Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.
Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.
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DOI：
10.1186/s12934-018-1027-3
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发表时间：
2018-11-19
影响因子：
6.4
通讯作者：
Pateraki I
中科院分区：
文献类型：
--
作者：
Forman V;Bjerg-Jensen N;Dyekjær JD;Møller BL;Pateraki I
毛喉素是一种高价值的二萜类化合物，仅由唇形科植物毛喉鞘蕊花产生。今天，毛喉素被用于其腺苷酸环化酶激活特性的药物。目前，纯毛喉素的有限可用性阻碍了其充分利用，因此需要一种新的环境友好，可扩展和可持续的战略来生产毛喉素。最近，整个生物合成途径导致毛喉素被阐明。该途径的关键步骤由细胞色素P450酶（CYP）催化，其已被证明是该途径的限制步骤。在这里，我们研究是否CYP的底物识别位点（SRS）的蛋白质工程可以提高其对毛喉素在酵母中的生物合成效率。作为概念的证明，我们设计了负责毛喉素途径的第一个推定的氧化步骤的酶：由CYP 76 AH 15催化的13 R-甘露酰氧化物转化为11-氧代-13 R-甘露酰氧化物。四个CYP 76 AH 15的变体，工程在SRS地区，产生了至少两倍的增加11-氧代-13 R-甘露酰氧化物时，在酵母细胞生长在微量滴定板中表达。在SRS 1区域突变的变体A99 I中观察到最高滴度（5.6倍增加）。双重或三重CYP 76 AH 15突变体变体产生具有优化性能的额外酶。CYP 76 AH 15在植物体内可由米替雷烯合成ferruginol。在这项工作中，我们表明，突变体影响11-氧代-13 R-甘露酰氧化物的合成，不影响ferruginol生产，反之亦然。利用表现最好的变体A99 I在酵母细胞中重建毛喉素生物合成途径。尽管与天然CYP 76 AH 15相比，这些菌株显示出11-氧代-甘露酰氧化物产量增加和其他途径中间体的蓄积增加，但观察到毛喉素产量较低。如CYP 76 AH 15所证明的，植物CYP的SRS区域的定点诱变可能是提高这些酶的性能的有效和靶向的方法。虽然在这项工作中，我们已经成功地实现了更高的效率和特异性的第一步的途径，进一步的工作是必要的，以增加毛喉素在酵母细胞的整体生产。本文的在线版本（10.1186/s12934-018-1027-3）包含补充材料，可供授权用户使用。
Forskolin is a high-value diterpenoid produced exclusively by the Lamiaceae plant Coleus forskohlii. Today forskolin is used pharmaceutically for its adenyl-cyclase activating properties. The limited availability of pure forskolin is currently hindering its full utilization, thus a new environmentally friendly, scalable and sustainable strategy is needed for forskolin production. Recently, the entire biosynthetic pathway leading to forskolin was elucidated. The key steps of the pathway are catalyzed by cytochrome P450 enzymes (CYPs), which have been shown to be the limiting steps of the pathway. Here we study whether protein engineering of the substrate recognition sites (SRSs) of CYPs can improve their efficiency towards forskolin biosynthesis in yeast. As a proof of concept, we engineered the enzyme responsible for the first putative oxygenation step of the forskolin pathway: the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide, catalyzed by the CYP76AH15. Four CYP76AH15 variants—engineered in the SRS regions—yielded at least a twofold increase of 11-oxo-13R-manoyl oxide when expressed in yeast cells grown in microtiter plates. The highest titers (5.6-fold increase) were observed with the variant A99I, mutated in the SRS1 region. Double or triple CYP76AH15 mutant variants resulted in additional enzymes with optimized performances. Moreover, in planta CYP76AH15 can synthesize ferruginol from miltiradiene. In this work, we showed that the mutants affecting 11-oxo-13R-manoyl oxide synthesis, do not affect ferruginol production, and vice versa. The best performing variant, A99I, was utilized to reconstruct the forskolin biosynthetic pathway in yeast cells. Although these strains showed increased 11-oxo-manoyl oxide production and higher accumulation of other pathway intermediates compared to the native CYP76AH15, lower production of forskolin was observed. As demonstrated for CYP76AH15, site-directed mutagenesis of SRS regions of plant CYPs may be an efficient and targeted approach to increase the performance of these enzymes. Although in this work we have managed to achieve higher efficiency and specificity of the first CYP of the pathway, further work is necessary in order to increase the overall production of forskolin in yeast cells. The online version of this article (10.1186/s12934-018-1027-3) contains supplementary material, which is available to authorized users.
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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：
3.8
作者：
Dai, Zhubo;Liu, Yi;Zhang, Xueli
通讯作者：
Zhang, Xueli
影响因子：
16.6
作者：
El-Awaad I;Bocola M;Beuerle T;Liu B;Beerhues L
通讯作者：
Beerhues L
影响因子：
15
作者：
Baylon, Javier L.;Lenov, Ivan L.;Sligar, Stephen G.;Tajkhorshid, Emad
通讯作者：
Tajkhorshid, Emad
影响因子：
3.8
作者：
Jensen, Kenneth;Osmani, Sarah Anne;Moller, Birger Lindberg
通讯作者：
Moller, Birger Lindberg