Vitamin B₁₂ (cobalamin) is crucial for various cellular metabolic processes, including DNA synthesis and mitochondrial function. It exists in several forms, with methylcobalamin (MeCbl) and AdoCbl being the bioactive forms. Cyanocobalamin (CNCbl) is a stable, industrially produced form. Historically, vitamin B₁₂ production relied on the challenging chemical synthesis developed by Woodward and Eschemoser. Currently, microbial fermentation using strains like *Pseudomonas denitrificans* is the primary method. However, these methods have limitations, including lengthy fermentation times and limited genetic engineering tools for optimization. Therefore, alternative production methods are needed. Cell-free enzymatic systems offer an advantageous alternative to metabolic engineering, providing flexibility, efficient separation and purification, and high tolerance to toxic intermediates. While cell-free synthesis of various compounds, such as fatty acids and monoterpenes, has been achieved, the de novo synthesis of cobalamin remained a significant challenge due to its intricate and lengthy biosynthetic pathway. This study aims to overcome this challenge by reconstituting a synthetic cell-free platform for AdoCbl synthesis, utilizing 5-ALA as the starting substrate.

Previous research has explored the enzymatic synthesis of various vitamin B₁₂ intermediates. For example, hydrogenobyrate (HBA), a metal-free corrinoid precursor, has been synthesized from 5-ALA via 17 enzyme-catalyzed reactions. In vitro reconstitution of nucleotide loop assembly (NLA), assembling the lower ligand, has also been reported. Additionally, cell-free synthesis of precorrin-2, a key precursor, has been demonstrated. However, these studies did not achieve the complete de novo synthesis of cobalamin due to the complexity of the pathway, involving dozens of steps. The current study builds upon this previous work by aiming for the complete cell-free synthesis of AdoCbl, addressing the challenges posed by the long and complex biosynthetic pathway.

This study designed a cell-free platform for AdoCbl synthesis from 5-ALA, utilizing the aerobic pathway. The pathway was divided into five synthetic modules (precursor, HBA, AdoCby, AdoCbl, and two branch modules) and five cofactor regeneration modules. Each module was individually optimized using a design-test-optimize cycle. The precursor module's challenge was the oxidation of uroporphyrinogen III to uroporphyrin III, which was addressed by using β-mercaptoethanol. The HBA module faced feedback inhibition from S-adenosylhomocysteine (SAH), which was mitigated using methionine adenosyltransferase (MetK) and SAH hydrolase (MtnN). The AdoCby module required optimizing the cobalt-chelation reaction and developing a stop-flow detection method for adenosylcobyrate (AdoCby). The cofactor regeneration modules addressed the supply of SAM, NADH, ATP, L-glutamine, and 5-ALA, while reducing SAH and polyphosphate accumulation. Key enzymes, including transmembrane enzymes (CbiB and CobS), were expressed using a SIMPLEX complex to improve stability. AdoCbl was detected using UPLC-MS, and a cyanide-free method was developed for conversion to CNCbl for improved stability. Enzyme purification, intermediate purification, reaction system setups, and analytical methods are detailed in the methods section of the original paper.

The study successfully reconstituted a cell-free platform for AdoCbl synthesis using 36 enzymes from various microorganisms. The complete pathway, encompassing 32 steps, was divided into modules, allowing for individual optimization. Significant improvements were achieved by addressing the oxidation of intermediates (using β-mercaptoethanol) and feedback inhibition (using MetK and MtnN). A stop-flow detection method was developed to monitor AdoCby. A customized cofactor regeneration system further enhanced the system's efficiency. The optimized system produced 417.41 µg/L AdoCbl from 5-ALA and 5.78 mg/L from purified HBA. The use of a batch reaction mode (synthesizing HBA first, then adding enzymes for downstream reactions) was key to improving yields. The transmembrane enzymes CbiB and CobS were expressed more effectively using the SIMPLEX complex. A cyanide-free method for converting AdoCbl to CNCbl for detection was developed.

This study demonstrates a successful cell-free synthesis of AdoCbl, offering a potential alternative to microbial fermentation for vitamin B₁₂ production. The modular approach and optimization strategies highlight the power of synthetic biology in tackling complex metabolic pathways. The achieved titers, while not yet competitive with industrial fermentation in terms of absolute yield, show promise for future development. The strategies of optimizing individual modules, addressing feedback inhibition, and developing a cofactor regeneration system provide valuable insights for constructing other cell-free systems for complex molecule production. The improved yield using the batch reaction method highlights the importance of carefully considering metabolic flux and reaction equilibrium in cell-free systems.

This research successfully constructed a cell-free system capable of synthesizing AdoCbl from 5-ALA, achieving a significant increase in titer compared to previous attempts. The modular design and optimization strategies provide a valuable template for future cell-free synthesis of other complex natural compounds. Future work should focus on further titer improvement through optimization of enzyme kinetics, cofactor regeneration, and reaction conditions. Exploring alternative, less expensive cofactor regeneration methods could enhance the economic viability of this approach.

While this study demonstrates a significant advancement in cell-free vitamin B₁₂ synthesis, further improvements are needed to make it industrially competitive. The current system requires a relatively high concentration of purified enzymes and cofactors, which increases the cost. Optimizing the cofactor regeneration system to minimize the use of expensive reagents and exploring enzyme immobilization strategies to improve enzyme stability and reusability are crucial for future industrial applications. The instability of certain intermediates also needs to be addressed for larger-scale production.