Evolution-guided optimization of biosynthetic pathways
Evolution-guided optimization of biosynthetic pathways
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DOI：
10.1073/pnas.1409523111
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发表时间：
2014-12-16
影响因子：
11.1
通讯作者：
Church, George M.
中科院分区：
文献类型：
--
作者：
Raman, Srivatsan;Rogers, Jameson K.;Church, George M.
化学生产的工程生物合成途径需要广泛优化宿主细胞代谢机械。由于指定先验的最佳设计是具有挑战性的，因此代谢工程师通常需要构建和评估大量途径变体。我们报告了一种一般策略，该策略结合了针对性的全基因组诱变，以生成途径变体，并进化以富含稀有高生产者。我们通过使用响应化学物质的传感器域来控制选择性条件下生存所需的记者基因，将目标化学物质的细胞内存在转化为细胞的适应性优势。由于人工选择倾向于扩大非生产性的作弊者，因此我们设计了一个负面选择方案，以消除作弊者，同时保留图书馆的多样性。该方案使我们能够执行多个回合的进化（每轮相似，每发10（9）个牢房），每轮后的作弊者的遗留最小。基于通过通量平衡分析鉴定的候选基因，我们使用靶向全基因组诱变来改变参与纳林宁和葡萄糖酸的产生的途径基因的表达。通过多达四轮的进化，我们分别将Naringenin和Glucaric Acid的产生增加了36倍和22倍。葡萄糖中的纳林宁蛋白产生（61 mg/L）是先前报道的最高滴度的两倍以上。进化菌株的全基因组测序揭示了可能受益于生产的其他非靶向突变，这表明了新的优化途径。
Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing similar to 10(9) cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36- and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Whole-genome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.