The Biology of Natural Polymers Accelerates and Expands the Science of Biomacromolecules: A Focus on Structural Proteins

Abstract

This Perspective explores the use of biomacromolecules in natural materials synthesized by living organisms, such as spider silk, in the development of sustainable synthetic materials. Currently employed synthetic polymers lack the hierarchical complexity and unique properties of natural materials composed of biomacromolecules. By understanding the composition of these natural materials, it may be able to reproduce their properties synthetically. Additionally, research directions involving the use of renewable resources such as nitrogen and carbon dioxide from the air and seawater to develop biomacromolecules such as spider silk and biopolyester via photosynthetic organisms are reviewed. Next-generation biomacromolecule research will aid in the creation of a sustainable global society, advancing fields such as biomanufacturing, agriculture, aquaculture, and other industries.

I. Introduction

The various physical and biological properties and functions of naturally occurring polymers and composite materials have not yet been able to be replicated in synthetic polymers and artificial materials.¹⁻³ Many of the unique properties of these natural materials depend on the hierarchical structure, including the primary structure (chemical structure), of the biopolymers produced by a particular species. If we can elucidate these hierarchical structures and clarify the mechanisms that contribute to the physical properties of natural materials, it will be possible for humans to artificially mimic the unique physical properties of natural polymers and biopolymers. While we have learned from silkworm silk and developed nylon, no polymer that combines the toughness, lightness, and interfacial affinity of spider silk has been developed. Thus, it is necessary to move away from the current biomimetic science research and establish an academic field where biological materials can be artificially created and used in a practical manner and to establish a process where materials used by humans do not put the global environment and the natural environment at risk and do not compete with food and energy production.

In nature, there are many attractive biomacromolecules, such as protein,⁴ polyhydroxyalkanoate,^5,6 natural rubber,^7,8 cellulose,⁹⁻¹² lignin,^13,14 chitin,¹⁵⁻¹⁷ and nucleic acid.¹⁸⁻²¹ Among them, I have been continuously studying biomacromolecules synthesized by living organisms, such as proteins, biopolyesters, and natural rubber.^1,3,22−25 In recent years, I have attempted to develop new processes for the artificial production of biomacromolecules such as cellulose, polyhydroxyalkanoate (PHA), and those found in spider silk and natural rubber by using inexhaustible resources (nitrogen and carbon dioxide) from air and seawater and photosynthetic organisms (Figure 1).^26,27 If this series of studies reveals promising results, then it will be possible to prevent resource depletion and reduce greenhouse gas emissions while simultaneously establishing a material cycle that is symbiotic with nature. While building a production process based on the fixation of nitrogen and carbon dioxide via photosynthetic organisms, we aim to design molecules and materials that are stable during use and return to the natural environment after use. This process of recycling materials is important for low-resource countries and regions. Moreover, this process does not place a burden on the global environment by utilizing carbon and nitrogen fixation via photosynthetic organisms. I suggest that the creation of next-generation biomanufacturing industries, as well as chemical industries, will lead to the realization of a harmonious global society through organic linkages with next-generation agriculture, animal husbandry, fisheries, and forestry industries. In this Perspective, structural proteins are introduced as representative examples of biopolymers, and the decomposition of carbon dioxide and biomacromolecule biosynthesis from carbon dioxide are outlined.

Figure 1.

Overview of the design and sustainable biosynthesis of spider silk-based materials. Using carbon dioxide and nitrogen, artificial silk was synthesized from marine red photosynthetic bacteria (left figure).²⁸⁻³⁰ The spinning mechanism of spiders has been elucidated at the molecular level, and a hierarchical structure similar to spider silk was successfully reproduced for the first time in the world (lower right figure).³¹⁻³⁵ A database of spider silk proteins was constructed, and the use of material informatics enabled the development and commercialization of artificial silk with excellent water resistance (upper right figure).^36,37 Copyright 2020 AAAS.

