材中发现一种细胞色素P450的基因和酶时所使用的方法组合，该细胞色素P450负责（Z）-檀香醇生物合成中定义香味的最后一步。
