Tyrosine Mutation in the Characteristic Motif of the Amorphous Region of Spidroin for Self-Assembly Capability Enhancement.

Spidroin, with robust mechanical performance and good biocompatibility, could fulfill broad applications in material science and biomedical fields. Development of miniature spidroin has made abundant fiber production economically feasible, but the mechanical properties of artificial silk still fall short of natural silk. The mechanism behind mechanical properties of spidroin usually focuses on ß-microcrystalline regions; the effect of amorphous regions was barely studied. In this study, residue tyrosines (Y) were designed to replace asparagine (N)/glutamic acid (Q) in the characteristic motifs (GGX)n in amorphous regions for performance enhancement of spidroin; the mutants presented lower free energy and significantly exhibited stronger van der Waals and electrostatic interactions, which might result from π-π stacking interactions between the phenyl rings in the side chain of tyrosine. Additionally, the soluble expressions of wild-type spidroin and mutant spidroin were achieved when heterologously expressed in E. coli, with yields of 560 mg/L (2REP), 590 mg/L (2REPM), 240 mg/L (4REP), and 280 mg/L (4REPM). Significantly, secondary structure analysis confirmed that the mutant spidroin more avidly forms more ß-sheets than the wild-type spidroin, and aggregation morphology suggested that mutant spidroin displayed better self-assembly capacity and was easier to form artificial spider silk fibers; in particular, self-assembled 4REPM nanofibrils had an average modulus of 11.2 ± 0.35 GPa, about 2 times higher than self-assembled B. mori silk nanofibrils and almost the same as that of native spider dragline silk fibers (10-15 GPa). Thus, we first demonstrated a new influence mechanism of the amorphous region's characteristic motif on the self-assembly and material properties of spidroin. Our study provides a reference for the design of high-performance material proteins and their heterologous preparation.

Kirigami triboelectric spider fibroin microneedle patches for comprehensive joint management.

Joint injuries are among the leading causes of disability. Present concentrations were focused on oral drugs and surgical treatment, which brings severe and unnecessary difficulties for patients. Smart patches with high flexibility and intelligent drug control-release capacity are greatly desirable for efficient joint management. Herein, we present a novel kirigami spider fibroin-based microneedle triboelectric nanogenerator (KSM-TENG) patch with distinctive features for comprehensive joint management. The microneedle patch consists of two parts: the superfine tips and the flexible backing base, which endow it with great mechanical strength to penetrate the skin and enough flexibility to fit different bends. Besides, the spider fibroin-based MNs served as a positive triboelectric material to generate electrical stimulation, thereby forcing drug release from needles within 720 min. Especially, kirigami structures could also transform the flat patch into three dimensions, which could impart the patch with flexible properties to accommodate the complicated processes produced by joint motion. Benefiting from these traits, the KSM-TENG patch presents excellent performance in inhibiting the inflammatory response and promoting wound healing in mice models. The results indicated that the mice possessed only 2% wound area and the paw thickness was reduced from 10.5 mm to 6.2 mm after treatment with the KSM-TENG patch, which further demonstrates the therapeutic effect of joints in vivo. Thus, it is believed that the proposed novel KSM-TENG patch is valuable in the field of comprehensive treatments and personalized clinical applications.

Kirigami-inspired artificial spidroin microneedles for wound patches.

Intelligent wound management has important potential for promoting the recovery of chronic wounds caused by diabetes. Here, inspired by the field of kirigami, smart patterned high-stretch microneedle dressings (KPMDs) based on gene-modified spider silk proteins were developed to achieve sensitive biochemical and physiological sensing. The spider silk protein (spidroin) has excellent tensile properties, ductility, toughness and biocompatibility. Notably, the kirigami method-prepared kirigami structure of the spidroin MN dressing had a high tensile strength , while its ductility reached approximately 800 %. Moreover, the unique optical properties of photonic crystals allow for fluorescence enhancement, providing KPMD with color-sensitive properties suitable for wound management and clinical guidance. Furthermore, to improve the sensitivity of KPMD-s to motion monitoring, a microelectronic matrix was integrated on its surface. These distinct material properties suggest that this research lays the foundation for a new generation of high-performance biomimetic diatomaceous earth materials for application.

[Primary Structure Characterization and Biosynthesis of Spider Silk Proteins for Multifunctional Biomaterials].

Spider silk is a natural fiber known as "biosteel" with the strongest composite performance, such as high tensile strength and toughness. It is also equipped with excellent biocompatibility and shape memory ability, thus shows great potential in many fields such as biomedicine and tissue engineering. Spider silk is composed of macromolecular spidroin with rich structural diversity. The characteristics of the primary structure of natural spidroin, such as the high repeatability of amino acids in the core repetitive region, the high content of specific amino acids, the large molecular weight, and the high GC content of the spidroin gene, have brought great difficulties in heterologous expression. This review discusses focuses on the relationship between the featured motifs of the microcrystalline region in the repetitive unit of spidroin and its structure, as well as the spinning performance and the heterologous expression. The optimization design for the sequence of spidroin combined with heterologous expression strategy has greatly promoted the development of the biosynthesis of spider silk proteins. This review may facilitate the rational design and efficient synthesis of recombinant spidroin.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy.

