Engineering of silk proteins for materials applications
Graphical abstract
Natural silk as a green biopolymer.
Recombinant silk proteins can be engineered at the molecular level, produced in different host organisms, and processed into different morphologies for new technical or medical applications.
Introduction
Silk is defined as a class of fibrous proteins, which is extracorporally applied by animals of the phylum of arthropods, including the classes of Insecta, Arachnida, and Myriapoda [1,2]. Among them, silkworms and spiders are the most prominent silk producers. Since millions of years, silk fibers have evolved diversely to be used as protective shelter, dispersal, prey capture, cocoons and for reproduction [1]. Also, humans have utilized silk since thousands of years for several applications highlighting its importance in culture, economy, and science [3]. The cocoon silk of the domesticated silk moth Bombyx mori (mulberry silk) is most commonly used for textile applications [2,3], while silks from spiders have been often applied for medical purposes such as wound dressings and sutures [4]. These examples already show a broad usability for many applications, since silks often reveal excellent mechanical properties such as high strength and extensibility resulting in a great toughness (Figure 1) in combination with good biocompatibility, biodegradability, non-toxicity, and low immunogenicity when brought in contact with the human body [5, 6, 7]. So far, no other natural or synthetic fibrous material can accomplish these remarkable mechanical characteristics, which are based on the hierarchical setup of silk proteins and the strict control of structural arrangements during fiber assembly [8]. Despite the diversity of silks and their sequences from different organisms, common structural patterns can be seen. Generally, the proteinaceous silk core is composed of fibrils, which are oriented along the fiber axis and contain nanometer-sized crystallites embedded in an amorphous matrix [3]. These crystals comprise tightly stacked anti-parallel β-sheet stretches, which contribute to the silk’s tensile strength and high toughness [8]. Additionally, β-spirals, β-turns, random coils, and helices form an amorphous region and provide flexibility and elasticity to the fiber [1,8]. Besides the relative ratio and distribution of crystalline and amorphous parts, the nanoconfinement of β-sheet crystals is a key factor in obtaining good mechanical properties [9]. It was detected that nanometer-sized crystals reveal higher toughness than larger β-sheet crystals [9]. At molecular level, structural motifs are given by the protein’s sequence, showing a highly repetitive core domain flanked by highly conserved non-repetitive terminal domains. The core domain contains alternating crystalline domains and amorphous regions and is responsible for the mechanical properties of the final fiber [10], while the non-repetitive termini are essential for storage of silk proteins at high concentrations and are playing an important role for fiber assembly by acting as molecular switches sensing pH-changes, mechano-changes, and changes in ionic strength and composition [11].
Utilization of natural animal-based materials such as silk in applications often entails several drawbacks, for example, impurities, batch-to-batch fluctuations in quality and quantity, or difficulties in domesticating the animals (e.g. in case of spiders) [8,12••]. In recent years, several biotechnological production routes have been developed to establish an alternative source of silk on an industrial scale, ensuring constant quality and biological safety [8,12••]. Genetic engineering of silk proteins allows to design a multitude of future-oriented ecological materials with tunable features for defined applications and best possible performance [13, 14, 15]. Knowledge concerning the sequence-structure-function relationship of the proteins is essential for the engineering process in order to avoid a negative impact on the physical properties [14,16•]. Great progress has been made concerning the modification of proteins by changing the amino acid composition or utilizing the ability to generate fusion constructs with other materials (mainly peptides and proteins) with synergistic properties [14]. Such functionalization of the underlying silk proteins enables the development of customized functional materials.
Living in an increasingly resource-constrained world, the development of bio-based materials with low environmental footprint is indispensable. Bioinspired silk materials can replace traditional synthetic polymers, especially when they are functionalized for explicit applications, and consequently contribute to green fabrication and to circular economy in terms of sustainability and performance [17]. Innovative solutions have been developed by interdisciplinary research teams to recombinantly produce functionalized silk proteins at large scale and in high quality. Thereby, several commercial applications have emerged over time in several market segments, such as cosmetics, regenerative medicine, or textile fabrication (Table 1).
Here, we review recent advances in engineering of recombinant silk proteins at the molecular level to achieve novel functions. Accordingly, we pay special attention to genetic modifications to establish new characteristics of the silk proteins. Moreover, we highlight current limitations and present approaches to overcome some of these challenges in the near future.
Section snippets
Functionalization of silk proteins using molecular engineering
Functionalized recombinant silk proteins are currently investigated concerning their application as green biopolymers by researchers within different disciplines. Molecular engineering techniques are applied to design bio-based silk proteins with tunable features (Table 2). While the present review covers molecular engineering of recombinant silk proteins, silk materials also can be functionalized at the macroscopic level resulting in composite materials. Briefly, this can be accomplished by
Current limitations in recombinant production of engineered silk proteins
So far, great progress has been made in the development of engineering approaches for silk proteins, but several limitations still exist concerning their production. The extent of modifications such as the incorporation of functional tags in a silk protein domain is limited. Structural properties of silk regarding self-assembly into higher ordered structures need to be maintained to guarantee the functional properties within the final material [16•]. Computational simulations would provide
Applications of functionalized recombinant silk proteins
Functionalization of silk proteins enables the development of a multitude of materials with unprecedented versatilities for example for medical applications including diagnostics, biosensoring, or regeneration of soft and hard tissues (Table 2). New functionalized materials combining physical silk properties and biologically active peptides comprising for example, specific cell binding motifs achieved proper cell attachment and proliferation suitable for tissue engineering [12••]. Incorporation
Outlook
Great progress has been made in the development of engineered silk materials for a variety of applications. Understanding structure-function relations of silk proteins using computational simulations will very likely guide engineering of artificial silk in the future. Utilizing technologies such as intein splicing can overcome protein size limitations within biotechnological production, and further enable the fabrication of new recombinant silk proteins with additional properties, even in
Conflict of interest statement
T.S. is co-founder of the company AMSilk GmbH.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
Financial support is provided by the Collaborative Research CenterTRR 225 TP C01.
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2022, Industrial Crops and ProductsCitation Excerpt :Silk is one of the major fabrics that must be dry-cleaned (Wang et al., 2019). In addition to various applications in biomedicine, ecology, and electricity, silk fibers are excellent materials for high-end textiles because of excellent properties such as comfort, strength, elasticity, moisture absorbency, anti-static charge (Liang et al., 2021; Saric and Scheibel, 2019; Zheng et al., 2018). Due to the poor wet stabilities, silk fabrics must be dry-cleaned at great expense to retain those excellent properties (Kan, 2014; Mu et al., 2020b; Wen et al., 2022).
Expanding the chemical repertoire of protein-based polymers for drug-delivery applications
2022, Advanced Drug Delivery ReviewsCitation Excerpt :Silkworm and dragline silk are characterized by assembly of the hydrophobic domains into crystalline β-sheet structures, the extent of which can affect the properties of the resulting biomaterial such as the tensile strength and biodegradability [25,26]. Although the recombinant expression of high molecular-weight silk proteins is not trivial [10,11,27,28], several SLPs, which comprise repetitive domains derived from various silk proteins, can be recombinantly produced [6]. Naturally harvested silk and SLPs have been utilized to form fibers, films, hydrogels, and nanoparticles for drug-delivery applications.