The Exploration of Escherichia coli as a Host for Vitamin B12 Biosynthesis: Engineering Pathway Flux, Cobalt Uptake and Reporter Systems

Vitamin B12 (cobalamin) is nature’s most structurally complicated vitamin. It is essential for humans where it is utilised by two vitamin B12-dependent enzymes: methylmalonyl-CoA mutase and methionine synthase. Vitamin B12 deficiency leads to a broad range of symptoms including tiredness, depression and headaches, though if severe can lead to organ failure and death. As a nutrient, vitamin B12 is produced commercially through fermentation and

requires the inclusion of cobalt within the large-scale fermentation bioreactors, which poses environmental risk concerns. Bacteria that have been used for the commercial production of vitamin B12 include Pseudomonas denitrificans, Propionobacterium shermanii and Sinorhizobium meliloti but these are all fairly slow growing and require specialise fermentation conditions. The era of synthetic biology has ushered in an optimism that biological processes can be improved and adapted for useful purposes through the rational design, build, test, redesign engineering cycle of problem solving. The advent of CRISPR-Cas9 technology has accelerated this viewpoint with Escherichia coli often used as the delivery chassis for this kind of biotechnological advancement. E. coli has a proven track record for biotechnological applications, is fast growing, has established fermentation protocols and represents a natural

model for a synthetic biology project on vitamin B12 production.

Although E. coli does not make vitamin B12 de novo, it has previously been used for the heterologous production of many vitamin B12 biosynthetic enzymes. Consequently, it was decided to clone the complete pathway for vitamin B12 biosynthesis into E. coli allowing the nutrient to be synthesised de novo. This strain is at an early design stage and is ready for further optimisation to increase the level of B12 production to industrial levels with

improvements on issues concerning environmental concerns.

The research in this thesis has initiated the first cycles of engineering improvements on a vitamin B12 producing strain of E. coli. Investigations were performed on the heme-branching enzyme, HemE, in order to try and reduce heme synthesis and promote B12 synthesis. Mutant enzymes of HemE were generated, characterised and incorporated into the B12-producing strain. However, the resultant strains did not improve B12 production. Cobalt acquisition and

incorporation into the B12 -producing strain was also studied. Here, a number of cobalt transporters were investigated for their effectiveness at cobalt uptake and one particular transporter was identified. Potential cobalt exporters were also inactivated and the combined effect of improved cobalt uptake and reduced cobalt export resulted in a strain that required much less cobalt to be added to the growth medium. This general principal holds great

potential not only for the engineering of B12-producing strains, but also for any protein that requires a heavy metal. Finally, a rapid reporter system/screen to help in the identification Abstract iii of strains that have enhanced B12-production levels was investigated. This involved the

development of a B12-responsive riboswitch mediated indicator strain. The limitations of this strain are discussed.