An Investigation into the Relationship Between Adenine Modification and Cobamide Lower Base Synthesis

Cobalamin, also known as vitamin B12 is a widely used cofactor in cellular metabolism but is only synthesized in a few species of prokaryotes. Interestingly, cobalamin in the large intestine is not bioavailable. But 25% of sequenced human intestinal bacteria have the capacity to produce corrinoids and 80% produce gene products that potentially require corrinoids, leading to some questions about the diversity of cobamides. This project specifically takes interest in adenine and 2-methyladenine as lower ligands in cobamides. Which are present in some biomes such as the large intestine and are even produced by the flora which reside there. Even though they appear frequently in samples, little is known about the precursors to these lower ligands. RlmN is a dual specificity methyl transferase which methylates adenine residues on tRNA and rRNA. In this project, we seek to investigate the effects of adenine supplementation and the deletion of this adenine methyltransferase on the lower ligands of cobamides synthesized by genetically modified E. coli. This project demonstrated that the deletion of the rlmN gene was possible via CRISPR Cas9 gene deletion facilitated by homology directed repair with donor DNA. Additionally, it was demonstrated that the deletion may affect the growth rate of the deletion strain but is ultimately non-lethal. Adenine supplementation appeared to impact the variety of lower ligand incorporated to form a cobamide in the absence of DMB. Without supplemented adenine, as an intracellular booster or externally with powder, 2-methyladenine appears to be the main preferred lower ligand to be incorporated into a cobamide and produces the highest ratio of cobamides. Contrastingly, when supplemented with adenine, with an intracellular booster or externally, adenine becomes the main lower ligand of the cobamides produced by ratio. Interestingly, the addition of an intracellular adenine booster results in the loss of GDP cobinimide production. This project demonstrates the that the precursors for 2-methyladenine as a lower base are of intracellular origin, possibly from rlmN methylated t-RNA. Additionally, we have demonstrated that cobamides are preferentially produced when supplemented with adenine, even in the absence of DMB.