Synthetic protein condensates for cellular and metabolic engineering.

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.

Direct and Efficient Incorporation of DOPA into Resilin-Like Proteins Enables Cross-Linking into Tunable Hydrogels.

3,4-Dihydroxyphenylalanine (DOPA), a naturally occurring yet noncanonical amino acid, endows protein polymers with diverse chemical reactivities and novel functionalities. Although many efforts have been made to incorporate DOPA into proteins, the incorporation efficiency and production titer remain low and severely hinder the exploration of these peculiar proteins for biomaterial fabrication. Here, we report an efficient biosynthetic strategy to produce large amounts of DOPA-incorporated structural proteins for the fabrication of hydrogels with tunable mechanical properties. First, synthetic genes were constructed that encode repetitive resilin-like proteins (RLPs) with varying proportions of tyrosine residues and molecular weights (Mw). Decoding of these genes into RLPs incorporated with DOPA was achieved via mis-aminoacylation of DOPA by endogenous tyrosyl-tRNA synthetase (TyrRS) in recombinant Escherichia coli cells. By developing a stoichiometry-guided two-phase culture strategy, we achieved independent control of the bacterial growth and protein synthesis phases. This enabled hyperproduction of the DOPA-incorporated RLPs at gram-per-liter levels and with a high DOPA incorporation yield of 76-85%. The purified DOPA-containing RLPs were then successfully cross-linked into bulk hydrogels via facile DOPA-Fe3+ complexations. Interestingly, these hydrogels exhibited viscoelastic and self-healing properties that are highly dependent on the catechol content and Mw of the RLPs. Finally, exploration of the molecular cross-linking mechanisms revealed that higher DOPA contents of the proteins would result in the concomitant occurrence of metal coordination and oxidative covalent cross-linking. In summary, our results suggest a useful platform to generate DOPA-functionalized protein materials and provide deeper insights into the gelation systems based on DOPA chemistry.

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers.

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-1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m-3. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.

Formation and functionalization of membraneless compartments in Escherichia coli.

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.

Fibrous Structure and Stiffness of Designer Protein Hydrogels Synergize to Regulate Endothelial Differentiation of Bone Marrow Mesenchymal Stem Cells.

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.

Spatially Directed Biosynthesis of Quantum Dots via Spidroin Templating in Escherichia coli.

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 ß-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.

Controllable Fibrillization Reinforces Genetically Engineered Rubberlike Protein Hydrogels.

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.

Development of Adhesive and Conductive Resilin-Based Hydrogels for Wearable Sensors.

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.

Engineering tunable biosensors for monitoring putrescine in Escherichia coli.

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.

Combination of traditional mutation and metabolic engineering to enhance ansamitocin P-3 production in Actinosynnema pretiosum.

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.

Rationally Designed Redox-Sensitive Protein Hydrogels with Tunable Mechanical Properties.

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.

Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

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.

Direct biosynthesis of adipic acid from a synthetic pathway in recombinant Escherichia coli.

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.

Designer protein compartments for microbial metabolic engineering.

Protein compartments are distinct structures assembled in living cells via self-assembly or phase separation of specific proteins. Significant efforts have been made to discover their molecular structures and formation mechanisms, as well as their fundamental roles in spatiotemporal control of cellular metabolism. Here, we review the design and construction of synthetic protein compartments for spatial organization of target metabolic pathways toward increased efficiency and specificity. In particular, we highlight the compartmentalization strategies and recent examples to speed up desirable metabolic reactions, to reduce the accumulation of toxic metabolic intermediates, and to switch competing metabolic pathways. We also identify the most important challenges that need to be addressed for exploitation of these designer compartments as a versatile toolkit in metabolic reprogramming.

Modulating Polyalanine Motifs of Synthetic Spidroin for Controllable Preassembly and Strong Fiber Formation.

