Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering.
Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering.
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DOI：
10.1007/s00253-009-2219-x
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发表时间：
2010-02
影响因子：
5
通讯作者：
Peters, Reuben J.
中科院分区：
文献类型：
--
作者：
Morrone, Dana;Lowry, Luke;Determan, Mara K.;Hershey, David M.;Xu, Meimei;Peters, Reuben J.
异源微生物宿主生物体中的工程生物合成途径通过掺入假定的生物合成酶和由此产生的新代谢物的表征，提供了一种阐明途径的优雅方法。我们之前在大肠杆菌中的工作证明了一种简单的模块化方法来设计生产与拉丹烷相关的二萜（20 个碳）天然产物的可行性。然而，产量有限（<0.1 mg/L），可能是由于依赖类异戊二烯前体二甲基烯丙基二磷酸和异戊烯基二磷酸的内源生产。在这里，我们报告了异源甲羟戊酸途径（MEV）或内源甲基赤藓糖醇磷酸途径（MEP）的增强与我们的模块化代谢工程系统的结合。随着MEP途径的增强，发现丰富培养基中丙酮酸的补充和三个基因（idi、dxs和dxr）的同时过表达导致二萜产量的最大增加，表明该途径内的分布式代谢控制。在生物反应器生长的培养物中掺入异源 MEV 途径导致的产量显着高于 MEP 途径增强。我们已经建立了合适的生长条件，使大肠杆菌培养物的二萜产量水平范围为 10 至 >100 mg/L。这些量足以进行核磁共振分析，从而能够表征酶产物，从而阐明途径。此外，这些结果表明我们简便的模块化平台的二萜产量提高了 1,000 倍以上，MEP 途径增强提供了具有成本效益的替代方案和合理的产量。最后，我们在此重申，这种模块化方法是可扩展的，并且应该很容易适应任何萜类天然产品的生产。本文的在线版本 (doi:10.1007/s00253-009-2219-x) 包含补充材料，可供授权用户使用。
Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product. The online version of this article (doi:10.1007/s00253-009-2219-x) contains supplementary material, which is available to authorized users.
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