Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by ¹³C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future.

乙酰辅酶A是地球上所有生命的基本代谢产物，也是各种工业化学品和天然产物生物合成的关键起点。在这里，我们通过重新利用乙醇醛合酶和乙酰磷酸合酶来设计和构建合成乙酰辅酶A（SACA）途径。首先，我们设计并设计了乙醇醛合酶，以将催化活性提高70倍以上，从而将两个甲醛分子缩合为一个乙醇醛。第二，我们重新利用磷酸酮醇酶将乙醇醛转化为乙酰磷酸的能力。我们证明了SACA途径在体外的可行性，实现了约50％的碳收率，并通过¹³证实了SACA途径C标记的代谢物。最后，通过使用乙醇醛，甲醛和甲醇作为补充碳源的细胞生长来验证SACA途径。事实证明，SACA途径是乙酰辅酶A生物合成的最短，不依赖ATP，不耗碳且对氧不敏感的途径，这为将来从一碳资源生产乙酰辅酶A衍生的化学品提供了可能性。