Engineering of a Synthetic Metabolic Pathway for the Carbon-Conserving Production of Value-Added Compounds from Ethylene Glycol

由于日益增长的环境问题，开发有效利用二氧化碳（CO2）生产增值化学品的生化过程受到越来越多的关注。目前的技术依赖于（电）化学将二氧化碳还原为合成气、甲酸盐或甲醇，所有这些都可以作为微生物生产过程中的底物。目前，甲醇是发酵过程中最常用的c1 -碳底物。然而，在遗传上可适应的微生物中，甲醇同化途径的实施和优化受到阻碍，因为它们在很大程度上与宿主生物的自然代谢重叠。这使得很难确保在再生甲醛接受分子（通常是单磷酸核酮糖或甘氨酸）和将捕获的碳转移到产物之间进行适当的碳通量重新分配。为了解决这一问题，最近描述了一种合成代谢途径，该途径通过特征中间体乙醇醛（GA）将甲醇转化为乙酰辅酶a。GA是由两个甲醛分子直接产生的，因此，优雅地绕过了再生甲醛接受分子的需要。在目前的项目建议中，我们提出了扩展GA产生的合成代谢途径面板的想法。具体来说，我们提出了一种碳保护合成代谢途径，将GA转化为四碳化合物2-氧-4-羟基丁酸，从中可以衍生出包括苏氨酸和2,4-二羟基丁酸在内的几种增值产品。合成途径由四种酶活性组成，其中两种以前没有描述过，将在本项目中进行工程设计。虽然所提出的途径与甲醇的使用完全兼容，但我们将使用乙二醇（EG）作为底物来证明该途径的功能。在项目描述中讨论了用EG代替甲醇的技术优势。

Due to growing environmental concerns, the development of biochemical processes for efficient utilization of carbon dioxide (CO2) for the production of value-added chemicals receives growing attention. Current technologies rely on (electro-)chemical reduction of CO2 to synthesis gas, formate or methanol, all of which can serve as substrates in microbial production processes. At present, methanol is the most popular C1-carbon substrate for use in fermentation processes. However, the implementation and optimization of methanol assimilation pathways in genetically amenable microorganisms is hampered by the fact that they overlap in large part with the natural metabolism of the host organisms. This makes it difficult to assure proper carbon flux repartitioning between regenerating a formaldehyde-accepting molecule, typically ribulose monophosphate or glycine, and diverting captured carbon towards the product. Addressing this problem, a synthetic metabolic pathway has recently been described which converts methanol to acetyl-CoA via the characteristic intermediate glycolaldehyde (GA). GA is directly produced from two molecules of formaldehyde, thus, elegantly by-passing the need of regenerating a formaldehyde-accepting molecule. With the present project proposal, we are putting forward the idea of extending the panel of synthetic metabolic pathways emanating from GA. Specifically, we propose a carbon-conserving synthetic metabolic pathway which converts GA into the four-carbon compound 2-oxo-4-hydroxybutyric acid from which several value-added products including threonine and 2,4-dihydroxybutyric acid can be derived. The synthetic pathway is composed of four enzymatic activities, out of which two have not been previously described and shall be engineered in this project. Although the proposed pathway is entirely compatible with the use of methanol, we shall demonstrate the function of the pathway using ethylene glycol (EG) as the substrate. The technological advantages of using EG instead of methanol are discussed in the project description.