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.
Kim
Jin-Seog
JS
Sustainable Chemical Technologies Division, Korea Research Institute of Chemical Technology, Daejeon, Korea.
Lim
He Kyoung
HK
Lee
Myung Hwan
MH
Park
Ji-Hye
JH
Hwang
Eul Chul
EC
Moon
Byung Ju
BJ
Lee
Seon-Woo
SW
D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008969:Molecular Sequence Data; D012275:Oryza; D010860:Pigments, Biological / Q000378:metabolism; D011166:Porphyrins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D004269:DNA, Bacterial; D010860:Pigments, Biological; D011166:Porphyrins