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Protein condensates are distinct structures assembled in living cells that concentrate molecules via phase separation in a confined subcellular compartment. In the past decade, remarkable advances have been made to discover the fundamental roles of the condensates in spatiotemporal control of cellular metabolism and physiology and to reveal the molecular principles, components and driving forces that underlie their formation. Here we review the unique properties of the condensates, the promise and hurdles for harnessing them toward purposeful design and manipulation of biological functions in living cells. In particular, we highlight recent advances in mining and understanding the proteinaceous components for creating designer condensates, along with the engineering approaches to manipulate their material properties and biological functions. With these advances, a greater variety of complex organelle-like structures can be built for diverse applications, with unprecedented effects on synthetic biology.

Spider dragline silk is a remarkable protein fiber that is mechanically superior to almost any other natural or synthetic material. As a sustainable supply of natural dragline silk is not feasible, recombinant production of silk fibers with native-like mechanical properties and non-native physiochemical functions is highly desirable for various applications. Here, we report a new strategy for simultaneous functionalization and reinforcement of recombinant spider silk fibers by confined nanoparticle formation. First, a mimic silk protein (N16C) of spider

Spatially directed synthesis of quantum dots (QDs) is intriguing yet challenging in organisms, due to the dispersed feature of templating biomolecules and precursors. Whether this task could be accomplished by biomolecular condensates, an emerging type of membraneless compartments in cells remains unknown. Here we report synthetic protein condensates for templated synthesis of QDs in bacterium Escherichia coli. This was realized by overexpression of spider silk protein to bind precursor ions and recruit other necessary components, which induced the spidroin to form more β-sheet structures for assembly and maturation of the protein condensates. This in turn enabled formation and co-localization of the fluorescent QDs to "light up" the condensates, and alleviated cytotoxicity of the precursor heavy metal ions and resulting QDs. Thus, our results suggest a new strategy for nanostructure synthesis and deposition in subcellular compartments with great potential for in situ applications.

Despite considerable progress having been made in thermosensitive protein hydrogels, regulating their thermal transitions remains a challenge due to the intricate molecular structures and interactions of the underlying protein polymers. Here we report a genetic fusion strategy to tune the unique dual thermal transitions of the C-terminal domain (CTD) of spider major ampullate spidroin 1, and explore the regulation mechanism by biophysical characterization and molecular dynamics simulations. We found that the fusion of elastin-like polypeptides (ELPs) tuned the dual transition temperatures of CTD to a physiologically relevant window, by introducing extra hydrogen bonding at low temperatures and hydrophobic interactions at high temperatures. The resulting hydrogels constructed from the fusion proteins were demonstrated to be a promising vehicle for cell preservation and delivery. This study provides insights on the regulation of the dual thermosensitive protein hydrogels and suggests a potential application of the hydrogels for consolidated cell storage and delivery.

Matrix stiffness and fibrous structure provided by the native extracellular matrix have been increasingly appreciated as important cues in regulating cell behaviors. Recapitulating these physical cues for cell fate regulation remains a challenge due to the inherent difficulties in making mimetic hydrogels with well-defined compositions, tunable stiffness, and structures. Here, we present two series of fibrous and porous hydrogels with tunable stiffness based on genetically engineered resilin-silk-like and resilin-like protein polymers. Using these hydrogels as substrates, the mechanoresponses of bone marrow mesenchymal stem cells to stiffness and fibrous structure were systematically studied. For both hydrogel series, increasing compression modulus from 8.5 to 14.5 and 23 kPa consistently promoted cell proliferation and differentiation. Nonetheless, the promoting effects were more pronounced on the fibrous gels than their porous counterparts at all three stiffness levels. More interestingly, even the softest fibrous gel (8.5 kPa) allowed the stem cells to exhibit higher endothelial differentiation capability than the toughest porous gel (23 kPa). The predominant role of fibrous structure on the synergistic regulation of endothelial differentiation was further explored. It was found that the stiffness signal activated Yes-associated protein (YAP), the main regulator of endothelial differentiation, via spreading of focal adhesions, whereas fibrous structure reinforced YAP activation by promoting the maturation of focal adhesions and associated F-actin alignment. Therefore, our results shed light on the interplay of physical cues in regulating stem cells and may guide the fabrication of designer proteinaceous matrices toward regenerative medicine.

