Synthetic protein scaffolds provide modular control over metabolic flux
Synthetic protein scaffolds provide modular control over metabolic flux
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DOI：
10.1038/nbt.1557
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发表时间：
2009-08-01
影响因子：
46.9
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
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作者：
Dueber, John E.;Wu, Gabriel C.;Keasling, Jay D.
由与生产宿主不同的酶构建的工程代谢途径经常遭受通量失衡，因为它们通常缺乏自然代谢特征的调节机制。为了增加感兴趣途径中每个成分的有效浓度，我们构建了合成蛋白质支架，以可设计的方式在空间上招募代谢酶。承载后生动物信号蛋白相互作用结构域的支架会产生带有其同源肽配体标记的途径酶。这些结构域的天然模块化使我们能够优化三种甲羟戊酸生物合成酶的化学计量学，从而在低酶表达和减少代谢负荷的情况下，将产物滴度提高77倍。在没有合成复合物的情况下，尽管滴度很高（0.5 g/I），但使用其中一种相同的支架将葡萄糖酸的产量提高了三倍。这些策略应该被证明可推广到其他代谢途径，并可编程微调途径通量。
Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/I) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.