Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in , which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

中文翻译：

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通过正交基因表达控制迭代途径的代谢通量优化：在β-氧化逆转中的应用

平衡途径基因的相对表达以尽量减少通量瓶颈和代谢负担是代谢工程的关键挑战之一。这对于迭代途径尤其重要，例如反向β-氧化（rBOX）途径，该途径需要控制多个节点的通量分配以实现目标产物的高效合成。在这里，我们开发了一种基于质粒的诱导系统，用于正交控制基因表达（称为 TriO 系统），并证明了其在 rBOX 途径中的实用性。通过以即插即用的方式轻松构建 TriO 载体，我们同时探索了酶选择和相对表达水平的解决方案空间。值得注意的是，不同的个体表达水平导致产物特异性发生显着变化，范围从不产生到所需产物理论产量约 90% 的最佳性能。我们从甘油中获得了 6.3 g/L 丁酸、2.2 g/L 丁醇和 4.0 g/L 己酸的滴度，这超过了之前使用等效酶组合报道的最佳滴度。由于在替代终止路径和高阶迭代中观察到了类似的系统行为，因此我们设想我们的方法将广泛适用于除 rBOX 之外的其他迭代路径。考虑到使用组合启动子和 RBS 文库的高通量、自动化菌株构建对于许多研究人员（尤其是学术界）来说仍然遥不可及，像 TriO 系统这样的工具可以通过降低成本、时间和基础设施要求，使通路设计的测试和评估民主化。