Enzymatic Synthesis of 3-Hydroxypyruvate and Pyruvate from CO ₂ -Derived C1 Compounds

Enzymatic transformation is an attractive strategy for converting C1 compounds derived from CO2 into high-value products. However, its large-scale application is hampered by the necessity of costly cofactors such as ATP, NADH, and tetrahydrofolate. Here, we designed two costly cofactor-free enzymatic pathways that require only 5 enzymes and chemically synthesized cheap cofactor TPP for the production of platform compounds 3-hydroxypyruvate (3-HP) and pyruvate from formaldehyde. We screened and characterized two key enzymes, dihydroxyacetone oxidase catalyzing dihydroxyacetone oxidation and glyoxalase III catalyzing 3-hydroxypyruvaldehyde isomerization, in the designed pathways. We also engineered glyoxalase III SAV0551 toward stereoselectively isomerizing 3-hydroxypyruvaldehyde into l-glycerate to improve the production of 3-HP from formaldehyde. Then, we combined methanol oxidation with alcohol oxidase and the two designed pathways for enzymatic synthesis of 3-HP and pyruvate from CO2-derived methanol. The final concentrations of 3-HP and pyruvate reached 50.2 and 49.6 mM, with synthesis rates of 7.2 mmol C L–1 h–1 and 5.5 mmol C L–1 h–1, respectively. In addition, by coupling transketolase and pyruvate decarboxylase into the enzymatic system, we also achieved the biosynthesis of d-sedoheptulose-7-phosphate and acetoin from CO2-derived 3-HP and pyruvate with concentrations of 46.9 and 23.6 mM, respectively.