Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.

工程生物技术微生物以甲醇作为生物生产原料是合成代谢界的主要目标。在这里，我们旨在重新设计天然丝氨酸循环以在大肠杆菌中实施。我们提出了基于两个混杂甲醛醛缩醛酶反应的高丝氨酸循环，作为高级途径设计。就生物量产量，热力学适应性以及与宿主内源性代谢的整合而言，高丝氨酸循环有望优于丝氨酸循环及其变体。即使与RuMP循环相比，RuMP循环是最有效的天然甲醇同化途径，高丝氨酸循环也有望支持多种产品的更高收率。我们在体内测试通过构建几种大肠杆菌基因缺失菌株来实现高丝氨酸循环的可行性，这些菌株的生长与不同途径片段的活性有关。使用这种方法，我们证明了所有必需的混杂酶都具有足够的活性，能够使营养缺陷型菌株生长。因此，我们的发现确定了一种新型的代谢解决方案，该解决方案为优化的甲基营养平台开辟了道路。