Membraneless organelles formed by liquid-liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid-liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.

Electrostatic interaction is a promising mechanism to expand the range of physiochemical properties of hydrogel materials. However, the versatility of such materials is still limited because of the...

Spider dragline silk is a remarkable fiber made of unique proteins-spidroins-secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (65.8%) and titer (554.7 mg L

Spider dragline silk is a remarkable fiber made by spiders from an aqueous solution of spidroins, and this feat is largely attributed to the tripartite domain architecture of the silk proteins leading to the hierarchical assembly at the nano- and microscales. Although individual amino- and carboxy-terminal domains have been proposed to relate to silk protein assembly, their tentative synergizing roles in recombinant spidroin storage and spinning into synthetic fibers remain elusive. Here, we show biosynthesis and self-assembly of a mimic spidroin composed of amino- and carboxy-terminal domains bracketing 16 consensus repeats of the core region from spider Trichonephila clavipes. The presence of both termini was found essential for self-assembly of the mimic spidroin termed N16C into fibril-like (rather than canonical micellar) nanostructures in concentrated aqueous dope and ordered alignment of these nanofibrils upon extrusion into an acidic coagulation bath. This ultimately led to continuous, macroscopic fibers with a tensile fracture toughness of 100.9 ± 13.2 MJ m-3, which is comparable to that of their natural counterparts. We also found that the recombinant proteins lacking one or both termini were unable to similarly preassemble into fibrillar nanostructures in dopes and thus yielded inferior fiber properties. This work thereby highlights the synergizing role of terminal domains in the storage and processing of recombinant analogues into tough synthetic fibers.

Integrating multifunctionality such as stretchability, adhesiveness, and electroconductivity on a single protein hydrogel is highly desirable for various applications, and remains a challenge. Here we present the development of such multifunctional hydrogels based on resilin, a natural rubber-like material with remarkable extensibility and resilience. First, genetically engineered reslin-like proteins (RLPs) with varying molecular weight were biosynthesized to tune mechanical strength and stiffness of the cross-linked RLP hydrogels. Second, glycerol was incorporated into the hydrogels to endow adhesive properties. Next, a graphene-RLP conjugate was synthesized for cross-linking with the unmodified, pristine RLP to form an integrated network. The obtained hybrid hydrogel could be stretched to over four times of its original length, and self-adhered to diverse substrate surfaces due to its high adhesion strength of ∼24 kPa. Furthermore, the hybrid hydrogel showed high sensitivity, with a gauge factor of 3.4 at 200% strain, and was capable of real-time monitoring human activities such as finger bending, swallowing, and phonating. Due to these favorable attributes, the graphene/resilin hybrid hydrogel was a promising material for use in wearable sensors. In addition, the above material design and functionalization strategy may provide intriguing opportunities to generate innovative materials for broad applications.

Electron beam lithography (EBL) is renowned to provide fabrication resolution in the deep nanometer scale. One major limitation of current EBL techniques is their incapability of arbitrary 3d nanofabrication. Resolution, structure integrity and functionalization are among the most important factors. Here we report all-aqueous-based, high-fidelity manufacturing of functional, arbitrary 3d nanostructures at a resolution of sub-15 nm using our developed voltage-regulated 3d EBL. Creating arbitrary 3d structures of high resolution and high strength at nanoscale is enabled by genetically engineering recombinant spider silk proteins as the resist. The ability to quantitatively define structural transitions with energetic electrons at different depths within the 3d protein matrix enables polymorphic spider silk proteins to be shaped approaching the molecular level. Furthermore, genetic or mesoscopic modification of spider silk proteins provides the opportunity to embed and stabilize physiochemical and/or biological functions within as-fabricated 3d nanostructures. Our approach empowers the rapid and flexible fabrication of heterogeneously functionalized and hierarchically structured 3d nanocomponents and nanodevices, offering opportunities in biomimetics, therapeutic devices and nanoscale robotics., Electron beam lithography (EBL) is renowned to provide fabrication resolution in the deep nanometer scale but their incapability of arbitrary 3D nanofabrication poses a major limitation to the technique. Here, the authors demonstrate a manufacturing technique of functional 3d nanostructures at a resolution of sub-15 nm using voltage-regulated 3d EBL.

