The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea. Here, using genetic and metabolic engineering, we generate an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. In vitro and/or in vivo analysis of genes involved in adenosylcobinamide phosphate biosynthesis from Rhodobacter capsulatus suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increase the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg g−1 DCW via metabolic engineering and optimization of fermentation conditions. Beyond our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.
The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ~250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.
The class II clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR–Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.
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