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首页> 外文会议>Biochemical and molecular engineering XX: the next generation of biochemical engineering: from nanoscale to industrial scale >POST-TRANSLATIONAL STRATEGIES FOR ENHANCING BIOSYNTHETIC PATHWAY EXPRESSION AND ACTIVITY.
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POST-TRANSLATIONAL STRATEGIES FOR ENHANCING BIOSYNTHETIC PATHWAY EXPRESSION AND ACTIVITY.

机译：翻译后战略，以增强生物合成途径的表达和活性。

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The past decade has seen considerable advancements in synthetic biology tools for controlling protein and biosynthetic pathway expression. For example, low cost DNA synthesis and the standardization of genetic parts has enabled high through pathway refactoring, and riboswitches and ribosome binding site engineering has enabled the fine tuning of translational rates. As a posttranslation strategy, synthetic protein scaffolds that co-localize pathway enzymes and enhance chemical biosynthesis have also been demonstrated. In this work, we demonstrate that controlled intracellular trafficking of membrane-associated enzymes can also be used to tune pathway expression and enhance activity. Using yeast ester biosynthesis as a model system, we demonstrate two separate approaches to post-translational control over pathway expression and function. In the first, we have engineered mitochondrial and lipid droplet targeting in an alcohol-O-acyltransferase, Eeb1. Eeb1 natively localizes to mitochondria, expression levels are low, and increased transcript levels do not produce corresponding increases in expression and function. Positive correlations between transcription and translation were achieved in the engineered mutant, which traffics to the ER and then to lipid droplets as cells reach stationary phase. In comparison to the wild type, lipid droplet targeting increases expression levels by more than 20-fold. Our second approach builds on the concept of enzyme co-localization. The short chain alcohol-O-acetyltransferase Atf1 natively localizes to the surface of lipid droplets, which is necessary for high activity. A synthetic protein scaffold based on the plant protein oleosin was engineered to co-localize upstream enzymes of the ester biosynthesis pathway to the surface of lipid droplets. Optimization of the scaffold architecture and enzyme expression levels enhanced ester biosynthesis by nearly 2-fold. Combined, these two examples demonstrate new post translation strategies with the potential to provide subcellular engineering solutions to other membrane-bound pathways.
机译：在过去的十年中，用于控制蛋白质和生物合成途径表达的合成生物学工具取得了长足的进步。例如，低成本的DNA合成和基因部分的标准化已实现了高途径的重构，而核糖开关和核糖体结合位点工程已使翻译速率得以微调。作为翻译后策略，也已证明了共定位途径酶并增强化学生物合成的合成蛋白支架。在这项工作中，我们证明受控的细胞内膜相关酶的运输也可以用于调整途径表达和增强活性。使用酵母酯生物合成作为模型系统，我们演示了两种单独的途径来翻译后控制途径的表达和功能。首先，我们设计了针对乙醇-O-酰基转移酶Eeb1的线粒体和脂质滴。 Eeb1本地化为线粒体，表达水平较低，并且转录水平升高不会产生相应的表达和功能增强。在工程突变体中实现了转录与翻译之间的正相关，该突变体在细胞到达固定相时向ER转运，然后向脂质滴转运。与野生型相比，脂质滴靶向将表达水平提高了20倍以上。我们的第二种方法基于酶共定位的概念。短链醇-O-乙酰基转移酶Atf1自然地位于脂质液滴的表面，这对于高活性是必需的。基于植物蛋白油质蛋白的合成蛋白支架经过工程改造，可将酯生物合成途径的上游酶共定位在脂质液滴的表面。支架结构和酶表达水平的优化将酯的生物合成提高了近2倍。结合起来，这两个例子说明了新的翻译后策略，具有为其他膜结合途径提供亚细胞工程解决方案的潜力。

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