Rubberlike protein hydrogels are unique in their remarkable stretchability and resilience but are usually low in strength due to the largely unstructured nature of the constitutive protein chains, which limits their applications. Thus, reinforcing protein hydrogels while retaining their rubberlike properties is of great interest and has remained difficult to achieve. Here, we propose a fibrillization strategy to reinforce hydrogels from engineered protein copolymers with photo-cross-linkable resilin-like blocks and fibrillizable silklike blocks. First, the designer copolymers with an increased ratio of the silk to resilin blocks were photochemically cross-linked into rubberlike hydrogels with reinforced mechanical properties. The increased silk-to-resilin ratio also enabled self-assembly of the resulting copolymers into fibrils in a time-dependent manner. This allowed controllable fibrillization of the copolymer solutions at the supramolecular level for subsequent photo-cross-linking into reinforced hydrogels. Alternatively, the as-prepared chemically cross-linked hydrogels could be reinforced at the material level by inducing fibrillization of the constitutive protein chains. Finally, we demonstrated the advantage of reinforcing these hydrogels for use as piezoresistive sensors to achieve an expanded pressure detection range. We anticipate that this strategy may provide intriguing opportunities to generate robust rubberlike biomaterials for broad applications.

Genetically engineered protein polymers, which can combine different unique peptide sequences from natural protein materials, offer great opportunities for making advanced materials with well-defined structures and properties. Here we report for the first time biosynthesis and self-assembly of a recombinant resilin-silk (RS) copolymer consisting of repeating units of silk and resilin blocks. The copolymer in aqueous solution self-assembled into nanoparticles, and the assembled nanoparticles further form nano- to microscale fibers in a time-dependent manner at body temperature, whereas such fibers were not formed upon incubation of the copolymer at either low or high temperatures. In contrast, a resilin-like polypeptide without the silk blocks exhibited a typical thermoresponsive dual-phase transition behavior and was incapable of self-assembling into fibers. More interestingly, the microscale fibers self-assembled from a moderately concentrated RS solution (20 wt %) could interact to give a self-supporting, semitransparent hydrogel with elastic modulus at approximately 195 Pa. Furthermore, photo-cross-linking of either freshly prepared or annealed RS copolymer led to the formation of stiff hydrogels and the material mechanical property was superior upon annealing of the RS solution for a longer time up to 4 h, with elastic modulus ranging from 2.9 to 7.0 kPa. These results not only shed light on the fundamental hierarchical assembly mechanism of a new family of genetically engineered RS copolymer but also suggest future opportunities for these thermoresponsive polymers in fabrication of hydrogel materials with tunable mechanical properties for diverse applications.

Recombinant protein polymers that mimic the structures and functions of natural proteins and those tailor-designed with new properties provide a family of uniquely tunable and functional materials. However, the diversity of genetically engineered protein polymers is still limited. As a powerful engine for the creation of new biological devices and systems, synthetic biology is promising to tackle the challenges that exist in conventional studies on protein polymers. Here we review the advances in design and biosynthesis of advanced protein materials by synthetic biology approaches. In particular, we highlight their roles in expanding the variety of designer protein polymers and creating programmable materials with live cells.

Growing concerns of environmental pollution and fossil resource shortage are major driving forces for bio‐based production of chemicals traditionally from petrochemical industry. Dicarboxylic acids (DCAs) are important platform chemicals with large market and wide applications, and here the recent advances in bio‐based production of straight‐chain DCAs longer than C4 from biological approaches, especially by synthetic biology, are reviewed. A couple of pathways were recently designed and demonstrated for producing DCAs, even those ranging from C5 to C15, by employing respective starting units, extending units, and appropriate enzymes. Furthermore, in order to achieve higher production of DCAs, enormous efforts were made in engineering microbial hosts that harbored the biosynthetic pathways and in improving properties of biocatalytic elements to enhance metabolic fluxes toward target DCAs. Here we summarize and discuss the current advantages and limitations of related pathways, and also provide perspectives on synthetic pathway design and optimization for hyper‐production of DCAs.

