Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein

Background Fluorinases play a unique role in the production of fluorine-containing organic molecules by biological methods. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates. Results A gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused fluorinase was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase displayed on the surface showed good stability while retaining the catalytic activity. The engineered E.coli with surface-displayed fluorinase could be cultured to obtain a larger cell density, which was beneficial for industrial application. And 55% yield of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) was achieved by using the whole-cell catalyst. Conclusions Here, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of E.coli and maintained its catalytic activity. The surface display provides a new solution for the industrial application of biological fluorination. Graphical Abstract

Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein

BackgroundFluorinases play a unique role in producing fluorinated organic molecules through a biological method. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.ResultsA gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused protein was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and Immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase-containing surface display system with improved whole-cell catalytic efficiency and stability showed low growth pressure on the protein expressing host. The conversion rate of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) achieved 55%.ConclusionsHere, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of Escherichia coli and maintained its catalytic activity. The surface display offers a new solution for the industrial application of biological fluorination.

Complete Genome Sequence of Pseudomonas syringae BIM B-268, a Plant Pathogen Strain Used for In Vitro Testing of Agricultural Bacteriophage Efficiency

Pseudomonas syringae BIM B-268 is the strain used for in vitro testing of the efficiency of Multiphage, a bacteriophage-based biopesticide produced in Belarus. The genome sequence of this strain consists of a single circular chromosome harboring the genes encoding the ice nucleation protein, syringopeptin biosynthesis, and types III and VI secretion systems.

Evaluation of a Temperature-Induced Chitosanase Bacterial Cell Surface Display System for the Efficient Production of Chitooligosaccharides

Objective To establish a temperature-induced chitosanase bacterial cell surface display system to produce chitooligosaccharides (COSs) efficiently for industrial applications. Results Temperature-inducible chitosanase CSN46A bacterial surface display systems containing one or two copies of ice nucleation protein (InaQ-N) as anchoring motifs were successfully constructed on the basis of Escherichia coli and named as InaQ-N-CSN46A and 2InaQ-N-CSN46A. The specific enzyme activity of 2InaQ-N-CSN46A reached 886.33±0.81 U/g cell dry weight, which was 45.6% higher than that of InaQ-N-CSN46A. However, few proteins were detected in 2InaQ-N-CSN46A hydrolysis system. Therefore, 2InaQ-N-CSN46A had higher hydrolysis efficiency and stability than InaQ-N-CSN46A. GPC revealed that under the optimum enzymatic hydrolysis temperature, the final products were mainly chitobiose and chitotriose. Chitopentaose accumulated (77.62%) when the hydrolysis temperature reached 60 ℃. FTIR and NMR analysis demonstrated that the structures of the two hydrolysis products were consistent with those of COSs.Conclusions In this study, chitosanase was expressed on the surfaces of E. coli by increasing induction temperature, and chitosan was hydrolysed directly without enzyme purification steps. This study provided a novel strategy for industrial COSs production.

Water Structure on Mica Surfaces: Investigating the Effect of Cations

We studied thin films of water at the mica-air interface using infrared spectroscopy and molecular dynamics simulations. We investigate the influence of ions on interfacial water by exchanging the naturally occurring K⁺ ion with H⁺/Na⁺, Ca²⁺, and Mg²⁺. The experiments do not show a difference in the bulk structure (<i>i. e.</i> in the infrared spectra), but indicate that water is more strongly attracted to the Mg²⁺ mica. The simulations reveal that the cation-water interactions significantly influence the microscopic arrangement of water on mica. Our results indicate that the divalent cations result in strong water-mica interactions, which leads to longer hydrogen bond lifetimes and larger hydrogen bonded clusters of interfacial water molecules. These results have implications for surface-mediated processes such as heterogeneous ice nucleation, protein assembly and catalysis.

Water Structure on Mica Surfaces: Investigating the Effect of Cations

We studied thin films of water at the mica-air interface using infrared spectroscopy and molecular dynamics simulations. We investigate the influence of ions on interfacial water by exchanging the naturally occurring K⁺ ion with H⁺/Na⁺, Ca²⁺, and Mg²⁺. The experiments do not show a difference in the bulk structure (<i>i. e.</i> in the infrared spectra), but indicate that water is more strongly attracted to the Mg²⁺ mica. The simulations reveal that the cation-water interactions significantly influence the microscopic arrangement of water on mica. Our results indicate that the divalent cations result in strong water-mica interactions, which leads to longer hydrogen bond lifetimes and larger hydrogen bonded clusters of interfacial water molecules. These results have implications for surface-mediated processes such as heterogeneous ice nucleation, protein assembly and catalysis.

Applying In Silico Approaches for Designing a Chimeric InaV/N-DFPase Protein and Evaluating its Binding with Diisopropyl-Fluorophosphate

The N-terminal domain of the ice-nucleation protein InaV (InaV-N) ofPseudomonas syringaewas applied to display the DFPase on the cell surface.In silicotechniques were used to generate a model in order to examine the possibility of DFPase exhibition on the cell surface. The secondary and tertiary structures of a chimeric protein were determined and then, the predicted model was subjected to several repeated cycles of stereochemical evaluation and energy minimization. The homology-modeled structure of the InaV/N-DFPase protein was docked to DFP. The optimizedinaV/N-dfpasegene was translated to 519 amino acids. The minimum free energy of the best-predicted secondary structures was formed by RNA molecules (-215.45 kcal/mol). SOPMA analysis results showed that the main helix peak corresponded to the anchor fragment. Validation of the 3D model indicated that 86.1% of amino acid residues were incorporated into the favored regions. The moldock score was 360.22 for DFP. Results of this study indicated that according toin silicoanalysis, all of these findings were effective in targeting DFPase.

Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ

The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Å by microfocus X-ray crystallography and to 0.9 Å by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals.