Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.
Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.
Campos
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N
Department de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, C/Martí i Franquès 1, 08028 Barcelona, Spain. campos@sun.bq.ub.es
Rodríguez-Concepción
M
M
Sauret-Güeto
S
S
Gallego
F
F
Lois
L M
LM
Boronat
A
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D005818:Genetic Engineering; D045782:Hemiterpenes; D006801:Humans; D008798:Mevalonic Acid / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D009876:Operon / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010641:Phenotype; D019761:Phosphorus-Oxygen Lyases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D013403:Sugar Phosphates / Q000378:metabolism*
C114232:2-C-methylerythritol 4-phosphate; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D013403:Sugar Phosphates; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C404502:IspE protein, E coli; C021796:mevalonate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase; C400183:4-diphosphocytidyl-2-C--methylerythritol synthase; D009713:Nucleotidyltransferases; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase; D019761:Phosphorus-Oxygen Lyases; C400181:ISPF protein, E coli; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D004896:Erythritol; D008798:Mevalonic Acid