title: High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis.
abstract: Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 micro mol/liter, which is close to industrial vitamin B(12) production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of E. coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Saint Paul, Minnesota 55108, USA.
authors.forename: Seok Joon
authors.initials: SJ
authors.lastname: Kwon
authors.forename: Arjo L
authors.initials: AL
authors.lastname: de Boer
authors.forename: Ralf
authors.initials: R
authors.lastname: Petri
authors.forename: Claudia
authors.initials: C
authors.lastname: Schmidt-Dannert
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005294:Ferrochelatase / Q000378:metabolism; D005818:Genetic Engineering; D006418:Heme / Q000096:biosynthesis*; D006877:Hydroxamic Acids / Q000378:metabolism; D011166:Porphyrins / Q000096:biosynthesis*
chemical_list: D006877:Hydroxamic Acids; D011166:Porphyrins; D006418:Heme; C010896:coprogen; D000624:5-Aminolevulinate Synthetase; D005294:Ferrochelatase