Vitamin A deficiency remains a severe global health issue,which creates a need to biofortify crops with provitamin A carotenoids(PACs).Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored.Here,we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves,Arabidopsis seeds,and citrus callus cells,using a fungal(Neurospora crassa)carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs,including β-carotene.This strategy led to the accumulation of significant amounts of phytoene and γ-and β-carotene,in addition to fungal,health-promoting carotenes with 13 conjugated double bonds,such as the PAC torulene,in the cytosol.Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production.Engineered carotenes accumulate in cytosolic lipid droplets(CLDs),which represent a novel sequestering sink for storing these pigments in plant cytosol.Importantly,β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidialβ-carotene.Moreover,engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids,including retinal,the aldehyde corresponding to vitamin A.Collectively,our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues,especially in lipid-storing seeds,and thus paves the way for further optimization of carotenoid biofortification in crops.
为了提高麸皮中总阿魏酸的释放量,进而研发出具有替抗功能的麸皮发酵饲料,试验对好食脉孢菌发酵麸皮产总阿魏酸的工艺进行了优化,比较分析了不同溶剂对总阿魏酸的提取效果,检测了不同的总阿魏酸提取液对大肠杆菌和金黄色葡萄球菌的抑菌效果。结果表明:好食脉孢菌发酵麸皮产总阿魏酸工艺参数优化组合为:在14 m L液体培养基中添加葡萄糖93.90 mg,尿素31.20 mg,Mg^(2+)1.60 mg。在100 m L烧杯中加入10 g麸皮和14 m L液体培养基,接种好食脉孢菌孢子悬液1.00 mL,混匀,30℃发酵72 h后总阿魏酸的释放量为(1.18±0.02)mg/g,是未优化的2.9倍。以75%乙醇为提取溶剂,发酵麸皮中总阿魏酸提取量达到(1.42±0.04)mg/g。用75%乙醇提取的总阿魏酸对金黄色葡萄球菌抑菌效果较好,而用乙酸乙酯提取的总阿魏酸对大肠杆菌的抑菌效果较好。
水稻油菜轮作制度是华中地区最具代表性的耕作制度,但水稻收获后的秸秆清理和还田效果会影响后续油菜的播种和收获。利用微生物降解秸秆是解决这一问题的有效途径之一。前期研究发现,粗糙脉孢菌接种至水稻秸秆,培养48 h时秸秆有明显降解现象,因此,本研究通过转录组测序技术研究分析比较接种在PDA培养基上和接种在水稻秸秆上培养48 h时粗糙脉孢菌的差异表达基因,以探讨其降解机理。结果表明,两者间共存在3329个显著差异表达基因,GO(gene ontology)功能注释分析发现,这些基因主要富集在生物过程类,表达差异较大的基因主要参于核苷酸代谢、蛋白质代谢及与内膜系统;KEGG(Kyoto Encyclopedia of Genes and Genomes)通路富集分析发现,这些差异表达基因的功能主要涉及半乳糖代谢、果糖和甘露糖代谢、氧化磷酸化、次生代谢产物的生物合成和核糖体,在这些代谢途径中筛选到的与降解秸秆相关的酶系多数为上调表达。在这些代谢途径中,推测粗糙脉孢菌主要通过半乳糖代谢、果糖和甘露糖代谢来降解水稻秸秆。以上研究可为更高效地利用粗糙脉孢菌降解秸秆提供新的理论依据和一定的技术支持。
