背景回顾：Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the color and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the Carotenoid Cleavage Dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. 

主要研究：To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison to CsCCD2L, in citrus callus engineered by Agrobacterium-mediated super-transformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. 

结果1-最优组合：We demonstrate that co-expression of GjCCD4a with phytoene synthase and β-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus.

结果2-GjCCD4a底物与产物：By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β-carotene, in planta, and produced crocetin dialdehyde via C30 β-apocarotenoid intermediate. GjCCD4a also cleaved C27 β-apocarotenoids, providing a new route for C17-dialdehyde biosynthesis. 

结果3-GjCCD4a功能：Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. 

结论：In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.

 摘 要 

藏红花素（crocins）是一类有益的抗氧化剂，并且具有抗癌活性，与藏红花苦素（picrocrocin）一起赋予了香料植物藏红花的颜色和味道。藏红花素形成于藏红花酸二醛，在番红花中由类胡萝卜素裂解双加氧酶CsCCD2L催化玉米黄素获得；而来自另外一种富含藏红花素的植物栀子花的GjCCD4a则能够在细菌中催化包括玉米黄素在内的不同类胡萝卜素形成藏红花酸二醛。为了建立一个可持续生产藏红花素的生物技术平台，作者利用柑橘愈伤组织和烟草叶片为材料，通过农杆菌介导的稳定遗传转化和瞬时转化方法，比较了GjCCD4a和CsCCD2L的酶活。作者发现，共表达GjCCD4a和八氢番茄红素合酶编码基因PSY及β-胡萝卜素羟化酶编码基因BCH，是一个在柑橘愈伤组织中异源合成藏红花酸二醛、藏红花素以及藏红花苦素的最佳基因组合。通过对脱辅基类胡萝卜素的定量以及体外实验，作者发现GjCCD4a能够在植物体内剪切β-胡萝卜素，并通过C30 β-脱辅基类胡萝卜素的中间产物生成藏红花酸二醛。GjCCD4a同样还能够剪切C27 β-脱辅基类胡萝卜素，为C17-二醛的生物合成提供了一个新的途径。过表达GjCCD4a的柑橘愈伤株系在类有色体的质体中含有更多的质体小球，并且八氢番茄红素、C17:0脂肪酸（FA）、C18:1 cis-9和C22:0 FA酯类物质含量更高。GjCCD4a在烟草叶片中表现出更广的底物特异性，并且对底物的催化效率更高，从而导致积累的藏红花素干重高达1.6 mg/g。综上，本文的研究结果建立了一个在植物体内研究CCD酶活的系统平台，同时也建立了一个以绿色和非绿色组织或器官为生物材料，高效生产抗癌活性物质藏红花素的生物技术平台。

 Salim Al-Babili