Chemical Analysis of Hybrid Fungal Megasynthases

杂合真菌大合成酶的化学分析

• 批准号: EP/F066104/1
• 负责人: Russell Cox
• 金额: $ 89.25万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FF066104%2F1
• 关键词: Chemical; Analysis; Hybrid; Fungal; Megasynthases

Fungi produce a diverse array of biologically active compounds with roles as pharmaceuticals, agrochemicals and toxins. These include drugs such as the penicillins for treating bacterial infections, anticholesterol compounds such as the statins, and psychoactive drugs such as xenovulene. A major class of these compounds are the polyketides. In fungi polyketides are synthesised by giant multifunctional proteins known as polyketide synthases (PKS) - in turn these giant proteins are encoded by very large PKS genes. We have developed ways of isolating PKS genes from any given fungi which are involved in the biosynthesis of specific chemical compounds. For example we have found PKS gene clusters involved in the biosynthesis of the anticholesterol compound squalestatin, the mycotoxin fusarin A, the pigment tenellin and the psychoactive drug xenovulene from different fungal species. These PKS genes have been transferred to a new fungal host and switched on so that new chemical compounds are made. Despite very similar gene sequences, the four PKS genes encode proteins which can make very different chemical compounds. Thus, a knowledge of the gene sequence for these PKS does not yet help in understanding the processes which occur during the catalysis of polyketide formation in fungi. The PKS proteins evidently carry out a complex series of highly programmed chemical steps. While it is possible to read from the sequence the steps which could be catalysed, the programme controlling the order in which the steps are used, and the number of times they are repeated, is cryptic. The aim of this project is to find out how the giant PKS proteins are programmed. We will take a chemical approach to this problem. BBSRC and EU funded work is currently underway to generate numerous genetic changes in the genes themselves. We expect the genetic changes to lead to the production of modified proteins, which in turn should make new chemical compounds. By detecting, purifying and analysing the chemical structure of these new compounds we will be able to reveal the effect of the genetic change on the programme of the PKS. We expect to generate many tens of genetic experiments and each of these will require the examination of tens of genetic clones in fungi for the production of new compounds. We thus expect to have to examine hundreds to thousands of chemical extracts. We will use a state-of-the-art instrument to automate many of the purification and analysis steps. This instrument will combine High Performance Liquid Chromatography (HPLC) with sensitive, but robust, detectors which will be able to detect new compounds by size (evaporative light scattering detector), mass (mass spectrometer) and ultraviolet light (uv). The instrument will also be able to do automated purification for small samples, and will thus assist the chemist in processing the many hundreds or thousands of samples. We will also use the facilities of the School of Chemistry such as high field NMR and high resolution MS for structural elucidation. The new knowledge chemical structures of the new compounds produced from the mutant PKS will then allow us to elucidate the chemical effect of the genetic changes. We hope to eventually understand the link between gene sequence and chemical compound. This will allow two major advances - the ability to engineer fungal PKS at will to produce new compounds; and the ability to predict what compound will be made by simply reading a gene sequence.

真菌产生多种多样的生物活性化合物，具有药物、农用化学品和毒素的作用。这些药物包括用于治疗细菌感染的青霉素类药物、他汀类药物等抗胆固醇化合物和异戊烯等精神活性药物。这些化合物的主要类别是聚酮化合物。在真菌中，聚酮化合物是由称为聚酮化合物脱氢酶（PKS）的巨型多功能蛋白质合成的，而这些巨型蛋白质又由非常大的PKS基因编码。我们已经开发了从参与特定化合物生物合成的任何给定真菌中分离PKS基因的方法。例如，我们已经发现PKS基因簇参与来自不同真菌物种的抗胆固醇化合物角鲨烯、真菌毒素镰孢菌素A、色素tenellin和精神活性药物xenovulene的生物合成。这些PKS基因被转移到一个新的真菌宿主中，并被打开，从而产生新的化合物。尽管基因序列非常相似，但这四个PKS基因编码的蛋白质可以产生非常不同的化合物。因此，这些PKS的基因序列的知识还没有帮助理解的过程中发生的聚酮化合物形成在真菌中的催化。PKS蛋白显然执行一系列复杂的高度编程的化学步骤。虽然可以从序列中读出可能被催化的步骤，但控制步骤使用顺序和重复次数的程序是神秘的。这个项目的目的是找出巨大的PKS蛋白是如何编程的。我们将用化学方法来解决这个问题。BBSRC和欧盟资助的工作目前正在进行中，以产生基因本身的许多遗传变化。我们希望遗传变化能导致蛋白质的改变，从而产生新的化合物。通过检测、纯化和分析这些新化合物的化学结构，我们将能够揭示遗传变化对PKS程序的影响。我们预计将产生数十个遗传实验，每个实验都需要检查真菌中的数十个遗传克隆，以生产新化合物。因此，我们预计必须检查数百到数千种化学提取物。我们将使用最先进的仪器来自动化许多纯化和分析步骤。该仪器将联合收割机高效液相色谱（HPLC）与灵敏但耐用的检测器相结合，检测器将能够通过大小（蒸发光散射检测器）、质量（质谱仪）和紫外光（UV）检测新化合物。该仪器还将能够对小样本进行自动纯化，从而帮助化学家处理数百或数千个样本。我们还将使用化学学院的设施，如高场NMR和高分辨率MS进行结构解析。从突变体PKS产生的新化合物的新知识化学结构将使我们能够阐明遗传变化的化学效应。我们希望最终能够理解基因序列和化合物之间的联系。这将带来两项重大进展--随意设计真菌PKS以产生新化合物的能力;以及通过简单地阅读基因序列来预测将产生什么化合物的能力。

项目成果

Identification of genes encoding squalestatin S1 biosynthesis and in vitro production of new squalestatin analogues

角鲨他汀 S1 生物合成编码基因的鉴定及新型角鲨他汀类似物的体外生产

• DOI: 10.15488/791
• 发表时间: 2016
• 作者: Bonsch B

Diverse and potentially manipulative signalling with ascarosides in the model nematode C. elegans.

• DOI: 10.1186/1471-2148-14-46
• 发表时间: 2014-03-11
• 期刊: BMC evolutionary biology
• 影响因子: 3.4
• 作者: Diaz SA;Brunet V;Lloyd-Jones GC;Spinner W;Wharam B;Viney M
• 通讯作者: Viney M

Oxidative dearomatisation: the key step of sorbicillinoid biosynthesis†Electronic supplementary information (ESI) available: Containing all experimental details. See DOI: 10.1039/c3sc52911hClick here for additional data file.

• DOI: 10.1039/c3sc52911h
• 发表时间: 2014-02-23
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Fahad AA;Abood A;Fisch KM;Osipow A;Davison J;Avramović M;Butts CP;Piel J;Simpson TJ;Cox RJ
• 通讯作者: Cox RJ

Kinetic characterisation of the FAD dependent monooxygenase TropB and investigation of its biotransformation potential

• DOI: 10.1039/c5ra06693j
• 发表时间: 2015-01-01
• 期刊: RSC ADVANCES
• 影响因子: 3.9
• 作者: Abood, Amira;Al-Fahad, Ahmed;Cox, Russell J.
• 通讯作者: Cox, Russell J.

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production.

• DOI: 10.1038/srep25202
• 发表时间: 2016-05-04
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Bailey AM;Alberti F;Kilaru S;Collins CM;de Mattos-Shipley K;Hartley AJ;Hayes P;Griffin A;Lazarus CM;Cox RJ;Willis CL;O'Dwyer K;Spence DW;Foster GD
• 通讯作者: Foster GD