II-i. Structural Proteins

For the past 10 years, I have focused on structural proteins, which are biological materials, as indispensable materials for a sustainable global society. A structural protein is a protein that possesses a characteristic amino acid sequence or motif with tandem repeats, according to previous literature.² Structural proteins can be fibrillar proteins but partially similar to intrinsically disordered proteins.³⁸⁻⁴⁰ In most cases, such motifs form higher-order structures via intermolecular or intramolecular interactions, resulting in biological materials with specific physical characteristics. However, considering amorphous protein materials, the requirement of a hierarchical structure is not included in this definition. Structural proteins in nature include spider silk, silkworm silk and other silks, collagen, elastin, resilin, reflectin, and keratin (Figure 2).⁴¹⁻⁵⁷ Moreover, biomineralization, glycosylation, and post-translational modifications can be combined to achieve even more diverse physical and biological properties.⁵⁸⁻⁶¹ Thus, it is important to understand materials in nature in terms of their molecular units and use this information to design innovative biopolymers and develop artificial materials. I expect to contribute to the construction of a sustainable society by creating biomacromolecular materials with both mechanical properties and high environmental degradability that fundamentally solve the problems caused by the use of polymeric materials, such as microplastics in the ocean and carbon dioxide emissions from petroleum raw materials and processes.

Figure 2.

Pictures of silk producers and silk threads. (a) Trichonephila clavata spider and threads; (b) Nephila pilipes spider on the author’s hand; other silk producers, (c) silkworms and (d) bagworms; (e) spider egg case; and (f) silkworm cocoons and silk threads. (g) Examples of structural proteins are found in nature. Their roles/functions in nature and typical repeat sequences are listed.² Copyright 2020 Springer-Nature.

II-ii. Spider Silk

Spider silk, which is composed of the structural protein silk, has attracted attention from various research fields and industries as a material that exhibits a variety of physical properties that cannot be achieved with existing structural materials.^37,62,63 Using spidroin, the main component of spider silks, as a model polymer, we have clarified its hierarchical structure (especially crystal formation) and the mechanisms of its physical properties (Figure 3).^23,64,65 The spider draglines are composed of multiple proteins called major ampullate spidroins (MaSps), but how many types of MaSps exist is still under debate worldwide.^37,62,63 Currently, many types have been reported, including MaSp1, MaSp2, and MaSp3, whereas spider-silk constituting element (SpiCE), which is present in trace amounts but has been found to significantly affect the mechanical properties, can be an essential component to design artificial spider silk.³⁷ I studied the self-assembly behavior of MaSp1 and MaSp2 via various techniques, and the interaction between MaSp leads to dimerization of the C-terminal domain,^32,66 followed by liquid–liquid phase separation (LLPS) to form a fibrillar network with N-terminal domain dimerization;^31−33,66 then, dehydration and shear stress occur to form a bundle-like structure of microfibrils.^31,32,67 The ionic conditions affect the self-assembly behavior of the terminal domains of MaSp; for example, chaotropic ions cause MaSp to have more random structures, but kosmotropic ions, including phosphate ions, induce more hydrogen bonding and self-assembly such as LLPS.^31,34,35 The effect of water molecules on the formation process of the beta-sheet structure has also been studied widely with various silks.^68,69 Since I have published other reviews on the spinning process of these spider silk threads,^23,64 I will not got into depth here. However, the molecular mechanism for the construction of traction threads involves a complex system with multiple proteins and is not fully understood. In the future, it will be necessary to address the self-assembly behavior of multiple proteins and the formation mechanism of hierarchical structures and to elucidate the mechanism underlying the unique mechanical properties resulting from the presence of heterogeneous proteins.

Figure 3.

Overview of studies on the self-assembly behaviors and structures of spider silk MaSp proteins.⁶⁵ Copyright 2024 Wiley-VCH GmbH.

