Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Pyruvate Synthase ; genetics ; metabolism ; Transketolase ; genetics ; metabolism

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.
Animals ; Antibodies/metabolism ; Cell Proliferation/drug effects ; Epithelial Cells/parasitology ; Host-Pathogen Interactions/drug effects/*physiology ; Iron/pharmacology ; Mice ; Pyruvate Synthase/*metabolism ; Rabbits ; Trace Elements/pharmacology ; Trichomonas Infections/*parasitology ; Trichomonas vaginalis/drug effects/genetics/metabolism/*pathogenicity

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.
Trichomonas vaginalis/*growth & development ; Reverse Transcriptase Polymerase Chain Reaction ; Pyruvate Synthase/genetics/metabolism ; Organelles/*enzymology/metabolism/*physiology ; Membrane Potentials ; Malate Dehydrogenase/genetics/metabolism ; Iron/*metabolism ; Hydrogenase/genetics/metabolism ; Hydrogen/*metabolism ; Humans ; Gene Expression Regulation, Enzymologic ; *Gene Expression Regulation ; Ferredoxins/genetics/metabolism ; Culture Media ; Animals