Abstract
The characterization of plant enzymes by expression in prokaryotic and eukaryotic (yeast and plants) heterologous hosts has widely been used in recent decades to elucidate metabolic pathways in plant secondary metabolism. Yeast and plant systems provide the cellular environment of a eukaryotic cell and the subcellular compartmentalization necessary to facilitate enzyme function. The expression of candidate genes in these cell systems and the identification of the resulting products guide the way for the identification of enzymes with new functions. However, in many cases, the detected compounds are not the direct enzyme products but are caused by unspecific subsequent reactions. Even if the mechanisms for these unspecific reactions are in many cases widely reported, there is a lack of overview of potential reactions that may occur to provide a guideline for researchers working on the characterization of new enzymes. Here, an across-the-board summary of rearrangement reactions of sesquiterpenes in metabolic pathway engineering is presented. The different kinds of unspecific reactions as well as their chemical and cellular background are explained and strategies how to spot and how to avoid these unspecific reactions are given. Also, a systematic approach of classification of unspecific reactions is introduced. It is hoped that this mini-review will stimulate a discussion on how to systematically classify unspecific reactions in metabolic engineering and to expand this approach to other classes of plant secondary metabolites.
Highlights
The sesquiterpenes (ST) are a subgroup of the terpenes with a C15 backbone comprised of three isoprene units; sesquiterpenes with a lactone moiety are called sesquiterpene lactones (STL).The elucidation and metabolic engineering of their biosynthetic pathways have made significant progress in the past decades, as many sesquiterpenes are of commercial interest as fragrances [1], biodiesel [2]or pharmaceuticals [3,4]
Acidic conditions are known to induce transannular cyclization in germacrenes (Figure 1b), which can lead to a great number of rearrangement products, mostly with the C10 ring of a germacrene cyclizing to two C6 rings [22]
Acid-induced rearrangement can occur in solid-phase microextraction gas chromatography (SPME-GC) analysis which was shown for the analysis of germacrene D [32] or when acidified solvents are used in high performance liquid chromatography (HPLC) analysis [10]
Summary
The sesquiterpenes (ST) are a subgroup of the terpenes with a C15 backbone comprised of three isoprene units; sesquiterpenes with a lactone moiety are called sesquiterpene lactones (STL). The elucidation and metabolic engineering of their biosynthetic pathways have made significant progress in the past decades, as many sesquiterpenes are of commercial interest as fragrances [1], biodiesel [2]. In the first step of terpene biosynthesis the carbon backbone is formed by a terpene synthase [19]. The intermediate is often modified by cytochrome P450 enzymes that introduce oxygen into the core backbone [20,21]. One of the challenges when expressing biosynthetic enzymes of sesquiterpenoid pathways is the differentiation of the direct enzyme product and artificial products that arise from unspecific subsequent reactions.
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