Biosensors can be a powerful tool for real-time monitoring of specific small molecules and for precise control of gene expression in biological systems. Thus, biosensors have attracted much attention for monitoring increasing number of molecules. However, strategies to tune the properties of biosensors remain less explored, which might restrict their wide applicability. Here we report the development of tunable biosensors for monitoring putrescine, an important member of biological polyamines, in Escherichia coli. The native putrescine-responsive PuuR repressor protein was employed as a sensing component, and its cognate operator was installed in engineered promoters to control the expression of downstream green fluorescent protein (GFP) mut3 as a reporter protein. The engineered biosensors were specific for putrescine, and the response time could be modulated by altering growth medium of the biosensor strains. In addition, the response dynamics and detection ranges of the biosensors can be tuned at the genetic level by modulation of PuuR expression, and by manipulation of the chromosomal genes involved in putrescine biosynthesis. To demonstrate utility of the biosensors, we were able to monitor the changes of endogenous putrescine levels caused by genetic manipulations. Furthermore, a link between the excretory putrescine titer and intracellular GFP fluorescence was established for an E. coli strain that was engineered for improved putrescine biosynthesis and excretion. This study provides a strategy for engineering synthetic biosensor circuit for monitoring and tuning the dynamics in sensing putrescine, which can be generally applicable for monitoring other chemicals through taking a similar approach in circuit design.

Synthetic biology provides a significant platform in creating novel pathways/organisms for producing useful compounds, while it remains a challenge to enhance the production efficiency. Recently we constructed a recombinant Escherichia coli for glutarate production using a synthetic α-ketoacid reduction pathway, in which α-ketoglutarate is reduced to 2-hydroxyglutarate then converted to glutarate. However, the production titer was low, which may be due to 1) oxygen-sensitive nature of 2-hydroxyglutaryl-CoA dehydratase (HgdABC) and 2) limited cell growth in anaerobic cultivation. Therefore, we developed an aerobic-anaerobic two-stage strategy by growing more cells aerobically, then shifting to anaerobic cultivation to ensure the functional HgdABC for glutarate biosynthesis. The two-stage cultivation resulted in higher production of glutarate and other two C5 dicarboxylic acids – glutaconate and 2-hydroxylglutarate than the original anaerobic process. Furthermore, we used an anaerobically-inducible nar promoter to improve the hgdABC expression responding to aerobic-anaerobic shift. Finally, the glutarate, glutaconate and 2-hydroxyglutarate titer was increased about 2, 5 and 3 times, reaching 11.6, 108.8 and 399.5 mg/L, respectively. The work demonstrated an effective strategy for ameliorating α-ketoacid reduction pathway to produce C5 dicarboxylic acids, as well as the potential of integration of bioprocess and metabolic engineering for enhancing chemicals production by an engineered microorganism.

Amino acid decarboxylases catalyze decarboxylation of amino acids into amines that possess wide industrial applications. As key enzymes in biobased production of industrially important amines such as cadaverine, putrescine and β-alanine, lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase have attracted increasing attention. To develop enzyme variants with superior catalytic properties, there is a great need for high-throughput assay of these decarboxylases. Here we report the development of assays based on the color change of pH indicator – chlorophenol red (CPR) or bromothymol blue (BTB) – in decarboxylation reactions, in which one proton was consumed per carboxylic group decarboxylated resulting in an increase in pH. First, two buffer-indicator pairs, 4-morpholineethanesulfonic acid (MES)-CPR and 3-morpholinopropanesulfonic acid (MOPS)-BTB, were chosen on the basis of their similar pKa values at approximately pH 6.0 and 7.0, both of which are physiologically relevant. Next, the effects of buffer strength and indicator concentration on absorbance changes were examined in assay mixtures with NaOH titration, which mimicked proton consumption in decarboxylation reactions. Finally, high-throughput quantification of lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase was achieved using a microplate format. These results suggest that our indicator assay system may have potential applications for screening diverse decarboxylases.

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.

Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coli with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans -enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite α-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans -glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coli resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola . The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.