As mentioned above, spider silk is a natural fiber that has high strength and toughness and is attracting attention as a biomaterial that can replace existing petroleum-derived polymer materials. Since spider silk has a wide range of physical properties depending on its type and protein composition, artificial spider silk materials are expected to be used for a variety of applications, including next-generation high-strength structural materials.^37,62,63 However, the types of natural spider silk proteins and the amino acid sequences that affect their physical properties have not been fully elucidated. There have been several reports on the relationship between the amino acid sequence and the physical properties of the proteins of traction threads, which show particularly high toughness among the multiple types of spider silk, but these reports are limited. An international research group including the author has been collecting various types of spiders distributed worldwide and constructing a database on the structure and physical properties of diverse spider silk proteins.³⁶ After the collection of more than 1000 different spider species from various regions of the world, including Asia, America, and Europe, RNA was extracted from the cells of each spider, and genetic information such as the type and amino acid sequence of the spider silk proteins in the spider silk was analyzed by Arakawa and his colleagues. They also measured 12 properties of traction threads collected from each spider, including mechanical properties (tensile strength, elongation rate, and toughness), thermal stability, and thread diameter. The collected spiders were phylogenetically classified, and a database was created by linking the genetic information (RNA base sequence and amino acid sequence) to the physical properties of the biomacromolecules using RNA. The spider silk proteins for which amino acid sequences have been identified thus far have been derived from 52 species of spiders, and the number of protein types is limited; however, spider silk protein data from many spider species was obtained from the database in this study. This is the first comprehensive database combining spider silk protein structures and spider silk properties and has been published as the Spider Silkome Database (Silkome, https://spider-silkome.org/).³⁶ Currently, various molecules are designed using Silkome, which uses the sequences associated with specific properties of interest. For example, the correlation between the amino acid sequence of the spider silk protein and hypercontraction, which indicates sensitivity to water, revealed that the glutamine-glutamine (diglutamine, QQ) motif and proline in MaSp2 were significantly positively correlated, indicating that these motifs are the main factors responsible for sensitivity to water.^65,70 By replacing these amino acids/motifs with hydrophobic amino acids, it was possible to develop artificial silk with excellent water resistance, which is a rare success story from the viewpoint of material informatics in polymers.⁶⁵ In the future, artificial spider silk materials can be created at will by rational molecular design on the basis of the prediction of physical properties from the amino acid sequence.

III-i. Precise Polymer Degradation

One of the attractive properties of structural proteins is the combination of biodegradability and mechanical properties. In this section, I discuss the biodegradability and degradation products of structural proteins. Polymer science textbooks generally include the synthesis, properties, structures, and functions of numerous polymers. This might be because many polymer textbooks originated from Principles of Polymer Chemistry by P. J. Flory.⁷¹ Even in the latest textbooks, there are few descriptions of polymer degradation, with only a few references to thermal degradation, photodegradation (photooxidation), oxidative degradation, and biodegradation. However, there is a strong trade-off relationship between physical properties and degradability, and it is difficult to reconcile functionality and mechanical properties with degradability. In fact, there is a demand for polymeric materials that can be quickly degraded after use by inserting some molecular switches (in response to external and/or multiple stimuli); many researchers are working on such materials, but they have not yet been realized. In this context, basic molecular theory is needed to precisely induce or inhibit the degradation of polymers without interfering with their physical properties and functions. In fact, the precise degradation of polymers is necessary and attractive in many fields. For example, in the design and synthesis of new polymers, prediction of the degradation behavior is needed, and in the study of deformation and disintegration, it is necessary to chemically understand the molecular cleavage that occurs at the fracture interface. In the natural environment, additionally, the design of monomers and polymers must consider physical disintegration, chemical hydrolysis and photodegradation, and enzymatic degradation and biological metabolism involving living organisms (Figure 4). Thus, a systematic understanding of polymer degradation is important for a wide variety of disciplines.

Figure 4.

Various possibilities for the precise degradation of polymers.

As described above, it is necessary to understand precise polymer degradation mechanisms, such as physical degradation, chemical cleavage, and biological metabolism, which will lead to the establishment of new polymer material design guidelines. To the best of our knowledge, biopolymers and biomacromolecules are indispensable in these material design guidelines worldwide. Considering the wide range of environments in which polymeric/biomacromolecular materials are used, it is scientifically important and an interesting research topic to clarify the molecular mechanisms that dynamically influence the physical properties and functions of biopolymers under the conditions of physical breakdown, chemical degradation, biodegradation, and biological metabolism in vivo and in the natural environment.^72,73 When this series of studies is accomplished, it is expected to be applicable in a wide range of academic fields for creating biomacromolecules with precisely designed and controlled degradability, such as polymers that are safe even when released into the environment, polymers that can be safely used in the body for a long time, and polymers that can be recycled in a closed-loop manner. One of the candidate biomacromolecules with such controllable biodegradability is structural proteins, including spider silk.

