Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein-protein interactions. Many different methods are being applied, each with strengths and weaknesses. We will present and evaluate several techniques that have been applied in providing evidence for the orchestration of the biosynthetic pathways of cyanogenic glucosides and glucosinolates in metabolons. These evolutionarily related pathways have ER-localized cytochromes P450 that are proposed to function as anchoring site for assembly of the enzymes into metabolons. Additionally, we have included commonly used techniques, even though they have not been used (yet) on these two pathways. In the review, special attention will be given to less-exploited fluorescence-based methods such as FCS and FLIM. Ultimately, understanding the orchestration of biosynthetic pathways may contribute to successful engineering in heterologous hosts.

中文翻译：

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如何证明代谢产物的存在？

生物合成途径中的顺序酶组织在代谢产物中。由于生物合成途径是由高度动态的蛋白质-蛋白质相互作用组成的，因此提供代谢激素存在的实验证据具有挑战性。正在应用许多不同的方法，每种方法都有其优点和缺点。我们将介绍和评估几种技术，这些技术已被用于为代谢产物中的氰基葡萄糖苷和芥子油苷的生物合成途径提供协调。这些进化相关的途径具有ER-定位的细胞色素P450，其被提议用作将酶组装成代谢产物的锚定位点。此外，即使尚未在这两个途径上使用它们，我们也包含了常用的技术。在评论中 将特别关注未开发的基于荧光的方法，例如FCS和FLIM。最终，了解生物合成途径的编排可能有助于异源宿主的成功工程化。