Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).
Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Shikimic Acid ; metabolism

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.
Escherichia coli ; Genes, Plant ; Phylogeny ; Senecio ; enzymology ; Sesquiterpenes ; metabolism

Phenylalanine hydroxylase (PAH) is a member of aromatic amino acid hydroxylase (AAAHs) family, and catalyze phenylalanine (Phe) into tyrosine (Tyr). Using immunological and RT-PCR methods to prove the existence of phenylalanine hydroxylase (PAH) gene in the brain of Neanthes japonica in protein and nucleic acid level. Using Western blotting to detect the pah immunogenicity of Neanthes japonica. Making paraffin sections and using immunohistochemical technique to identify the presence and distribution of the phenylalanine hydroxylase gene in the brain of Neanthes japonica. Clone pah gene from the brain of Neanthes japonica by RT-PCR, constructing plasmid and transferring into E. coli to amplification, picking a single homogeneous colony, double digesting then making sequence and comparing homology. Western blotting results showed that the expression of the protein is present in Neanthes japonica brain, immunohistochemistry technique results showed that phenylalanine hydroxylase mainly expressed in abdominal of forebrain, dorsal and sides of midbrain. RT-PCR technique results showed that the phenylalanine hydroxylase exist in the brain of Neanthes japonica and has a high homology with others animals. PAH is present in the lower organisms Neanthes japonica, in protein and nucleic acid level. Which provide the foundation for further study the evolution of aromatic amino acid hydroxylase genes in invertebrate.
Animals ; Blotting, Western ; Brain ; enzymology ; Escherichia coli ; metabolism ; Phenylalanine Hydroxylase ; genetics ; metabolism ; Polychaeta ; enzymology ; genetics

High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E. coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E. coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5 mg/mL, and the phytase activity (fermentation potency) exceeded 7.5 x 10(6) IU/mL, which was the highest among those of all kinds of recombinant strains reported now.
6-Phytase ; genetics ; metabolism ; Escherichia coli ; enzymology ; Escherichia coli Proteins ; genetics ; Fermentation ; Pichia ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; Escherichia coli Proteins ; genetics ; Industrial Microbiology ; Mutagenesis, Site-Directed ; Phosphoglycerate Dehydrogenase ; genetics ; Serine ; biosynthesis

The differences in metabolism in Escherichia coli DH5alpha and its acetate-tolerant mutant DA19 were analyzed based on the activity of key enzymes involved in central metabolism when both strains were continuously cultured in nitrogen source-limited defined media. The activity of glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) in DA19 increased as compared with those in DH5alpha, while acetate kinase (ACK) and phosphofructosekinase (PFK) decreased. These indicated that more carbon flux of DA19 entered the phosphopentose pathway (PPP) and less entered the glycolytic (EMP) pathway and acetic acid production (Ack-Pta) pathway. Therefore, the differences in activity of key enzyme coincided with increased cell yield based on consumed glucose (Y(X/G)) and decreased production of acetic acid and pyruvate of DA19. G6PDH and ICDH in DH5alpha were up-regulated by addition of adenine, while ACK and PFK were down-regulated. On the other hand, adenine had little effect on those in DA19. The enzymes except PFK in both strains were down-regulated by sodium acetate, especially the activity of ICDH in DH5alpha. These results suggested changed flux of central metabolic pathways were also consistent with the changes of growth properties and byproducts formation.
Acetate Kinase ; metabolism ; Acetates ; metabolism ; Culture Techniques ; Escherichia coli ; enzymology ; genetics ; metabolism ; Glucosephosphate Dehydrogenase ; metabolism ; Mutation

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
Amidohydrolases ; chemistry ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; Molecular Weight ; Nocardia ; enzymology ; Phylogeny

7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.
Chromatography, Affinity ; DNA Damage ; DNA Repair ; Escherichia coli/enzymology* ; Nucleosidases/isolation & purification* ; Sequence Analysis, Protein

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Endopeptidases ; genetics ; metabolism ; Escherichia coli ; genetics ; Plum Pox Virus ; enzymology ; genetics ; Protein Interaction Mapping ; methods ; Proteolysis

To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBI(Eu) and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').
Carotenoids ; biosynthesis ; genetics ; metabolism ; Deinococcus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Oxidoreductases ; metabolism ; Substrate Specificity