III-ii. Biodegradation into CO₂ and/or Natural Molecules at Their Natural Concentrations

Do you think that structural proteins such as silk are sustainable plastics or polymers? In the white paper from Chemical Sciences and Society Summit (CS3), London, 2019 (“Science to enable sustainable plastics”, https://www.rsc.org/news-events/articles/2020/jun/science-to-enable-sustainable-plastics/), structural proteins such as spider silk were also introduced as an environmentally degradable polymer. Examples of the use of plastics that are biodegradable, especially examples of the use of on-demand biodegradation that induces degradation after use, are very limited. Precision control of polymer degradation is very important not only for ensuring the properties of degradable polymers but also for extending the long-term stability of polymer materials. Currently, it is challenging to suppress the deterioration and decomposition of polymer materials and improve their stability for several decades, preventing their use as major structural materials like metals. However, it is possible to address these problems related to the long-term stability and controlled degradability of polymer materials by understanding the decomposition of polymers from a multilevel perspective.

Silk, as a structural protein and a natural polymer, is biodegradable, which makes it a promising alternative to synthetic petroleum-based polymers. I have studied the biodegradability of silk resins through biochemical oxygen demand (BOD) tests and confirmed their breakdown in natural environments (Figure 5). Compared with synthetic polymers or biodegradable polyesters, natural structural proteins and artificial silk materials (resins, films, gels, coatings, composites, and fibers) can be degraded in the natural environment,⁷⁴⁻⁷⁷ and the degradation products are nontoxic to model organisms, e.g., Daphnia magna.⁷⁸⁻⁸⁰ These degradation productions are unique compared with biodegradable polyesters and other materials.⁸⁰ Further tests were performed to assess the impact of coating silk films with fluorinated polypeptides, which increased water repellency without significantly affecting biodegradability.⁷⁶ Scanning electron microscopy (SEM) revealed that the coated films developed surface roughness and cracks when exposed to seawater over time, indicating successful degradation. These results suggest that fluorinated polypeptides can alter the material properties of silk without compromising environmental friendliness, paving the way for more sustainable polymer materials.

Figure 5.

Biodegradation and environmental effects of model silk fragments, namely, polymers and oligomers of Ala and different nylon units. (a) BOD tests of poly(AlaNylXAla) and polyAla in seawater at 25 °C.⁷⁸ (b) Daphnia magna used for the environmental toxicity test. (c) Kaplan–Meier survival curves for evaluating the effects of poly(AlaNylXAla) and polyAla on the aquatic model animal Daphnia magna at 20 °C. Control denotes the background values without any test samples.⁷⁸ Copyright 2020 Royal Society of Chemistry.

IV-i. Biomacromolecule Synthesis from CO₂

To realize a sustainable society and a circular economy, polymer scientists need to create biomacromolecular materials that are attractive from an environmental, social, and governance (ESG) perspective. To solve the social problems caused by polymeric materials such as plastics, it is important for both industry and academia to create environmentally degradable and recyclable polymer materials that are friendly to the earth by making full use of biotechnology as well as CO₂ and nitrogen as raw materials. In particular, I aim to achieve biopolymer production using photosynthetic organisms through a process with a low environmental impact that does not require organic solvents, high-temperature/pressure, or harsh conditions.^22,81,82 For example, the spinning process of artificial silk fibers using only aqueous solvents is progressing with an emphasis on zero-carbon processes.^{32,65,67,83−85} Not only are the final biopolymer products environmentally degradable, but the synthesis and process also do not use fossil resources as raw materials, which coincides with zero-carbon initiatives.

