Effect of blocking the haem synthesis pathway and weakening the haem synthesis pathway for sirohaem on the growth of and vitamin B12 synthesis in Ensifer adhaerens Casida A.

Abstract

To block and weaken the bacterial branched VB12 synthetic metabolic pathway, homologous recombination technology was used to knock out the sirohaem synthase gene cysG located in the chromosome and the endogenous A plasmid of the Ensifer adhaerens Casida A strain, and the expression of the uroporphyrinogen III decarboxylase gene hemE was weakened by weak promoter substitution. The growth of the engineered strains and the production of VB12 and haem were analysed and measured in the engineered strains, aiming to provide a new strategy for enhancement of VB12 biosynthesis. The results showed that the chromosomal cysG gene knockout strain ΔcysG, endogenous A plasmid cysG gene knockout strain ΔpAcysG and cysG gene double knockout strain ΔcysGΔpAcysG grew normally, with VB12 yield increases of 19.9%, 11.2%, and 27.4% compared to the starting strain, respectively. In the background of the cysG gene knockout strain, the expression of the hemE gene was weakened, resulting in the generation of the strain ΔcysGΔpAcysG-E-pdnaD, and the VB12 yield of ΔcysGΔpA cysG-E-pdnaD reached 114.17 ± 5.77mgL-1, an increase of 45.1% compared to the yield of the original strain. The above results indicate that the strategy of increasing VB12 production by knocking out the haem synthesis pathway and weakening the haem synthesis pathway is effective.