不同CCDs基因表达介导藏红花素在烟草中的异源合成

Crocin heterologous synthesis in tobacco mediated by expressions of different CCDs

类胡萝卜素双加氧酶(Carotnoid Cleavage Dioxygenase,CCD)是藏红花素生物合成途径中的关键酶。为探究不同CCDs在烟草底盘中的催化效率,推动藏红花素在烟草中高效异源合成,测定了烟草中内源CCDs,并选择来源于5个物种的6个与藏红花素合成相关的CCDs基因在本氏烟中进行瞬时表达,同时分析了不同CCDs的催化效率以及藏红花素异源合成对烟草类胡萝卜素代谢流的影响。结果表明,烟草中内源CCDs不能催化合成藏红花素,CsCCD2L、BdCCD4.1、BoCCD4-3和GjCCD4a的转化株系中实现了藏红花素的异源合成,不同CCDs的催化效率存在较大差异。其中,CsCCD2L和GjCCD4a具有较高的催化活性。藏红花素异源合成影响了烟草中番茄红素、β-胡萝卜素和玉米黄质代谢流,CCDs可能具有更广泛的代谢底物特异性。基于核转化构建了藏红花素异源合成稳定遗传材料,明确了藏红花素在烟草中高效异源合成的优化策略,为藏红花素烟草生物反应器开发奠定了重要基础。

Carotenoid Cleavage Dioxygenase(CCD)serves as a key enzyme in the crocin biosynthetic pathway.To investigate the catalytic efficiency of different CCDs in tobacco chassis and to promote high-efficient heterologous synthesis of crocin in tobacco,six CCD genes related to crocin synthesis from five species were selected for transient expression in Nicotiana benthamiana,and the catalytic efficiency of different CCDs and the influence of heterologous synthesis of crocin on the carotenoid metabolic flux in tobacco were analyzed.The results showed that endogenous CCDs in tobacco could not catalyze the synthesis of crocin,while heterologous synthesis of crocin was achieved in the transgenic lines of CsCCD2L,BdCCD4.1,BoCCD4-3,and GjCCD4a.There were significant differences in the catalytic efficiency of different CCDs,with CsCCD2L and GjCCD4a exhibiting higher catalytic activities.Heterologous synthesis of crocin affected the metabolic flux of lycopene,β-carotene,and zeaxanthin in tobacco,suggesting that CCDs may have broader metabolic substrate specificity.Stable genetic materials for the heterologous synthesis of crocin were constructed by nuclear transformation,and optimization strategies for the heterologous synthesis of crocin in tobacco were clarified,which laid a crucial foundation for the development of a tobacco bioreactor for crocin production.