Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design

Abstract

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.

Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives

“…Although the production and yield of target chemical were increased significantly ( Liu et al, 2019 ), the water-insoluble nature of fatty acids makes it difficult to handle and monitor the fermentation process. Several synthetic pathways for acetyl-CoA from one-carbon substrates have been constructed in E. coli ( Lu et al, 2019 ; Wang et al, 2019 ), but their efficiency remains far below what is required for effective production of chemicals and materials.…”

Growth of E. coli on formate and methanol via the reductive glycine pathway

“…This is in part due to unfavorable cultivation parameters (e.g., cell concentration and growth rate) and low efficiency of the relevant metabolic pathways 9 . Adapting a model biotechnological microorganism for growth on formate or methanol has therefore been a key goal of the synthetic biology community in the last decade [10][11][12][13][14][15][16][17][18][19][20][21] .…”

Syngas and Methanol‐Based Biorefinery Concepts

“…When necessary enzyme activities were heterologously expressed in E. coli, cells could not grow on minimal medium with methanol as sole carbon source, but cells were nonetheless able to assimilate up to 2 g L -1 methanol in the presence of yeast extract which served as a co-substrate. More recently, Lu and co-workers [75] proposed an alternative non-natural pathway for methanol assimilation (see Fig. 5B), in which formaldehyde is condensed into glycolaldehyde that is further converted into acetyl-P.…”

“…The formolase pathway (A) [74] employs two enzymes, a formolase (I) and a dihydroxyacetone kinase (II). The synthetic acetyl-CoA (SACA) pathway (B) [75] employs three enzymes, a glycolaldehyde synthase (III), acetyl-P synthase (IV) and acetyl phosphotransferase (V). The third pathway (C) employs three enzymes, a glycolaldehyde synthase (III), an aldehyde dehydrogenase (VI) and a glycolate dehydrogenase (VII).…”