A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage.

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1' site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1' specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1' specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM = 3.72 ± 0.04 mM-1∙s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2' site, but with an expanded P1' specificity. Structural simulation of C3 indicated a reconstructed S1' binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3CP, also provide new tools for other enzyme engineering.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Development of a Bacterial FhuD-Lysozyme-SsrA Mediated Autolytic (FLSA) System for Effective Release of Intracellular Products.

Developing effective bacterial autolytic systems for fast release of intracellular bioproducts could simplify purification procedures and help with the high throughput screening of mutant libraries in protein engineering. Here, we developed a fast and tightly regulated E. coli autolytic system, named the FhuD-lysozyme-SsrA mediated autolytic (FLSA) system, by integrating the secretion signal peptide, T7 lysozyme, and E. coli ClpX/P-SsrA protein degradation machinery. To decrease the cytotoxicity of leaky T7 lysozymes, the SsrA tag was fused to the C-terminus of T7 lysozyme to confer a tight regulation of its production. Using sfGFP as a reporter, we demonstrated that anchoring the Sec-Tat dual pathway signal peptide FhuD to the N-terminus of T7 lysozyme-SsrA could give the highest cell lysing efficiency. The optimization of the FLSA system indicated that weak alkaline conditions (pH 8.0) and 0.5% Triton X-100 could further increase the lysing efficiency by about 24%. The FLSA system was validated by efficient production of sfGFP and human growth hormone 1 (hGH1) in a shake flask, with a cell lytic efficiency of approximately 82% and 80%, respectively. Besides, the FLSA system was applied for large-scale fermentation, in which approximately 90% sGFP was released with a cell density OD600 of 110. Moreover, the FLSA system was also tested for α-amylase mutant library screening in microplates, and the results showed that intracellular α-amylase can be efficiently released out of cells for activity quantitation. In all, the FLSA system can facilitate the release of intracellular recombinant proteins into the cell culture medium, which has the potential to serve as an integrated system for large-scale production of recombinant targets and high throughput enzyme engineering in synthetic biology.

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly-Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly-Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2-P1↓P1' (Gly-Gly↓Ala) position using the YESS-PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly-Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1-P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1' position Ala of Smt3 was found to play a vital role in maintaining Ulp1's precise cleavage after the Gly-Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

A split protease-E. coli ClpXP system quantifies protein-protein interactions in Escherichia coli cells.

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.

N-Terminal Fused Signal Peptide Prompted Extracellular Production of a Bacillus-Derived Alkaline and Thermo Stable Xylanase in E. coli Through Cell Autolysis.

Xylanases are extensively used as industrial enzymes for its ability of hydrolyzing xylan to oligosaccharides. Here, XynHB, a thermo and alkaline stable xylanase derived from Bacillus pumilus HBP8, was extracellularly produced in E. coli cells through N-terminal-fused signal peptides. We found that the matured XynHB itself could be auto-secreted out of E. coli BL21(DE3) cells at a very low level, and two Sec-pathway signal peptides, PelB and OmpA, and one dual Sec-Tat-pathway signal peptide, FhuD, could effectively prompt its extracellular production up to 12-fold. Our results showed that PelB signal peptide led to the highest extracellular production of XynHB for approximately 54.1 µg/mL, and FhuD-fused XynHB possessed the highest specific activity of 1746.0 U/mg at 70 °C. Meanwhile, our studies also indicated that PelB- and FhuD-fused XynHB might disrupt E. coli cells' periplasm during their secretion process, thus causing cell lysis to facilitate their extracellular production. Moreover, further characterization revealed that the extracellular production of XynHB was not affected by the outer membrane permeability of E. coli cells. Our studies provided an advantageous strategy for the extracellular production of xylanase in E. coli, which may also be used for E. coli autolysis in the future.