Antibody-drug conjugates (ADCs), which combine the exquisite specificity of antibodies with the cell-killing ability of cytotoxic drug payloads, have emerged as an attractive means for treating cancers. All (pre)clinical ADCs employ biosynthesized cytotoxins as their ADC payload precursors (APPs). The cost-effective bioproduction of APPs is receiving great interest from both academia and industry. Given the lack of systematic overviews of the topic, we provide the current status of APPs and focus on their state-of-the-art bioproduction strategies, illustrated with typical examples and critical analyses. Challenges in further enhancing the bioproduction efficiency of APPs and other cytotoxins are also discussed. This research has implications for bioprocess and metabolic engineering, systems and synthetic biology, and biopharmaceutical drug discovery, development, and industrialization.

A variety of devices used for biomedical engineering have been fabricated using protein polymer because of their excellent properties, such as strength, toughness, biocompatibility, and biodegradability. In this study, we fabricated an optical waveguide using genetically engineered spider silk protein. This method has two significant advantages: (1) recombinant spider silk optical waveguide exhibits excellent optical and biological properties and (2) biosynthesis of spider silk protein can overcome the limitation to the research on spider silk optical waveguide due to the low yield of natural spider silk. In detail, two kinds of protein-based optical waveguides made from recombinant spider silk protein and regenerative silkworm silk protein were successfully prepared. Results suggested that the recombinant spider silk optical waveguide showed a smoother surface and a higher refractive index when compared with regenerative silkworm silk protein. The optical loss of recombinant spider silk optical waveguide was 0.8 ± 0.1 dB/cm in air and 1.9 ± 0.3 dB/cm in mouse muscles, which were significantly lower than those of regenerative silkworm silk optical waveguide. Moreover, recombinant spider silk optical waveguide can meet the demand to guide and efficiently deliver light through biological tissue. In addition, recombinant spider silk optical waveguide showed low toxicity to cells in vitro and low-level inflammatory reaction with surrounding tissue in vivo. Therefore, recombinant spider silk optical waveguide is a promising implantable device to guide and deliver light with low loss.

Protein films are an important class of materials for applications in biomedicine and biotechnology. The rational design of protein polymer sequence and selection of customized cross-linking offers unique opportunities to engineer desirable functionalities into these materials. Here we report the fabrication of a series of films with tunable physiochemical properties from genetically engineered silk-elastin-like proteins (SELPs). The SELPs were recombinantly biosynthesized with different ratios of silk-to-elastin blocks and periodic cysteine residues incorporated in the elastin blocks. A disulfide cross-linking method was developed for the preparation of the SELP films under mild oxidative conditions with a low concentration of hydrogen peroxide, in comparison with the physical cross-linking method used with the organic solvent methanol. Film properties were characterized for solubility, water absorption, hydrophilicity, surface roughness, and cyto-compatibility. The results indicated that customized cross-linking supported the fabrication of films from the SELP proteins with tunable features, including smooth, water stable film materials with cyto-compatibility. Interestingly, hydrogen peroxide oxidation was a preferred cross-linking method for the cysteine-containing SELPs with a low ratio of the silk-to-elastin blocks, whereas methanol treatment was suitable for fabricating films from the SELPs with a high ratio of silk-to-elastin blocks into stable films with rougher surfaces. We anticipate that an appropriate combination of polymer design and cross-linking might be a useful strategy for the preparation of protein films for diverse applications.

Protein hydrogels are an important class of materials for applications in biotechnology and medicine. The fine-tuning of their sequence, molecular weight, and stereochemistry offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here we report a new family of redox-sensitive protein hydrogels with controllable mechanical properties composed of recombinant silk-elastin-like protein polymers (SELPs). The SELPs were designed and synthesized with different ratios of silk-to-elastin blocks that incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally responsive in solution and rapidly formed hydrogels at body temperature under physiologically relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative gelation was performed at other physiologically relevant temperatures, and this resulted in hydrogels with tunable mechanical properties, thus, providing a secondary level of control over hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive features and tunable mechanical properties may be potentially useful as biomaterials with broad applications in controlled drug delivery and tissue engineering.

Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer for industrial and biomedical applications. Since farming spiders is not feasible due to their highly territorial nature, recombinant production of dragline silk proteins in a foreign host has received great attention. However, their production titer remains low, because efficient expression of very large, highly repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3–256.5 kDa were significantly elevated by down-shifting the induction temperature. The beneficial effect was found to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combination of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 g l−1 of a 201.6-kDa MaSp2 protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins.

