Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.
Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.
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DOI：
10.1021/ja107084d
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发表时间：
2010-10-06
影响因子：
15
通讯作者：
Tang Y
中科院分区：
文献类型：
--
作者：
Xu W;Cai X;Jung ME;Tang Y
广泛发现的真菌迭代PKS-NRPS杂合大合成酶是高度编程的生物合成机器，参与3-酰基特特拉姆酸和相关天然产物的合成。迭代PKS-NRPS的体外分析受到与获得这些大酶（>400 kDa）的纯的和功能性形式相关的困难的阻碍。我们成功地表达构巢曲霉Aspyridone合成酶（ApdA）从工程酿酒酵母菌株。ApdA及其烯酰还原酶配偶体ApdC的完整功能在体外和S.酿酒酵母与preaspyridone 7的生产。然后在体外检查PKS和NRPS模块的编程规则。PKS和NRPS之间的关键相互作用的解剖和重建的反式使用独立的模块。7的类似物通过来自不同来源的PKS和NRPS模块的异源组合来合成。我们的研究结果代表了迄今为止最大的多结构域酶重组，并为真菌天然产物的工程生物合成提供了新的机会。
The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3- acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS have been hampered by the difficulties associated with obtaining pure and functional forms of these large enzymes (>400 kDa). We successfully expressed Aspergillus nidulans aspyridone synthetase (ApdA) from an engineered Saccharomyces cerevisiae strain. The complete functions of ApdA and its enoylreductase partner ApdC are reconstituted in vitro and in S. cerevisiae with the production of preaspyridone 7. The programming rules of both the PKS and NRPS modules were then examined in vitro. The key interaction between the PKS and the NRPS was dissected and reconstituted in trans by using standalone modules. Analogs of 7 were synthesized through heterologous combinations of PKS and NRPS modules from different sources. Our results represent the largest, multidomain enzyme reconstituted to date; and offer new opportunities for engineered biosynthesis of fungal natural products.
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