Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
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DOI：
10.1371/journal.pone.0072871
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Mortensen UH
中科院分区：
文献类型：
--
作者：
Nielsen MT;Nielsen JB;Anyaogu DC;Holm DK;Nielsen KF;Larsen TO;Mortensen UH
真菌天然产物是生物活性分子的丰富来源。为了充分利用这一潜力，有必要将基因与代谢物联系起来。近年来，通过基因组测序，许多假定的生物合成途径的遗传信息已经成为可能。然而，缺乏可靠的方法来对大多数物种进行遗传操作，严重阻碍了途径的表征。在这里，我们提出了一种简单的基于聚合酶链式反应的异源重组完整基因簇的方法。具体地说，负责从土曲霉中产生地黄素的推测基因簇通过两步程序被转移到根状曲霉的表达平台上。通过聚合酶链式反应产生单个簇片段，并通过有效的用户融合进行组装，然后通过重新迭代的基因打靶进行转化和整合。在这两个物种之间成功地转移了包含在25kb DNA中的13个开放阅读框，使其能够在多齿拟青霉中合成地黄素。随后，通过遗传和化学分析对该簇中三个基因的功能进行了验证。具体来说，ATEG_08451(GeDC)编码一个聚酮合成酶，ATEG_08453(GedR)编码一个负责geodin基因簇激活的转录因子，ATEG_08460(GedL)编码一个催化溶血素转化为二氢地黄素的卤素酶。我们预计，我们将完整的生物合成途径转移到具有良好遗传工具箱的真菌上的方法，将有助于表征目前由真菌基因组测序项目发现的许多令人兴奋的次生代谢物产生途径。
Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.
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用户融合：一种快速有效的方法，用于同时融合和克隆多个PCR产品。
影响因子：
14.9
作者：
Geu-Flores, Fernando;Nour-Eldin, Hussam H.;Nielsen, Morten T.;Halkier, Barbara A.
通讯作者：
Halkier, Barbara A.
影响因子：
4.4
作者：
Chiang, Yi-Ming;Szewczyk, Edyta;Oakley, Berl R.
通讯作者：
Oakley, Berl R.
影响因子：
2.7
作者：
FUJII, I;EBIZUKA, Y;SANKAWA, U
通讯作者：
SANKAWA, U
DOI：
10.3891/acta.chem.scand.23-3493
发表时间：
1969-01-01
期刊：
Acta Chemica Scandinavica
影响因子：
--
作者：
GATENBECK S;MALMSTROM L
通讯作者：
MALMSTROM L
影响因子：
14.8
作者：
Bergmann, Sebastian;Schuemann, Julia;Hertweck, Christian
通讯作者：
Hertweck, Christian