To explore related risk factors of postoperative wound infection in lumbar spondylolisthesis.The clinical data of 296 patients with lumbar spondylolisthesis underwent posterior decompression and fusion from January 2013 to December 2017 were retrospectively analyzed. According to whether the wound was infected or not, the patients were divided into infection group and non-infection group. There were 29 patients in infection group including 13 males and 16 females with an average age of(67.03±1.01) years old and 267 patients in non-infection group including 91 males and 176 females with an average age of (52.32±16.08) years old. Gender, age, body mass index (BMI), smoking history, diabetes history, hormone use history, albumin level, operation time, ASA score, perioperative blood transfusion and drainage time were recorded. The multivariate Logistic regression analysis was carried out if univariate Logistic regression analysis of risk factors were statistical significant. At the same time the SPSS decision tree model was used to determine the stratified points of the continuous variables to further specify the independent risk factors.Postoperative wound infection occurred in 29 cases among all patients, with the incidence rate of 9.8% (29/296). There were significant differences in BMI, age, diabetes history, drainage time, hormone use history, albumin level and operation time between two groups(Age, diabetes history, hormone use history and drainage time are independent risk factors for postoperative wound infection. Age more than or equal to 64 years old, drainage tube retention time more than 48 h, complicated with diabetes mellitus and hormone use are high risk factors for postoperative wound infection.

Responsive protein hydrogels are known to respond to target external stimuli that cause changes in their properties, attracting considerable attention for diverse applications. Here we report the design and recombinant biosynthesis of protein copolymers via genetic fusion of repeating units of resilin with spider silk carboxyl-terminal (CT) domain. The resulting copolymers were thermoresponsive in aqueous solutions, and formed reversible hydrogels at low temperatures and irreversible hydrogels at high temperatures within minutes, a peculiar dual thermogelation feature endowed by the CT domain. The incorporation of resilin blocks upshifted the temperature range of reversible gelation and hydrogel stiffness, whereas the temperature of irreversible gelation was differentially affected by the length of the resilin blocks. In addition, sodium chloride and potassium phosphate at moderate concentrations downregulated both the reversible and irreversible gelation temperatures and hydrogel mechanical properties, proving the salts as another level of control over dual thermogelation. Surprisingly, the copolymers were prone to gelate at body temperature in a time-dependent manner, and the resulting hydrogels were pH-responsive to release a highly polar model drug in vitro. The newly developed resilin-CT copolymers and the multistimuli-responsive hydrogels may be potentially useful in biomedicine, such as for drug delivery.

To retrospective analysis the mid-term follow-up effect of hip joint replacement in elderly patients with failure of intertrochanteric fractures of the hip joint internal fixation.From December 2008 to December 2011, 32 elderly patients underwent arthroplasty after intertrochanteric fracture fixation failure, of which, 4 death cases were excluded from the study, and the remaining 28 cases were in the study group. The age of patients ranged from 69 to 83 years old with a mean of 75 years old. The time from the internal fixation to the hip replacement were 8 to 72 months. Among them, 6 patients were Evans I type, 11 patients were Evans II type, 9 patients were Evans III type, and 2 patients were Evans IV type. Nine cases showed fracture of the lateral plate before operation, while 15 cases were femoral head screw cut-out and 4 cases were screw loosening. Harris score was used to compare the changes of hip function before operation with the final follow-up. Imaging results(X-ray) and erythrocyte sedimentation rate(ESR) were performed during the follow-up.All patients were followed up from 4 to 7 years with an average of 5.3 years. Pain was significantly reduced or disappeared in patients compared with pre-operation. And hip function was significantly improved. Two cases had moderate pain after the physical activity and 4 cases had mild pain after the physical activity. At the final follow-up, 19 patients resumed free walking, 8 patients required walking with walking sticks, and 1 patient needed walking aid. The Harris scores improved from preoperative 34.9±2.4 to 83.4±5.7 at the final follow-up, among them, 15 cases were classified as excellent, 10 as good, 2 as fair, and 1 as poor. X-ray examination showed no prosthesis loosening and sinking fracture.Salvage THA surgery could improve the hip function and the quality of life for old patients with intertrochanteric fracture fixation failure, and the middle-term follow-up results support that.

