Production of vitamin B12 in escheria coli Ida Ruuth

Production of vitamin B12 Vitamin B12 is widely used in medical industry and food industry Need for large scale industrial production Usually produced by Pseudomonas denitrificans, Propionibacterium shermanii, or Sinorhizobium meliloti Long fermentation cycles, complex and expensive media requirements and lack of suitable genetic systems for strain engineering

E. coli Fast growing, no expensive or complex media requirements Well-known Easy to modify genetically Has ability to produce ALA via C5 pathway Precursor for vitamin B12  E. coli lacks about 20 genes required to synthesize the central ring structure of vitamin B12   Has no ability to synthesize vitamin B12 compounds de novo

Pathway of adenosylcobalamin Complex pathway Fusion enzyme CysG is shared between cobalamin and siroheme synthesis. Cobalamin riboswitches regulate cobalamin biosynthesis and transportation at the transcriptional or translational level  btuB gene of E. coli 

Production of vitamin B12 in E.coli More effective ALA supply Expression of HemA from R. palustris Building biosynthetic pathway by using pathway tuning and functional components Genes involved in B12 vitamin pathway into E. coli from S. typhimurium Optimization of pathways Regulation btuB gene

Challenges and Problems How introduced genes function in E. coli Complex pathway and complex structure of vitamin B12 Balance of expressed genes and enzymes Challenges in expressing a great amount of recombinant genes More than 20 genes are involved Balanced supply of precursors

Previous studies Expression of the operon pduBAF-pocR-cbiABCDETFGHJKLMNQOP- cobUST from S. typhimurium into E. coli strain  de novo cobalamin biosynthesis  By Ko et al. twenty-two were cloned via restriction and ligation into three compatible plasmids under the control of a T7 promoter and introduced into E. coli strain.  Vitamin B12 was produced de novo under both anaerobic and aerobic conditions

References Dmitry A. Rodionov et al. 2003. Comparative Genomics of the Vitamin B12 Metabolism and Regulation in Prokaryotes. The Journal of Biological Chemistry. 278, 41148-41159. DOI: 10.1074/jbc.M305837200 Huan Fang et al. 2016.Microbial production of vitamin B12: a review and future perspectives. Microbial Cell Factories. DOI: 10.1186/s12934-017-0631-y Zhang, L., Chen, J., Chen, N. et al. 2013. Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production. Biotechnol Lett. 35: 763. doi:10.1007/s10529-013-1143-4