Production of porphyrin intermediates in Escherichia coli carrying soil metagenomic genes

Tetrapyrrole pigments are important components of many biological processes, and many of them are produced primarily by microorganisms. We constructed a soil metagenomic library using rice paddy soil consisting of 107 000 fosmid clones with an average DNA insert size of 35 kb. We isolated a clone carrying genes in the porphyrin biosynthetic pathway based on function-driven screening of the library. Through subcloning and mutagenesis analysis, we showed that two genes from soil metagenome, gtrA and hemC, were responsible for pigmentation in Escherichia coli. HPLC and LC-MS analysis of the purified pigments from E. coli carrying pSY143 identified coproporphyrin III without metal as a major compound as well as some other minor porphyrin intermediates. As gtrA and hemC encode glutamyl-tRNA reductase and porphobilinogen deaminase, respectively, which are enzymes involved in the C₅ biosynthetic pathway for porphyrin intermediates, our results suggest that hemL, hemB, hemD, and hemE should be provided by the E. coli chromosome to generate a hybrid biosynthetic pathway for production of porphyrin intermediates using E. coli and metagenomic genes.