Lu, X; Liu, Y; Yang, Y; Wang, S; Wang, Q; Wang, X; Yan, Z; Cheng, J; Liu, C; Yang, X; Luo, H; Yang, S; Gou, J; Ye, L; Lu, L; Zhang, Z; Guo, Y; Nie, Y; Lin, J; Li, S; Tian, C; Cai, T; Zhuo, B; Ma, H; Wang, W; Ma, Y; Liu, Y; Li, Y; Jiang, H
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.