Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli
Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli
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DOI：
10.1016/j.ymben.2010.01.003
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发表时间：
2010-05-01
影响因子：
8.4
通讯作者：
Prather, Kristala L. Jones
中科院分区：
文献类型：
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作者：
Moon, Tae Seok;Dueber, John E.;Prather, Kristala L. Jones
代谢工程领域有可能以廉价和生态友好的方式生产各种各样的化学品。新型酶组合的异源表达有望提供新的或改进的合成途径，以大幅增加小分子的多样性。最近，我们构建了一种合成途径来生产D-葡糖二酸，一种被认为是生物质中“最高附加值的化学品”的分子，从葡萄糖开始。限制通过该途径的流量是第二个重组步骤，由肌醇加氧酶（MIOX）催化，其活性受到肌醇底物浓度的强烈影响。为了合成性地增加肌醇的有效浓度，由蛋白质-蛋白质相互作用结构域构建多肽支架，以将所有三种途径酶共定位在可设计的复合物中，如前所述（Dueber等人，2009年）。葡糖二酸滴度被认为是强烈影响的scaffoldinteraction结构域的目标上游Ino1酶的数量，而MIOX靶向结构域的数量增加的效果是不太显着。我们确定支架直接增加了特定的MIOX活性，并且葡糖二酸滴度与MIOX活性强烈相关。总体而言，我们观察到产物滴度比非支架化对照提高了约5倍，并且比先前报道的最高滴度提高了50%。这些结果进一步验证了这些合成支架作为代谢工程工具的实用性。(C)2010年爱思唯尔公司All rights reserved.
The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce D-glucaric acid, a molecule that has been deemed a "top-value added chemical'' from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffoldinteraction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering. (C) 2010 Elsevier Inc. All rights reserved.