The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus.
The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus.
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DOI：
10.1186/s12934-014-0095-2
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发表时间：
2014-08-12
影响因子：
6.4
通讯作者：
Naesby M
中科院分区：
文献类型：
--
作者：
Nielsen CA;Folly C;Hatsch A;Molt A;Schröder H;O'Connor SE;Naesby M
麦角生物碱是由许多丝状真菌产生的一组具有高度生物活性的分子。这些化合物已经被深入研究了几十年，主要是由于它们在受污染的食品和饲料中的有害影响，以及它们有益的制药和农业应用。麦角生物碱的生物合成通过共同的中间体裸花黄素-I进行，并且参与裸花黄素-I形成的关键酶EasE和EasC的研究依赖于真菌中的基因互补，而进一步表征受到EasE蛋白表达差的困难的阻碍。为了促进麦角生物碱的研究，并最终走向商业化生产，在单细胞酵母酿酒酵母中重建了生物合成途径的早期步骤。利用麦角生物碱产生菌Aspergilluslaugus的基因组序列预测麦角生物碱早期代谢途径基因的蛋白质编码序列。这些被克隆并在酵母中表达，导致共同中间体裸花黄素-I的从头生产。这允许进一步表征EasE和EasC，并且我们能够证明EasE的N-末端ER靶向信号如何对酵母中的活性至关重要。在EasC中发现的一个假定的过氧化物酶体靶向信号被证明是不必要的。与二硫键形成和ER蛋白折叠机制相关的宿主基因pdi 1或ero 1的过表达被证明增加了酵母中的chanoclavine-I生产。这也是当过表达宿主fad 1时的情况，已知fad 1参与辅因子的产生。深入了解麦角生物碱形成中的酶促步骤对于这类有效化合物的商业化生产和开发至关重要。我们在这里表明，EasE和EasC是必要的和足够的生产chanoclavine-I在酵母中，我们提供了重要的新信息，参与ER和蛋白质折叠的EasE的正确功能表达。此外，通过在酵母中重建裸花黄素-I生物合成途径，我们展示了该宿主的优势和潜力，不仅作为一个方便的模型系统，而且作为麦角生物碱生产的替代细胞工厂。本文的在线版本（doi：10.1186/s12934-014-0095-2）包含补充材料，可供授权用户使用。
Ergot alkaloids are a group of highly bioactive molecules produced by a number of filamentous fungi. These compounds have been intensely studied for decades, mainly due to their deleterious effects in contaminated food and feeds, but also for their beneficial pharmaceutical and agricultural applications. Biosynthesis of ergot alkaloids goes via the common intermediate chanoclavine-I, and studies of the key enzymes, EasE and EasC, involved in chanoclavine-I formation, have relied on gene complementation in fungi, whereas further characterization has been hampered by difficulties of poor EasE protein expression. In order to facilitate the study of ergot alkaloids, and eventually move towards commercial production, the early steps of the biosynthetic pathway were reconstituted in the unicellular yeast Saccharomyces cerevisiae. The genomic sequence from an ergot alkaloid producer, Aspergillus japonicus, was used to predict the protein encoding sequences of the early ergot alkaloid pathway genes. These were cloned and expressed in yeast, resulting in de novo production of the common intermediate chanoclavine-I. This allowed further characterization of EasE and EasC, and we were able to demonstrate how the N-terminal ER targeting signal of EasE is crucial for activity in yeast. A putative, peroxisomal targeting signal found in EasC was shown to be nonessential. Overexpression of host genes pdi1 or ero1, associated with disulphide bond formation and the ER protein folding machinery, was shown to increase chanoclavine-I production in yeast. This was also the case when overexpressing host fad1, known to be involved in co-factor generation. A thorough understanding of the enzymatic steps involved in ergot alkaloid formation is essential for commercial production and exploitation of this potent compound class. We show here that EasE and EasC are both necessary and sufficient for the production of chanoclavine-I in yeast, and we provide important new information about the involvement of ER and protein folding for proper functional expression of EasE. Moreover, by reconstructing the chanoclavine-I biosynthetic pathway in yeast we demonstrate the advantage and potential of this host, not only as a convenient model system, but also as an alternative cell factory for ergot alkaloid production. The online version of this article (doi:10.1186/s12934-014-0095-2) contains supplementary material, which is available to authorized users.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
4.8
作者：
Rigbers, Ole;Li, Shu-Ming
通讯作者：
Li, Shu-Ming
影响因子：
2.6
作者：
Flagfeldt, Dongmei Bai;Siewers, Verena;Nielsen, Jens
通讯作者：
Nielsen, Jens
DOI：
10.1016/s1099-4831(06)63002-2
发表时间：
2006-01-01
期刊：
ALKALOIDS: CHEMISTRY AND BIOLOGY, VOL 63
影响因子：
--
作者：
Schardl, Christopher L.;Panaccione, Daniel G.;Tudzynski, Paul
通讯作者：
Tudzynski, Paul
影响因子：
3.9
作者：
Lu Q;McAlister-Henn L
通讯作者：
McAlister-Henn L