Among photosynthetic organisms, purple photosynthetic bacteria are unique. In addition to CO₂ and nitrogen fixation, purple photosynthetic bacteria, as exemplified by PHA and spider silk protein production, have been reported to be beneficial for biopolymer production.^{26,28,82,86−89} Industrial fermentation and production technologies generally utilize heterotrophic microorganisms, which require sugar or vegetable oil as a carbon source and fresh water as a culture medium for the growth of heterotrophic microorganisms, and there are concerns that as the scale of production increases, there will be competition for human food and water resources. Marine purple photosynthetic bacteria do not need a carbon source and do not compete with freshwater resources when they carry out photosynthesis under anaerobic conditions to promote CO₂ fixation. Furthermore, biopolymer/biomacromolecular production with marine photosynthetic bacteria has advantages in terms of raw material costs and large-scale production. Recently, genetic modification technology for marine photosynthetic purple bacteria has made it possible to produce exogenous proteins and metabolites.^29,30,90 The author studied and developed a system to synthesize biopolymeric materials from purple photosynthetic bacteria via the use of inexhaustible seawater, CO₂, and nitrogen as starting materials (Figure 6).^26,27,88,91 This bioproduction using autotrophic microorganisms has some drawbacks, and it is necessary to meet the need for an electron donor since electrons cannot be extracted from water splitting.

Figure 6.

Photosynthetic bacterial biopolymer synthesis.

I constructed a basic technology for the artificial synthesis of structural protein materials and biopolymers, such as spider silk, using Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium isolated from seawater, as the main strain.^26,87,88R. sulfidophilum, which can be cultured under seawater conditions and has also been isolated from the seawaters in Japan, fixes carbon dioxide via photosynthesis and nitrogen via nitrogenase activity.⁹¹ So far, the common genetic recombination is by gene transfer via Escherichia coli, but the “peptide method” I developed has made it possible to prepare recombinants in a high-throughput manner. The peptide method is a gene delivery system that uses fusion peptides to introduce exogenous DNA/RNA into various organisms as well as specific organelles (Figure 7).^{30,90,92−100} The promoter, terminator, and selection marker genes used to express the desired structural protein by genetic recombination were also optimized, and an artificial silk, i.e., a biopolymer with a polyamide backbone, was successfully synthesized from R. sulfidophilum.²⁸ The process by which photosynthetic bicarbonate ions are metabolized into amino acids and the process by which nitrogen gas is incorporated into amino acids via nitrogenase activity have also been confirmed from ¹³C and ¹⁵N labeling experiments, providing scientific evidence of carbon dioxide and nitrogen fixation.⁹¹ Thus, the use of photosynthetic organisms with carbon dioxide and nitrogen fixation abilities resolves the issue of limited carbon and nitrogen sources and reveals the possibility of circular polymer production.

Figure 7.

Schematic of the peptide method.^22,98,101 A carrier peptide consisting of two domains is designed and synthesized and complexed with the molecule to be delivered, such as DNA or protein (upper left figure). Moreover, macropinocytosis-inducing peptides are processed into target organisms (upper right figure), enabling efficient transformation and functional modification of cells (lower figure).

IV-ii. Artificial Silk Production via Photosynthesis (Air Silk)

As described in the previous section, the biosynthesis of spider silk protein by marine purple photosynthetic bacteria was performed via genetic modification.²⁸ The key technology for the future is how to utilize carbon dioxide and nitrogen for material production. Recently, a collaborating team and I succeeded in creating a zero-carbon protein fiber (Air Silk) using marine purple photosynthetic bacteria (Figure 8).⁶⁵ These protein fibers were prepared in a similar manner to natural spider silk;⁶⁷ hence, the coagulations were performed with weak acidic buffers. Air Silk can be prepared without an organic solvent and strong acid/alkaline conditions and therefore is expected to contribute to a sustainable next-generation textile industry as a new alternative to synthetic fibers.

Figure 8.

Development of Air Silk using photosynthetic bacteria. A scheme for introducing a gene encoding MaSp2 from spider silk into marine red photosynthetic bacteria and biosynthesizing silk protein through fermentation and culture.²⁸ The aim is to create a new value-added fiber material by converting the obtained silk protein into fiber.⁶⁵ Copyright 2024 Wiley-VCH GmbH.

