An official website of the United States government
Silk proteins represent a unique class of biopolymers produced by arthropods that can be leveraged to form a wide range of biomaterials, including particles at the nano- and microscale. Silk-derived particles are stable and biodegradable into amino acids and can entrap and stabilize cargo for applications in healthcare, agriculture, and advanced materials. The utility of silk particles stems from their inherent protein folding, leading to crystalline domains that kinetically trap and stabilize cargoes. Organization and presence of these domains are dependent on synthesis methodology, protein composition (silk fibroin, silk sericin, silk composites), and encapsulated cargo molecules. Herein, we evaluate regenerated silk particles derived from Plodia interpunctella silk through mechanisms of phase separation and nanoprecipitation with and without small molecule encapsulants. Shifts in particle properties are expected to result from the silk fiber source, the mechanism of protein self-assembly between particle synthesis methods, and cargo molecule encapsulation. The morphology, crystallinity, size, dispersity, and zeta potential of particles derived from nondegummed P. interpunctella silk fibers exhibit distinct properties from degummed Bombyx mori silk fibroin particles. Plodia interpunctella silk particles are capable of encapsulating small molecules of varying size, hydrophobicity, and charge with high efficiency. This study investigates silk-based biomaterials derived from native, nondegummed P. interpunctella silk fibers and demonstrates that protein composition and structural motifs of P. interpunctella silk fibroin influence particle formation and properties. Use of nondegummed silk fibers reduces the processing time required to regenerate silk proteins into biomaterials, offering a promising platform for expanding the functional space of silk-based biomaterials.
more »
« less
Secretion‐Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface
Abstract Protein‐based biomaterials have played a key role in tissue engineering, and additional exciting applications as self‐healing materials and sustainable polymers are emerging. Over the past few decades, recombinant expression and production of various fibrous proteins from microbes have been demonstrated; however, the resulting proteins typically must then be purified and processed by humans to form usable fibers and materials. Here, we show that the Gram‐positive bacteriumBacillus subtiliscan be programmed to secrete silk through its translocon via an orthogonal signal peptide/peptidase pair. Surprisingly, we discover that this translocation mechanism drives the silk proteins to assemble into fibers spontaneously on the cell surface, in a process we call secretion‐catalyzed assembly (SCA). Secreted silk fibers form self‐healing hydrogels with minimal processing. Alternatively, the fibers retained on the membrane provide a facile route to create engineered living materials fromBacilluscells. This work provides a blueprint to achieve autonomous assembly of protein biomaterials in useful morphologies directly from microbial factories.
more »
« less
- Award ID(s):
- 2045844
- PAR ID:
- 10482659
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 62
- Issue:
- 37
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Renewable and degradable materials, formed using biopolymers as material precursors, are sought after in pharmaceutical, biomedical, and industrial fields. Silk-based biomaterials, primarily derived from the silk fibroin protein of the Bombyx mori (B. mori) silkworm, have advantageous mechanical properties, biocompatibility, and commercial availability. Recent efforts aim to expand the range of achievable silk-based biomaterial properties via alternative sources of silk proteins with different sequences and structures. These structural distinctions drive differences in physical and chemical properties of silk fibers, primarily due to the varying degree of crystallinity in the polymers. For the development of alternative silk-based materials, silk from Plodia interpunctella (P. interpunctella), a small agricultural pest that infests and damages food products via silk production, is evaluated. Early investigations have highlighted differences between P. interpunctella and B. mori silk fibroin proteins, however P. interpunctella silk still largely lacks characterization and optimization on both the silk fiber and bulk material level. This work evaluates the structural, thermal, mechanical, and cell-material properties of non-degummed and degummed P. interpunctella silk as a raw material for biomaterial fabrication and discusses the benefits and limitations of these proteins as new biopolymers. Observed properties are used to identify links between silk fibroin protein sequence and fiber function in addition to forming hypotheses in how P. interpunctella silk-based biomaterials will perform in comparison to other natural biopolymers. Future work aims to develop methods to process P. interpunctella silk into material formats, utilizing the material characteristics determined here as a baseline for shifts in material performance.more » « less
-
