High-level diterpene production by transient expression in Nicotiana benthamiana.
High-level diterpene production by transient expression in Nicotiana benthamiana.
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DOI：
10.1186/1746-4811-9-46
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发表时间：
2013-12-12
期刊：
影响因子：
5.1
通讯作者：
Tissier A
中科院分区：
文献类型：
--
作者：
Brückner K;Tissier A
植物萜烯脱氢酶的表征通常通过在大肠杆菌中产生重组酶来进行。由于溶解度和密码子使用问题，这通常是困难的。此外，靶向质体的植物萜烯脱氢酶，如二萜烯脱氢酶，必须以或多或少的经验方法缩短以改善表达。我们在这里报告了一个优化的农杆菌介导的瞬时表达测定在烟草本塞姆安植物二萜合酶的表达和产物分析。农杆菌介导的植物二萜苷酶在N.本塞姆那导致二萜类化合物在渗透的3天内积累，并在5天达到最大值。超过50%的产品被输出到叶片表面，从而通过降低提取物的复杂性大大方便了分析。通过表达三种不同的植物酶来测试该方法的稳健性，所述三种不同的植物酶是来自欧洲烟草的西柏三烯-醇合酶、来自蓖麻的casbene合酶和来自银杏的左旋海松二烯合酶。此外，来自番茄的1-脱氧-D-木酮糖-5-磷酸合酶和来自烟草的香叶基香叶基二磷酸合酶的共表达导致所产生的西柏三烯-醇的量增加3.5倍，最大产量达到2500 ng/cm 2。利用这种优化的二萜合酶表达和产物分析方法，可以获得一个N。Benthamiana将足以产生未知二萜的结构解析所需的量。该方法在植物二萜类化合物生物合成的基因功能发现、途径重构和代谢工程等方面也具有普遍意义。
Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.
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影响因子：
3.9
作者：
Hill, AM;Cane, DE;West, CA
通讯作者：
West, CA
影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
7.4
作者：
Hall, Dawn E.;Zerbe, Philipp;Bohlmann, Joerg
通讯作者：
Bohlmann, Joerg
DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
DOI：
10.1007/bf00331014
发表时间：
1986-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
KONCZ, C;SCHELL, J
通讯作者：
SCHELL, J