IV-iii. Potential of Biological Synthesis of Biomacromolecules

In the last 20 years, I have synthesized various biopolymers through various biological processes (Figure 9). Recently, using plant cells, intact plants, cyanobacteria, and marine purple photosynthetic bacteria, we established a platform for the artificial synthesis of PHA, a biomass plastic, and structural proteins such as spider silk.^{27,28,81,82,102,103}

Figure 9.

How can we design, synthesize, and process biomacromolecules? Schematic illustration of the author’s research strategy. Sustainable biomacromolecular design, biosynthesis, and material processing are needed to achieve optimized physical, chemical, and biological properties/functions.³

The synthesis method that I ultimately aimed to generate various biomacromolecules via photosynthetic organisms was performed using abundant raw materials such as air. As introduced above, the purple photosynthetic bacterial production is one of the successful examples. In addition to marine purple photosynthetic bacteria, my target photosynthetic organisms include plants, trees, cultured plant cells, algae, cyanobacteria, and photosynthetic bacteria.^104−112 Using the inexhaustible resources of air (CO₂ and nitrogen) and seawater and photosynthetic organisms, my research group has attempted to achieve new production processes for biopolymers and proteins such as spider silk, cellulose, natural rubber, and other types of biopolymers. If this kind of research is accomplished successfully, a material cycle that is symbiotic with nature can be established while reducing resource depletion and rising greenhouse gas emissions. By building a production process based on the fixation of nitrogen and carbon dioxide via photosynthetic organisms, the next step can be designing and developing molecules and materials that are stable during use and return to the natural environment after use. This ultimate resource recycling of materials can be important for low-resource countries and regions, especially in Japan, by reducing the burden on the environment through the use of carbon dioxide and nitrogen as raw materials. Instead of conventional petroleum-derived plastics and synthetic fibers, I would like to promote the spread of environmentally friendly zero-carbon materials and products in terms of raw materials, manufacturing methods, and life cycles.

V. Conclusions and Future Perspectives

While silkworm silk has been extensively studied in the context of nylon development, no polymer that combines the toughness, lightness, and interfacial affinity of spider silk has been developed. In addition, although natural rubber is widely used and indispensable to modern society, elucidation of the mechanisms underlying its physical properties, such as strain-induced crystallization and artificial reproduction of its mechanical properties, has not been achieved. To the best of the author’s knowledge, these scientific limitations may be because only the chemical structure has been elucidated and not the hierarchical or network structure. In the future, I would like to continue to investigate biomacromolecules, including their hierarchical structure, and clarify the relationship between structure and function to provide novel insights for the development of artificial biological materials.

Using spider silk as an example in this Perspective, it is important to understand biological materials in nature in terms of their biomacromolecules and other constituent units and to elucidate the relationships between their properties and molecular composition, which leads to sustainable material innovation. The creation of polymeric materials with both optimized physical properties and environmental degradability will fundamentally solve the problems caused by polymeric materials, such as microplastics in the ocean and carbon dioxide emissions from petroleum raw materials and processes, and contribute to the construction of a sustainable and circular society. On a slightly larger scale, it is necessary to move away from the current biomimetic research and establish an academic domain where biological materials can be created and used in a practical manner and establish a process where materials used by humans do not put the global environment and the natural environment at risk and do not compete with food and energy production. I am convinced that such an academic field is essential for a sustainable global society and can be important for low-resource countries and regions, including Japan. Although not mentioned in this Perspective, efforts are underway in the fish industry to develop zero-carbon fishery feed by generating optimized proteins via photosynthetic bacteria and in the agricultural sector to develop zero-carbon fertilizers such as amino acid-based nitrogen fertilizer.¹¹³ I hope to contribute to the development of a sustainable global society not only through chemical research but also through biology studies and through tight linkages with diverse fields such as next-generation aquaculture and agriculture.

Author Contributions

K.N. designed, wrote, and edited the manuscript.

This work was supported by the Japan Science and Technology Agency (JST) Exploratory Research for Advanced Technology (ERATO, JPMJER1602), JST COI-NEXT (Grant Number JPMJPF2114), and the MEXT Program: Data Creation and Utilization-Type Material Research and Development Project Grant Number JPMXP1122714694.

The author declares no competing financial interest.

Dedication

This Perspective is dedicated to the 25th Anniversary of Biomacromolecules.