Global warming and climate change, mainly caused by the emission of carbon dioxide, is becoming a serious problem, and it is imperative to develop efficient carbon capture and utilization technologies for utilizing CO₂ and its derived one-carbon (C1) compounds. The use of CO₂ for biomanufacturing of valuable commodities, including industrial chemicals and food ingredients, is of great importance in the transition from a fossil-dependent economy to a green and sustainable bioeconomy. In the traditional CO₂-fixation biological pathways regardless of natural or synthetic, they usually require expensive energy (ATP, NADPH) cofactors, display low thermodynamic driving forces, and have limited biosynthetic rates.

To debottleneck these limitations, we designed an Integrated ChemoEnzymatic CO₂ to Amino-acid Pathway, called ICE-CAP, that demands no cofactors in terms of ATP and NAD(P)H and can transform air-captured CO₂ as well as gaseous CO₂ into amino acids effectively. ICE-CAP is based on the engineered glycine cleavage system (GCS) coupled with the co-metabolism of energy-rich C1 compound methanol or formaldehyde. The thermodynamic analysis highlights the significant advantages of this combination, which is much better than the traditional reductive glycine pathway based on formic acid. In ICE-CAP, methanol can give rise to formaldehyde through methanol oxidase (A), then the self-condensation of formaldehyde and tetrahydrofolate (THF) generates a C1 donor: 5,10-CH2-THF, which afterward, combines with ammonium and CO₂ to produce glycine after the catalysis of aminomethyltransferase (T) and glycine decarboxylase (P), respectively. Glycine can be further extended to serine and pyruvate by serine hydroxymethyltransferase (S) and serine deaminase (D).

ICE-CAP has several unique features and advantages.

1. Excellent combination of biocompatible chemistry with enzymatic catalysis in one-pot synthesis. The key non-enzymatic reactions lie in DTT (dithiothreitol) dependent sulfur-reducing chemistry to regenerate the reduced form of lipoylated H protein from its oxidized version, and the spontaneous condensation between formaldehyde and THF (tetrahydrofolate). The use of DTT chemistry in setting up artificial electron transfer is able to replace L protein (dihydrolipoamide dehydrogenase), avoid the requirement of expensive NADH, enhance the biological thermodynamic driving force, turn the reversible GCS into irreversible glycine-synthesis direction, prevent the polymerization of carboxylase enzymes, and thus enhance glycine production rate dramatically in combination with the rGCS system.
2. Double C1 fixation strategy. ICE-CAP applies the incorporation of one electron-intensive C1 compound (methanol or formaldehyde) to capture the electron-deficient CO₂ molecules, which offers an efficient manner to make full use of the input energy. With this double C1 fixation strategy, we obtained a glycine production rate of 54 nmol/min/mg protein from methanol and CO₂, which is the highest reported so far. As methanol or formaldehyde can be obtained from CO₂ chemically, the two carbons of glycine can be both derived from CO_2.
3. Direct air capture and utilization. It is only the direct removal of CO₂ from air that can actually reduce the global atmospheric CO₂ concentration. Therefore, it is imperative to develop carbon capture and utilization (CCU) processes. Using air-captured CO₂ as feedstock, we achieved g/L level production of glycine, serine and pyruvate. The capture of CO₂ into bicarbonate and its following conversion to amino acids offer a promising solution for CO₂ valorization. Further development of ammonium hydroxide as a sorbent can generate ammonium bicarbonate that is a perfect carbon and nitrogen source for amino acid biosynthesis, which will further reduce the purification cost of sorbents and benefit this CCU process feasibility.