title: Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.
abstract: BACKGROUND
  Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12.
  
  
  RESULTS
  Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved.
  
  
  CONCLUSIONS
  By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station 77843-3255, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Roessner
authors.forename: J B
authors.initials: JB
authors.lastname: Spencer
authors.forename: N J
authors.initials: NJ
authors.lastname: Stolowich
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: G P
authors.initials: GP
authors.lastname: Nayar
authors.forename: P J
authors.initials: PJ
authors.lastname: Santander
authors.forename: C
authors.initials: C
authors.lastname: Pichon
authors.forename: C
authors.initials: C
authors.lastname: Min
authors.forename: M T
authors.initials: MT
authors.lastname: Holderman
authors.forename: A I
authors.initials: AI
authors.lastname: Scott
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014578:Uroporphyrins / Q000096:biosynthesis* / Q000737:chemistry; D014805:Vitamin B 12 / Q000096:biosynthesis*
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; D014578:Uroporphyrins; C066509:precorrin 6x; C072476:hydrogenobyrinic acid; D014805:Vitamin B 12