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.

Efficient Biosynthetic Fabrication of Spidroins with High Spinning Performance.

The unique 3D structure of spider silk protein (spidroin) determines the excellent mechanical properties of spidroin fiber, but the difficulty of heterologous expression and poor spinning performance of recombinant spider silk protein limit its application. A high-yield low-molecular-weight biomimetic spidroin (Amy-6rep) is obtained by sequence modification, and its excellent spinning performance is verified by electrospinning it for use as a nanogenerator. Amy-6rep increases the highly fibrogenic microcrystalline region in the core repeat region of natural spidroin with limited sequence length and replaces the polyalanine sequence with an amyloid polypeptide through structural similarity. Due to sequence modification, the expression of Amy-6rep increased by ≈200%, and the self-assembly performance of Amy-6rep significantly increased. After electrospinning with Amy-6rep, the nanofibers exhibit good tribopower generation capacity. In this paper, a biomimetic spidroin sequence design with high yield and good spinning performance is reported, and a strategy for electrospinning to produce an artificial nanogenerator is explored.

Immobilization and Characterization of a Processive Endoglucanase EG5C-1 from Bacillus subtilis on Melamine-Glutaraldehyde Dendrimer-Functionalized Magnetic Nanoparticles.

Exploring an appropriate immobilization approach to enhance catalytic activity and reusability of cellulase is of great importance to reduce the price of enzymes and promote the industrialization of cellulose-derived biochemicals. In this study, Fe3O4 magnetic nanoparticles (MNPs) were functionalized with meso-2,3-dimercaptosuccinic acid to introduce carboxyl groups on the surface (DMNPs). Then, melamine-glutaraldehyde dendrimer-like polymers were grafted on DMNPs to increase protein binding sites for the immobilization of processive endoglucanase EG5C-1. Moreover, this dendrimer-like structure was beneficial to protect the conformation of EG5C-1 and facilitate the interaction between substrate and active center. The loading capacity of the functionalized copolymers (MG-DMNPs) for EG5C-1 was about 195 mg/g, where more than 90% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal stability and increased tolerability over a broad pH range compared with the free one. Additionally, MG-DMNP/EG5C-1 biocomposite maintained approximately 80% of its initial hydrolysis productivity after five cycles of usage using filter paper as the substrate. Our results provided a promising approach for the functionalization of MNPs, enabling the immobilization of cellulases with a high loading capacity and excellent activity recovery.

Evolving the 3-O/6-O regiospecificity of a microbial glycosyltransferase for efficient production of ginsenoside Rh1 and unnatural ginsenoside.

Glycosyltransferase is a popular and promising enzyme to produce high-value-added natural products. Rare ginsenoside Rh1 and unnatural ginsenoside 3ß-O-Glc-PPT are promising candidates for drugs. Herein, the microbial glycosyltransferase UGTBL1 was able to catalyze the 20(S)-protopanaxatriol (PPT) 3-O/6-O-glycosylation with poor 6-O-regiospecificity. A structure-guided strategy of mutations involving loop engineering, PSPG motif evolution, and access tunnel engineering was proposed to engineer the enzyme UGTBL1. The variant I62R/M320H/P321Y/N170A from protein engineering achieved a great improvement in 6-O regioselectivity which increased from 10.98 % (WT) to 96.26 % and a booming conversion of 95.57 % for ginsenoside Rh1. A single mutant M320W showed an improved 3-O regioselectivity of 84.83 % and an increased conversion of 98.13 % for the 3ß-O-glc-PPT product. Molecular docking and molecular dynamics (MD) simulations were performed to elucidate the possible molecular basis of the regiospecificity and catalytic activity. The unprecedented high titer of ginsenoside Rh1 (20.48 g/L) and 3ß-O-Glc-PPT (18.04 g/L) was attained with high regioselectivity and yields using fed-batch cascade reactions from UDPG recycle, which was the highest yield reported to date. This work could provide an efficient and cost-effective approach to the valuable ginsenosides.

Discovery and Mechanistic Understanding of a Lipase from Rhizorhabdus dicambivorans for Efficient Ester Aminolysis in Aromatic Amines.

The formation of amide bonds via aminolysis of esters by lipases generates a diverse range of amide frameworks in biosynthetic chemistry. Few lipases have satisfactory activity towards bulky aromatic amines despite numerous attempts to improve the efficiency of this transformation. Here, we report the discovery of a new intracellular lipase (Ndbn) with a broad substrate scope. Ndbn turns over a range of esters and aromatic amines in the presence of water (2 %; v/v), producing a high yield of multiple valuable amides. Remarkably, a higher conversion rate was observed for the synthesis of amides from substrates with aromatic amine rather than aliphatic amines. Molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies showcase the mechanism for the preference for aromatic amines, including a more suitable orientation, shorter catalytic distances in the active site pocket and a lower reaction barrier for aromatic than for aliphatic amines. This unique lipase is thus a promising biocatalyst for the efficient synthesis of aromatic amides.