Elucidation of the anaerobic biosynthesis of vitamin B₁₂ in Bacillus megaterium

Vitamin B₁₂ (cobalamin) is one of Nature's most complex small molecules. It is a cobalt-containing modified tetrapyrrole that plays a number of key metabolic roles in many prokaryotic systems and is also essential to the biochemistry of higher animals. However, it is made by only certain bacteria, requiring around thirty enzymatic steps for its complete de novo construction. Surprisingly, the nutrient is synthesised by one of two related, though genetically distinct, pathways that represent aerobic or anaerobic routes. The anaerobic pathway has remained poorly characterised due to the instability of the pathway intermediates to oxygen and the low activity of enzymes, and is the focus of the research reported in this thesis. The Gram-positive aerobe Bacillus megaterium has previously been used for the commercial production of cobalamin and has a complete anaerobic pathway. Several genes, (termed chi for cobinamide biosynthesis) from the cobalamin biosynthetic pathway have been cloned and overexpressed individually within the host B. megaterium DSM319. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, where only limited yields (<5 %) of product had previously been obtained using crude-lysate based incubations. By studying the purified B. megaterium CbiH60 enzyme, an efficient method was developed to allow the generation of the ring-contracted product cobalt-factor IV in high yields. This breakthrough then permitted the characterisation of many of the down-stream steps in the pathway to be characterised by using other purified cobalamin biosynthetic enzymes from B. megaterium, by incubating combinations of purified enzymes with cobalt-factor IV and the appropriate cofactors (SAM, NADH). This has resulted in the successful step-by-step elucidation of the anaerobic cobalamin pathway, including the isolation of the long sought-after and elusive intermediates cobaltprecorrin- 5, -6A, -6B, -7 and -8. An enzyme-cocktail approach has also resulted in a full in vitro synthesis of cobyrinic acid (the first known stable intermediate in the pathway) from 5-aminolevulinic acid has been demonstrated, involving a full 15 enzymatic steps. The exposition of the anaerobic pathway has been interpreted with respect to the chemical logic of the metabolic process and the evolution of multifaceted biochemical pathways.