Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.
Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.
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DOI：
10.1111/pbi.13574
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发表时间：
2021-08
影响因子：
13.8
通讯作者：
Graham IA
中科院分区：
文献类型：
--
作者：
Forestier ECF;Czechowski T;Cording AC;Gilday AD;King AJ;Brown GD;Graham IA
为了使本氏烟草产生新的二萜类化合物，我们首先通过上调拟南芥来源的非甲氧丙酮酸(MEP)途径的关键基因来提高二萜类前体香叶基焦磷酸(GGPP)的产量。我们使用瞬时表达来评估8个MEP途径基因与GGPP合成酶和麻疯树酪蛋白合成酶(JcCAS)的组合，以确定从GGPP中生产酪宾的最佳组合。与单独瞬时表达JcCAS相比，AtDXS和AtHDR与AtGGPPS和JcCAS一起提高了酪宾产量410%。使用MoClo工具箱将这一组合克隆到一个单一的结构中，并稳定地整合到N.C.benthamiana基因组中。我们还创建了多基因构建物，用于稳定转化两个金龟子细胞色素P450基因JcCYP726A20和JcCYP71D495，当与JcCAS在本氏新月浑藻中瞬时表达时，这两个基因可以从酪烯中产生更复杂的二萜类jolkinol C。JcCYP726A20、JcCYP71D495和JcCAS的稳定转化不能产生任何可检测到的jolkinol C，直到这些基因与最适的前体途径基因共转化。其中一个稳定的纯合子株系被用来通过瞬时表达来评估一个由四个基因组成的类似烯醛还原酶的家族参与了jolkinol C的进一步转化，从而证明其中一个基因能够还原jolkinol C中的12，13-双键。这项工作突出了在稳定的转化子中优化前体供应以生产复杂的二萜类化合物的必要性，以及这些株系对新基因发现的价值。
To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up‐regulation of key genes of the non‐mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co‐transformed with the optimal set of precursor‐pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an ‘alkenal reductase’‐like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13‐double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.
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DOI：
10.1002/anie.201510650
发表时间：
2016-02-05
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Andersen-Ranberg J;Kongstad KT;Nielsen MT;Jensen NB;Pateraki I;Bach SS;Hamberger B;Zerbe P;Staerk D;Bohlmann J;Møller BL;Hamberger B
通讯作者：
Hamberger B
影响因子：
13.8
作者：
Forestier ECF;Czechowski T;Cording AC;Gilday AD;King AJ;Brown GD;Graham IA
通讯作者：
Graham IA
影响因子：
4.8
作者：
Estévez, JM;Cantero, A;León, P
通讯作者：
León, P
影响因子：
7.2
作者：
Lois, LM;Rodríguez-Concepción, M;Boronat, A
通讯作者：
Boronat, A
DOI：
10.1073/pnas.1607504113
发表时间：
2016-08-23
影响因子：
11.1
作者：
Luo, Dan;Callari, Roberta;Hamberger, Bjoern
通讯作者：
Hamberger, Bjoern