Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium.
Bottom Line: Similarly, the removal of the B(12) riboswitch upstream of the cbiXJCDETLFGAcysG(A)cbiYbtuR operon and the recombinant production of three different vitamin B(12) binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B(12)-dependent feedback inhibition.All these strategies increased cobalamin production in B. megaterium.Finally, combinations of these strategies enhanced the overall intracellular vitamin B(12) concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.
Affiliation: Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.Show MeSH
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Mentions: To evaluate the physiological consequences of ashemZ expression, we determined the coproporphyrinogen III : protoporphyrinogen IX as well as the heme and vitamin B12 ratio in a strain harbouring pWH1520ashemZ and compared it with an appropriate wild‐type control strain. For simplicity, coproporphyrinogen III and protoporphyrinogen IX were oxidized by hydrogen peroxide treatment to their corresponding porphyrins, which are more easily detectable via fluorescence spectroscopy. Coproporphyrin III shows two distinct emission peaks at 579 and 620 nm when excited by light of wavelength 409 nm while protoporphyrin IX is characterized by a single peak at 633 nm (Fig. 4A). Figure 4B shows a fluorescence spectrum of cell free extracts of B. megateriumstrain DSM509 carrying pWH1520ashemZ in comparison with a spectrum obtained from B. megaterium DSM509 carrying the empty vector pWH1520. A comparison of the peak maxima reveals that more coproporphyrinogen III is accumulated in the strain producing ashemZ (Fig. 4C). Furthermore, the accumulation of coproporphyrinogen III results in decreased heme formation in the ashemZ‐producing B. megaterium (data not shown). Higher levels of coproporphyrinogen III are also likely to inhibit the HemE reaction by product inhibition (Jones and Jordan, 1993). Significantly, though, this antisense approach led to an increase in the intracellular vitamin B12 concentration by 20% (0.05 µg l−1 OD578nm−1 and 0.31 µg l−1, respectively) (Table 1).
Affiliation: Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.