Article Figures & Data
Figures
- Fig. 1.
The MEP pathway of IPP and DMAPP biosynthesis inE. coli and genetically engineered synthesis of IPP from exogenously supplied mevalonate. Interrupted lines indicate not fully elucidated steps. Mvk, mevalonate kinase; Pmk, phosphomevalonate kinase; Mpd, mevalonate pyrophosphate decarboxylase; Dxs, DOXP synthase; Dxr, DOXP reductoisomerase; Ipi, IPP isomerase; GAP,d-glyceraldehyde 3-phosphate; P, phosphate; PP, pyrophosphate.
- Fig. 2.
Construction of the synthetic operon conferring the ability to utilize mevalonate for IPP synthesis. The genes coding for Mvk, Pmk, and Mpd were amplified from genomic yeast DNA, thereby introducing ribosome binding sites (indicated by gray lines) with the various primers (A, Mev-kin-Sc-for; B, Mev-kin-Sc-rev; C, Pmev-kin-Sc-for; D, Pmev-kin-Sc-rev; E, Decarb-Sc-for; F, Decarb-Sc-rev). The three PCR products were annealed at their overlapping regions defined by the specific primers and assembled in a second round of amplification using the outer primers. The synthetic operon was cloned into the pBAD vector.
- Fig. 3.
Alignment of the deduced amino acid sequence ofgcpE from E. coli and other organisms using the MEP pathway. Ecol, E. coli (Swiss-Prot accession no.P27433 ); Bsub, Bacillus subtilis (Swiss-Prot accession no.P54482 ); Pfal, Plasmodium falciparum (assembled from different sequences from The Institute for Genomic Research and Sanger databases and deposited in GenBank with accession no. AF323928 ); Syne,Synechocystis sp. strain PCC6803 (Protein Identification Resource accession no. S77159 ); Atha, Arabidopsis thaliana(GenBank accession no. BAB09833 ). Three dots indicate sequence insertion of 258 amino acids in the sequence of A. thalianaand of 304 amino acids in the sequence of P. falciparum with weak similarities to each other. Black and gray outlines indicate identical and similar amino acid residues, respectively.
- Fig. 4.
Replacement of the gcpE gene with a precisely engineered deletion. (A) Diagram of the gcpE region of the wild-type strain and the gcpE deletion mutant. Small arrows indicate the primer sites used for PCR analysis. Primers: A, Gcpe-con-N; B, Gcpe-con-C; C, ecolgcpefor; D, ecolgcperev. (B) Verification of the deletion of the gcpE gene by PCR. After selection for integrates of the gene replacement vector pKO3-ΔgcpE into the chromosome at 43°C, bacteria were plated at 30°C on sucrose medium and replica plated onto chloramphenicol plates. The chloramphenicol-sensitive, sucrose-resistant colonies were screened by PCR. The PCR product of 530 bp obtained using the primer pair A plus B of the gcpEmutant strain is the expected 1,070 bp smaller than the wild-type product of 1,600 bp. Using the primer pair C plus D, thegcpE gene (1,116 bp) was amplified in the wild-type strain, and no product was obtained in the gcpE mutant strain.
- Fig. 5.
Growth of the E. coli strains indicated in panel A (wt, wild type; wtΔdxr, dxr deletion mutant; wtΔgcpE, gcpE deletion mutant) on medium without (B) and with (C) mevalonate and after complementation of the mutant strains with episomal dxr and gcpE genes, respectively, without mevalonate (D).
Tables
- Table 1.
Accession numbers of GcpE homologues in various organisms
Organism GcpE accession no. Eubacteria Aquifex aeolicus sp O67496 Bacillus subtilis sp P54482 Chlamydia muridarum tr Q9PKY3 C. pneumoniae tr Q9Z8H0 C. trachomatis sp O84060 Escherichia coli sp P27433 Haemophilus influenzae spP44667 Helicobacter pylori tr Q9ZLL0 Mycobacterium tuberculosis sp O33350 Synechocystis strain PCC6803 pir S77159 Thermotoga maritima tr Q9WZZ3 Treponema pallidum sp O83460 Neisseria meningitidis tr Q9JZ40 Campylobacter jejuni tr Q9PPMI Deinococcus radiodurans tr Q9RXC9 Pseudomonas aeruginosa tr AAG07190 Vibrio cholerae tr Q9KTX1 Staphylococcus aureus Streptococcus pyogenes S. pneumoniae Borrelia burgdorferi Mycoplasma genitalium M. pneumoniae Rickettsia prowazekii Archaebacteria Archaeoglobus fulgidus Methanobacterium thermoautotrophicum Aeropyrum pernixK1 Methanococcus jannaschii Pyrococcus horikoshii Halobacterium sp. strain NRC-1 Pyrococcus abyssi Eucaryota Plasmodium falciparum gb AF323928 Arabidopsis thaliana gb BAB09833 Saccharomyces cerevisiae Drosophila melanogaster Caenorhabditis elegans Homo sapiens
