Bacterial heme synthetic pathway: determination of the bottleneck steps

Heme is essential for living organisms for its function involved in oxygen transfers and redox reactions in electron transfer systems, and bacteria synthesized heme is compatible for the heme in mice. Intracellular aminolevurinic acid (ALA) concentration, the precursor of heme, is a critical regulation signal in the heme synthetic pathway in bacteria. Rhodobacter sphaeroides-derived ALA synthase (hemA+flanking region, catalyzing ALA formation from succinyl-CoA and glycine as substrate through C4-pathway) and self-derived pantothenate kinase (coaA, the first enzyme of the pantothenic acid synthetic pathway) were expressed in Escherichia coli to overproduce bacterial heme. Though the expressions of hemA and coaA enabled the recombinant E. coli to increase ALA and CoA pool leading heme production, the amount of heme was only few microgram per biomass and low for industrial application. To decide the potential bottleneck step in the heme biosynthetic pathway, intermediates from the steady state cells were analyzed by LC-MS. Accumulations of coproporphyrinogen I and protoporphyrin IX indicated that heme production could be further enhanced by overcome the bottleneck steps of the spontaneous conversion of HMB to coproporphyrinogen I and ferrous ion incorporation of protoporphyrin IX to heme. Succeeding pathway engineering of E. coli is on the way.