In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.
Carrier Proteins ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Interferometry ; Membrane Proteins/metabolism*

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

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.
Escherichia coli/metabolism* ; Metabolic Engineering ; Homoserine/metabolism* ; Escherichia coli Proteins/metabolism* ; Fermentation

Shikimic acid is an intermediate metabolite in the synthesis of aromatic amino acids in Escherichia coli and a synthetic precursor of Tamiflu. The biosynthesis of shikimic acid requires blocking the downstream shikimic acid consuming pathway that leads to inefficient production and cell growth inhibition. In this study, a dynamic molecular switch was constructed by using growth phase-dependent promoters and degrons. This dynamic molecular switch was used to uncouple cell growth from shikimic acid synthesis, resulting in the production of 14.33 g/L shikimic acid after 72 h fermentation. These results show that the dynamic molecular switch could redirect the carbon flux by regulating the abundance of target enzymes, for better production.
Escherichia coli/genetics* ; Escherichia coli Proteins/genetics* ; Industrial Microbiology/methods* ; Metabolic Engineering ; Shikimic Acid/metabolism*

Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.
Bacterial Proteins/genetics* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Gene Expression Regulation, Bacterial

Glucosamine (GlcN), also called amino sugar, is a compound derived from the substitution of a hydroxyl group of glucose molecule with an amino group. GlcN finds a wide-range of applications in health food and pharmaceutical industries. In our previous research, a recombinant Escherichia coli-glms-gnal was constructed for the efficient production of GlcN and N-acetylglucosamine (GlcNAc), the latter can be readily deacetylated to GlcN under mild acidic conditions. However, the results indicated that the titer of GlcN and GlcNAc decreased significantly due to the transportation of GlcN and GlcNAc from the culture broth to the inside of cells. To alleviate or block the transportation process, nagE gene (encoding for the GlcNAc-specific transporter) and manX gene (encoding for the mannose transporter) were knocked out with the Red homologous recombination method, and two engineered strains, E. coli-glms-gna1-delta nagE (with nagE gene deletion) and E. coli-glms-gna1-delta nagE-delta manX (with nagE and manX genes deletion), were successfully constructed. The two strains were cultured in a 7-L fermentor for the production of GlcN and GlcNAc. The maximal GlcN concentration of control strain E. coli-glms-gnal reached 4.06 g/L, and the maximal GlcNAc concentration reached 41.46 g/L. The maximal GlcN and GlcNAc concentration of E. coli-glms-gna1-delta nagE reached 4.38 g/L and 71.80 g/L, respectively, which were 1.08-fold and 1.70-fold of those of E. coli-glms-gnal, respectively. The maximal GlcN and GlcNAc concentration of E. coli-glms-gnal-delta nagE-delta manX reached 4.82 g/L and 118.78 g/L, respectively, which were 1.20-fold and 2.86-fold of those of E. coli-glms-gnal, respectively. These results suggested that the deletion of nagE and manX could significantly increase the extracellular accumulation of GlcN and GlcNAc. The results obtained here maybe useful for the microbial GlcN production in an industrial scale.
Acetylglucosamine ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Gene Knockout Techniques ; Glucosamine ; biosynthesis ; genetics ; Repressor Proteins ; 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

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K_m and k_cat/K_m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.
Recombinant Proteins/metabolism* ; Paenibacillus/metabolism* ; Mannose/metabolism* ; Escherichia coli/metabolism* ; Mannitol/metabolism*

We studied the ability of lpdA gene knockout Escherichia coli to ferment different sugars in mineral salts medium for the production of pyruvate. The sugars studied were glucose, fructose, xylose and mannose at a concentration of 10 g/L. At the same time, effect of inoculum size on lpdA fermentation with glucose was studied. The strain was able to use all sugars for biomass generation and pyruvate production. The lpdA knockout mutant converted glucose, fructose, xylose and mannose to pyruvate with yields of 0.884 g/g, 0.802 g/g, 0.817 g/g and 0.808 g/L, respectively. The pyruvate accumulation curve coupled with cell growth except for mannose as carbon source. When the inoculation size increased, the rate of glucose consumption, pyruvate accumulation and cell growth increased but lower pyruvate concentration. This study demonstrates that E. coli lpdA mutant has the potential to produce pyruvic acid from xylose and mannose.
Carbon ; metabolism ; Dihydrolipoamide Dehydrogenase ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Fermentation ; Fructose ; metabolism ; Gene Knockout Techniques ; Glucose ; metabolism ; Mannose ; metabolism ; Pyruvic Acid ; metabolism ; Xylose ; metabolism