Forestier Edith C F, Brown Geoffrey D, Harvey David, Larson Tony R, Graham Ian A
Department of Biology, Centre for Novel Agricultural Products, University of York, York, United Kingdom.
Department of Chemistry, University of Reading, Reading, United Kingdom.
Front Plant Sci. 2021 Oct 20;12:757186. doi: 10.3389/fpls.2021.757186. eCollection 2021.
Diterpene biosynthesis commonly originates with the methylerythritol phosphate (MEP) pathway in chloroplasts, leading to the C substrate, geranylgeranyl pyrophosphate (GGPP). The previous work demonstrated that over-expression of genes responsible for the first and last steps in the MEP pathway in combination with () and () is optimal for increasing flux through to casbene in . When the gene responsible for the last step in the MEP pathway, (), is removed from this combination, casbene is still produced but at lower amounts. Here, we report the unexpected finding that this reduced gene combination also results in the production of 16-hydroxy-casbene (16-OH-casbene), consistent with the presence of 16-hydroxy-geranylgeranyl phosphate (16-OH-GGPP) in the same material. Indirect evidence suggests the latter is formed as a result of elevated levels of 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) caused by a bottleneck at the HDR step responsible for conversion of HMBPP to dimethylallyl pyrophosphate (DMAPP). Over-expression of a from produces 16-hydroxy-geranyllinalool (16-OH-geranyllinalool) when transiently expressed with the same reduced combination of MEP pathway genes in . This work highlights the importance of pathway flux control in metabolic pathway engineering and the possibility of increasing terpene diversity through synthetic biology.
二萜生物合成通常起始于叶绿体中的甲基赤藓糖醇磷酸（MEP）途径，生成C底物香叶基香叶基焦磷酸（GGPP）。先前的研究表明，在[具体植物名称]中，MEP途径第一步和最后一步相关基因与[基因1]和[基因2]组合过表达，对于增加通向卡斯贝烯的通量最为理想。当从该组合中去除MEP途径最后一步相关基因[基因名称]时，仍会产生卡斯贝烯，但产量较低。在此，我们报告了一个意外发现，即这种基因组合减少的情况还会导致16-羟基卡斯贝烯（16-OH-卡斯贝烯）的产生，这与同一材料中存在16-羟基香叶基香叶基磷酸（16-OH-GGPP）一致。间接证据表明，后者是由于负责将4-羟基-3-甲基-丁-2-烯基焦磷酸（HMBPP）转化为二甲基烯丙基焦磷酸（DMAPP）的HDR步骤出现瓶颈，导致HMBPP水平升高而形成的。当[植物名称]的[基因名称]与[具体植物名称]中MEP途径基因相同的减少组合瞬时表达时，会产生16-羟基香叶基芳樟醇（16-OH-香叶基芳樟醇）。这项工作突出了代谢途径工程中途径通量控制的重要性，以及通过合成生物学增加萜类多样性的可能性。
