1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.

Synthetic biology is contributing to the advancement of the global net-negative carbon economy, with emphasis on formate as a member of the one-carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within 2 months. This effort resulted in two mutant strains, designated as M25-70 and M25-14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the upregulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine-based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Y. lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase-based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Y. lipolytica.

Formic acid (HCOOH) and dihydrogen (H2) are characteristic products of enterobacterial mixed-acid fermentation, with H2 generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H2, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H2 and CO2 by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H2 production in enterobacteria.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

The photosynthetic autotrophic production of microalgae is limited by the effective supply of carbon and light energy, and the production efficiency is lower than the theoretical value. Represented by methanol, C1 compounds have been industrially produced by artificial photosynthesis with a solar energy efficiency over 10%, but the complexity of artificial products is weak. Here, based on a construction of chloroplast factory, green microalgae Chlamydomonas reinhardtii CC137c was modified for the bioconversion of formate for biomass production. By screening the optimal combination of chloroplast transport peptides, the cabII-1 cTP1 fusion formate dehydrogenase showed significant enhancement on the conversion of formate with a better performance in the maintenance of light reaction activity. This work provided a new way to obtain bioproducts from solar energy and CO2 with potentially higher-than-nature efficiency by the artificial-natural hybrid photosynthesis.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 μg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 μg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 μg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method. KEY POINTS: • Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes • Performance as an F-Tag is not restricted to particular structural motifs • Separation from untagged protein benefits from low foam stability and foam washings.