The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C-C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is first provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond formation for the transformation of CO₂, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.

Microbial protein production from acetate represents a promising route for sustainable protein supply, yet its efficiency is constrained by limited understanding of carbon–nitrogen metabolic coordination. In this study, nitrogen availability was systematically varied to investigate its role in regulating biomass composition and protein biosynthesis in Yarrowia lipolytica. Nitrogen limitation markedly reduced cell growth and protein accumulation (19.56% of dry cell weight) while increasing lipid content (up to 34.16%), indicating a altered protein and lipid accumulation under different nitrogen conditions. Transcriptomic analysis revealed a global downregulation of anabolic pathways under nitrogen limitation, accompanied by a shift in nitrogen assimilation from the glutamate dehydrogenase (GDH) pathway to the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway, as well as significant upregulation of genes related to ammonium and amino acid transport. Guided by these findings, metabolic engineering of key nitrogen assimilation pathways was performed. Strains harboring additional copies of GDH and GS expression cassettes showed increased protein content from 48.52% to 55.77% and improved amino acid composition, whereas strains with an additional copy of the GOGAT gene exhibited reduced growth and protein accumulation. These results demonstrate that nitrogen availability regulates biomass composition through coordinated control of nitrogen transport and assimilation, and that balanced upregulation of GDH and GS genes is an effective strategy to improve microbial protein production from acetate, supporting the development of efficient fermentation processes using low-cost carbon sources.

Microbial protein production from acetate represents a promising route for sustainable protein supply, yet its efficiency is constrained by limited understanding of carbon–nitrogen metabolic coordination. In this study, nitrogen availability was systematically varied to investigate its role in regulating carbon partitioning and protein biosynthesis in Yarrowia lipolytica. Nitrogen limitation markedly reduced cell growth and protein accumulation (19.56% of dry cell weight) while increasing lipid content (up to 34.16%), indicating a redistribution of carbon flux from protein to lipid synthesis. Transcriptomic analysis revealed a global downregulation of anabolic pathways under nitrogen limitation, accompanied by a shift in nitrogen assimilation from the glutamate dehydrogenase (GDH) pathway to the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway, as well as significant upregulation of genes related to ammonium and amino acid transport. Guided by these findings, metabolic engineering of key nitrogen assimilation pathways was performed. Co-overexpression of GDH and GS increased protein content from 48.52% to 55.77% and improved amino acid composition, whereas GOGAT overexpression impaired growth and protein accumulation. These results demonstrate that nitrogen availability governs carbon allocation through coordinated regulation of nitrogen transport and assimilation, and that balanced enhancement of GDH and GS is an effective strategy to improve protein production from acetate, supporting the development of sustainable fermentation processes using CO₂-derived substrates.

Acrylic acid is a bulk chemical predominantly produced via fossil-based processes. Here, we repurpose and engineer a thiamine diphosphate (ThDP)-dependent enzyme to catalyze direct \({{{\rm{H}}}}_{2}{{{\rm{PO}}}}_{4}^{-}\) elimination from dihydroxyacetone phosphate (DHAP) for acrylic acid synthesis. Quantum chemical calculations reveal the catalytic mechanism involving isomerization, enol-ThDP intermediate formation, \({{{\rm{H}}}}_{2}{{{\rm{PO}}}}_{4}^{-}\) elimination, and hydrolysis. Using an automated high-throughput screening platform, we perform directed evolution and obtain a 13.6-fold improved variant, designated acrylic acid synthase (AAS). We establish an enzymatic methanol-to-acrylic acid (MAAP) pathway, achieving a titer of 4.3 g L⁻¹, a productivity of 268.9 mg L⁻¹ h⁻¹, and 99.5% conversion. This work establishes a high-efficiency biomanufacturing paradigm for bulk acrylic acid, and expands the catalytic repertoire of ThDP-dependent enzymes for sustainable one-carbon conversion.