The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production.

This study aims to preserve fresh catfish meat by using Porphyra yezoensis extract, chitosan and lactic acid Nisin. The composite preservative obtained by sensory evaluation can effectively maintain the color, odor and texture of fresh catfish meat, as well as inhibit bacterial growth. Results show that treatment using a preservative solution (Porphyra yezoensis extract 10%, Nisin 0.2% and chitosan 15%) extended the shelf life of the fresh catfish meat from 12 h to 24 h when stored at room temperature and from 6 d to 9 d when stored at 4°C. These results provide a practical method of preserving fresh catfish meat.

In this work, the effect of N,N'-dicyclohexylcarbodiimide (DCCD) on ginsenoside biosynthesis in suspension cultures of Panax ginseng cells was investigated. The optimal concentration and timing of DCCD addition were found to be 10 μM and on day 4 of cultivation. Under this condition, the maximal content of total ginsenosides increased to 3.0-fold that of untreated control, and the contents of Rg-group (Rg1 and Re) ginsenosides and Rb1 were 2.5- and 8.9-fold higher, respectively, which coincided with elevated activities of protopanaxatriol biosynthetic enzyme protopanaxadiol 6-hydroxylase and UDPG-ginsenoside Rd glucosyltransferase that converts Rd to Rb1. In addition, DCCD treatment induced the activity of defense response enzyme, phenylalanine ammonia lyase. To gain a better understanding of the molecular processes underlying the elicitation, we examined nitric oxide (NO) content and expression levels of the triterpene biosynthetic genes encoding squalene synthase (sqs), squalene epoxidase (se), and dammarenediol-II synthase (ds). It was found that DCCD up-regulated NO generation and transcription levels of sqs, se and ds. Interestingly, these effects of DCCD were compromised by an NO biosynthetic inhibitor, while an NO donor alone recapitulated the elicitation effect of DCCD on ginsenoside biosynthesis. These results suggest that DCCD may induce the ginsenoside biosynthesis via NO signaling in the P. ginseng cells. The information obtained might also be helpful to hyperproduction of valuable secondary metabolites in other plant cell cultures.

Silk protein fibres produced by silkworms and spiders are renowned for their unparalleled mechanical strength and extensibility arising from their high-β-sheet crystal contents as natural materials. Investigation of β-sheet-oriented conformational transitions in silk proteins at the nanoscale remains a challenge using conventional imaging techniques given their limitations in chemical sensitivity or limited spatial resolution. Here, we report on electron-regulated nanoscale polymorphic transitions in silk proteins revealed by near-field infrared imaging and nano-spectroscopy at resolutions approaching the molecular level. The ability to locally probe nanoscale protein structural transitions combined with nanometre-precision electron-beam lithography offers us the capability to finely control the structure of silk proteins in two and three dimensions. Our work paves the way for unlocking essential nanoscopic protein structures and critical conditions for electron-induced conformational transitions, offering new rules to design protein-based nanoarchitectures., National Science Foundation (U.S.) (1563422), National Science Foundation (U.S.) (1562915)

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity ( k cat / K m ) for the OP pesticide ethyl -paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10 5 -fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.

Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.

Precise patterning of polymer-based biomaterials for functional bio-nanostructures has extensive applications including biosensing, tissue engineering, and regenerative medicine. Remarkable progress is made in both top-down (based on lithographic methods) and bottom-up (via self-assembly) approaches with natural and synthetic biopolymers. However, most methods only yield 2D and pseudo-3D structures with restricted geometries and functionalities. Here, it is reported that precise nanostructuring on genetically engineered spider silk by accurately directing ion and electron beam interactions with the protein's matrix at the nanoscale to create well-defined 2D bionanopatterns and further assemble 3D bionanoarchitectures with shape and function on demand, termed "Protein Bricks." The added control over protein sequence and molecular weight of recombinant spider silk via genetic engineering provides unprecedented lithographic resolution (approaching the molecular limit), sharpness, and biological functions compared to natural proteins. This approach provides a facile method for patterning and immobilizing functional molecules within nanoscopic, hierarchical protein structures, which sheds light on a wide range of biomedical applications such as structure-enhanced fluorescence and biomimetic microenvironments for controlling cell fate.

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.

Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.