Silk fibers are produced by a wide variety of insects. The silkworm Bombyx mori (Bombyx) was domesticated because the physical properties of its silk fibers were amenable to the production of fine textiles. Subsequently, engineers have regenerated silk fibroin to form biomaterials. The monocular focus on Bombyx silk has underutilized the expanse of diverse silk proteins produced by more than 100,000 other arthropods. This vast array of silk fibers could be utilized for biomedical engineering challenges if sufficient rearing and purification processes are developed. Herein, we show that the moth, Plodia interpunctella (Plodia), represents an alternative silk source that is easily reared in highly regulated culture environments allowing for greater consistency in the silk produced. We controlled the temperature, resource availability (larvae/gram diet), and population density (larvae/mL) with the goal of increasing silk fiber production and improving homogeneity in Plodia silk proteins. We determined that higher temperatures accelerated insect growth and reduced life cycle length. Furthermore, we established initial protocols for the production of Plodia silk with optimal silk production occurring at 24 °C, with a resource availability of 10 larvae/gram and a population density of 0.72 larvae/mL. Population density was shown to be the most prominent driving force of Plodia silk mat formation among the three parameters assessed. Future work will need to link gene expression, protein production and purification, and resulting mechanical properties as a function of environmental cues to further transition Plodia silk into regenerated silk fibroin biomaterials.more » « less
-
Cann, Isaac (Ed.)ABSTRACT Industrial bioproduction of proteins, particularly protein-based materials (PBMs) like spider silk and elastin proteins, is rapidly expanding. PBMs often have high molecular weights and are highly repetitive, transcribed from long and repetitive mRNAs that are prone to degradation in microbial hosts. As a result, recombinant expression of PBMs often has low protein yields. In this study, we engineered a circular mRNA expression system to enhance mRNA stability and protein expression. The system uses self-cleaving ribozymes to form circular mRNA structures and a pair of insulation RNA loops to improve protein translation. When tested using a green fluorescent protein (GFP) reporter, the engineered circular mRNA enhanced GFP expression by 1.5-fold compared to expression from a linear construct. mRNA circularization was further confirmed using reverse transcription followed by DNA amplification and sequencing. We also demonstrate the effectiveness of circular mRNA in enhancing the expression of various material proteins, including a 96-mer repeat ofNephila clavipesdragline silk, a titin repeat, a mussel foot protein oligomer, and an silk-amyloid repeat, resulting in up to 2.5-fold increase in protein yield. Additionally, the circular mRNA system also improved the stability of the PBM-encoding plasmid. Overall, the circular RNA expression system enhances both the expression level and plasmid stability and is suitable for various protein production applications.IMPORTANCEIndustrial bioproduction of complex proteins is limited by unstable expression. Long and repetitive proteins have unstable expression and often yield truncated products that will change the properties of the final materials. We show that by using a self-circularizing mRNA system, the expression is stabilized to not only increase yields but also prevent truncated products. The ability to produce full-length proteins consistently and control their size offers precise control over protein properties, making it highly relevant for products with specific mechanical properties. The study showcases the potential for scaling up protein production in industrial bioreactors under challenging conditions. The findings contribute to synthetic biology tools and offer new avenues for manufacturing bioproducts at an industrial scale.more » « less
-
Abstract BackgroundThe increasing prevalence of plastic waste combined with the inefficiencies of mechanical recycling has inspired interest in processes that can convert these waste streams into value-added biomaterials. To date, the microbial conversion of plastic substrates into biomaterials has been predominantly limited to polyhydroxyalkanoates production. Expanding the capabilities of these microbial conversion platforms to include a greater diversity of products generated from plastic waste streams can serve to promote the adoption of these technologies at a larger scale and encourage a more sustainable materials economy. ResultsHerein, we report the development of a new strain ofPseudomonasbacteria capable of converting depolymerized polyethylene into high value bespoke recombinant protein products. Using hexadecane, a proxy for depolymerized polyethylene, as a sole carbon nutrient source, we optimized media compositions that facilitate robust biomass growth above 1 × 109 cfu/ml, with results suggesting the benefits of lower hydrocarbon concentrations and the use of NH4Cl as a nitrogen source. We genomically integrated recombinant genes for green fluorescent protein and spider dragline-inspired silk protein, and we showed their expression inPseudomonas aeruginosa, reaching titers of approximately 10 mg/L when hexadecane was used as the sole carbon source. Lastly, we demonstrated that chemically depolymerized polyethylene, comprised of a mixture of branched and unbranched alkanes, could be converted into silk protein byPseudomonas aeruginosaat titers of 11.3 ± 1.1 mg/L. ConclusionThis work demonstrates a microbial platform for the conversion of a both alkanes and plastic-derived substrates to recombinant, protein-based materials. The findings in this work can serve as a basis for future endeavors seeking to upcycle recalcitrant plastic wastes into value-added recombinant proteins.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/anie.202305178
