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Modular Engineering of l-Tyrosine Production in Escherichia coli

机译:大肠杆菌中左旋酪氨酸生产的模块化工程

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摘要

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.
机译:从葡萄糖有效地生物合成l-酪氨酸对于使生物生产在经济上可行是必要的。为此,我们通过在两个质粒上编码将赤藓糖-4-磷酸酯(E4P)和磷酸烯醇丙酮酸(PEP)转化为1-酪氨酸的酶,设计并构建了在大肠杆菌MG1655中生产1-酪氨酸的模块化生物合成途径。靶向蛋白质组学和代谢物谱分析指导了合理的工程设计,以提高l-酪氨酸的产量并确定途径瓶颈。通过修饰质粒拷贝数,启动子强度,基因密码子使用以及操纵子中基因的位置,可以缓解该途径中的瓶颈。一个主要的瓶颈是由于奎宁酸盐/ shi草酸酯脱氢酶(YdiB)的双功能活性,导致中间体中间体脱氢奎宁酸酯(DHQ)和脱氢shi草酸酯(DHS)以及副产物奎宁酸酯的积累。通过用其对等物AroE代替YdiB可以缓解这一瓶颈,从而产生了超过700毫克/升的iki草酸酯。由于脱氢奎宁酸合酶(AroB)的低表达,sh草酸酯生产中的另一个瓶颈通过优化基因的前15个密码子得以缓解。通过用其同功酶AroL代替the草酸酯激酶AroK,可以改善草酸酯向L-酪氨酸的转化,该酶同功酶AroL有效地消耗了该途径上半部分形成的所有中间体。在蛋白质和代谢物测量的指导下,由两个中等拷贝数的双操纵子质粒组成的最佳生产者被优化为以理论收率的80%产生> 2 g / L的L-酪氨酸。这项工作证明了靶向蛋白质组学和代谢物谱分析在途径构建和优化中的实用性,应该适用于其他代谢途径。

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