Spider dragline (major ampullate) silk is one of the toughest known fibers in nature and exhibits an excellent combination of high tensile strength and elasticity. Increasing evidence has indicated that preassembly plays a crucial role in facilitating the proper assembly of silk fibers by bridging the mesoscale gap between spidroin molecules and the final strong fibers. However, it remains challenging to control the preassembly of spidroins and investigate its influence on fiber structural and mechanical properties. In this study, we explored to bridge this gap by modulating the polyalanine (polyA) motifs in repetitive region of spidroins to tune their preassemblies in aqueous dope solutions. Three biomimetic silk proteins with varying numbers of alanine residues in polyA motif and comparable molecular weights were designed and biosynthesized, termed as N16C-5A, N15C-8A, and N13C-12A, respectively. It was found that all three proteins could form nanofibril assemblies in the concentrated aqueous dopes, but the size and structural stability of the fibrils were distinct from each other. The silk protein N15C-8A with 8 alanine residues in polyA motif allowed for the formation of stable nanofibril assemblies with a length of approximately 200 nm, which were not prone to disassemble or aggregate as that of N16C-5A and N13C-12A. More interestingly, the stable fibril assembly of N15C-8A enabled spinning of simultaneously strong (623.3 MPa) and tough (107.1 MJ m-3) synthetic fibers with fine molecular orientation and close interface packing of fibril bundles. This work highlights that modulation of polyA motifs is a feasible way to tune the morphology and stability of the spidroin preassemblies in dope solutions, thus controlling the structural and mechanical properties of the resulting fibers.

Spatiotemporal Organization of Functional Cargoes by Light-Switchable Condensation in Escherichia coli Cells.

Biomolecular condensates are dynamic subcellular compartments that lack surrounding membranes and can spatiotemporally organize the cellular biochemistry of eukaryotic cells. However, such dynamic organization has not been realized in prokaryotes that naturally lack organelles, and strategies are urgently needed for dynamic biomolecular compartmentalization. Here we develop a light-switchable condensate system for on-demand dynamic organization of functional cargoes in the model prokaryotic Escherichia coli cells. The condensate system consists of two modularly designed and genetically encoded fusions that contain a condensation-enabling scaffold and a functional cargo fused to the blue light-responsive heterodimerization pair, iLID and SspB, respectively. By appropriately controlling the biogenesis of the protein fusions, the condensate system allows rapid recruitment and release of cargo proteins within seconds in response to light, and this process is also reversible and repeatable. Finally, the system is demonstrated to dynamically control the subcellular localization of a cell division inhibitor, SulA, which enables the reversible regulation of cell morphologies. Therefore, this study provides a new strategy to dynamically control cellular processes by harnessing light-controlled condensates in prokaryotic cells.

Programmable adhesion and morphing of protein hydrogels for underwater robots.

Soft robots capable of efficiently implementing tasks in fluid-immersed environments hold great promise for diverse applications. However, it remains challenging to achieve robotization that relies on dynamic underwater adhesion and morphing capability. Here we propose the construction of such robots with designer protein materials. Firstly, a resilin-like protein is complexed with polyoxometalate anions to form hydrogels that can rapidly switch between soft adhesive and stiff non-adhesive states in aqueous environments in response to small temperature variation. To realize remote control over dynamic adhesion and morphing, Fe3O4 nanoparticles are then integrated into the hydrogels to form soft robots with photothermal and magnetic responsiveness. These robots are demonstrated to undertake complex tasks including repairing artificial blood vessel, capturing and delivering multiple cargoes in water under cooperative control of infrared light and magnetic field. These findings pave an avenue for the creation of protein-based underwater robots with on-demand functionalities.

Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

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.

Synthetic biology-guided design and biosynthesis of protein polymers for delivery.

Vehicles derived from genetically engineered protein polymers have gained momentum in the field of biomedical engineering due to their unique designability, remarkable biocompatibility and excellent biodegradability. However, the design and production of these protein polymers with on-demand sequences and supramolecular architectures remain underexplored, particularly from a synthetic biology perspective. In this review, we summarize the state-of-the art strategies for constructing the highly repetitive genes encoding the protein polymers, and highlight the advanced approaches for metabolically engineering expression hosts towards high-level biosynthesis of the target protein polymers. Finally, we showcase the typical protein polymers utilized to fabricate delivery vehicles.