Design and utility of oligonucleotide gene probes for fungal polyketide synthases
Design and utility of oligonucleotide gene probes for fungal polyketide synthases
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DOI：
10.1016/s1074-5521(00)90064-4
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发表时间：
2001-02-01
影响因子：
--
通讯作者：
Cox, RJ
中科院分区：
文献类型：
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作者：
Nicholson, TP;Rudd, BAM;Cox, RJ
背景资料：聚酮化合物生物合成的分子生物学的最新进展已经允许聚酮化合物脱氢酶的工程化和新型聚酮化合物的生物（“组合”）合成。如果可以利用更多样的聚酮化合物脱氢酶（PKS），则可以预期这些化合物中的额外结构多样性。真菌聚酮化合物的结构高度可变，反映了真菌PKS复合物的结构和功能可能存在广泛的差异。相对较少的真菌真菌的pesticases已被调查，也许是因为缺乏合适的遗传技术可用于分离和操纵基因簇从不同的主机。我们着手设计一个通用的方法，用于检测特定的PKS基因从fungies.Results：我们研究了已知的真菌和细菌的聚酮酶的序列数据，以及从细菌，真菌和脊椎动物的脂肪酸脱氢酶的序列数据，以确定高序列保守的区域。使用单独的结构域如β-酮脂酰基转移酶（KS）、β-酮还原酶（KR）和甲基转移酶（MeT），我们确定了特定的短（约7个氨基酸）序列，其显示出对特定功能结构域（例如，参与产生部分还原代谢物的真菌KR结构域;参与产生高度还原代谢物的真菌KS结构域等）的高度保守性.设计与这些特异性同源性区域相匹配的简并PCR引物，并将该引物用于与来自许多已知聚酮化合物产生物种的真菌基因组DNA的PCR反应中。对从这些反应中获得的产物进行测序，结果表明是来自尚未发现的PKS基因簇的片段。该片段可用于与同源或异源真菌基因组DNA.Conclusions的印迹实验：一些序列的发现新的真菌PKS基因簇具有很高的实用性。该序列似乎对特定类型的真菌聚酮化合物（即非还原、部分还原或高度还原的KS结构域）具有特异性。我们还开发了适合于扩增编码聚酮化合物C-甲基转移酶结构域的真菌基因片段的引物。使用这些特异性引物序列扩增的基因组片段可用于印迹实验，并具有很高的潜力，作为新的真菌PKS基因簇的最终克隆的艾滋病。(C)2001爱思唯尔科技有限公司版权所有。
Background: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi.Results: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as beta -ketoacylsynthases (KS), beta -ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA.Conclusions: A number of sequences are presented which have high utility for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters. (C) 2001 Elsevier Science Ltd. All rights reserved.