Table of Content

31 August 2022, Volume 3 Issue 4

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Current advance in engineered living materials

Yuxiang WANG, Xialing WU, Wenbin ZHANG

2022, 3(4):  621-625.  doi:10.12211/2096-8280.2021-083

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Engineered living materials are an emerging research field at the interface between synthetic biology and materials science (especially, polymer science). While materials science provides the fundamental idea about materials construction and a deep understanding on structure-property relationship, synthetic biology affords the possibility to engineer living organisms to fit into the needs of materials. With the development, crossover, and integration of synthetic biology and materials science, novel responsive engineered living materials have been cotinuously emerging. Recently, an engineered living material based on semi-interpenetrating polymer network was reported by the teams of Dr. Dai Zhuojun at Shenzhen Institute of Advanced Technology and Prof. You Lingchong at Duke University. This research used the genetic circuit to control the density-dependent cell lysis and the subsequent release of reactive functional proteins which then polymerize in situ to form a semi-interpenetrating network with the chitosan matrix and anchor the effector proteins. The resulting capsule not only protects the cells from the environment, but also becomes resilient to environmental perturbations. This modular approach to engineered living materials holds great promise for diverse applications such as active biological therapy.

Invited Review



Biofilm matrixes-from soft matters to engineered materials

Runtao ZHU, Chao ZHONG, Zhuojun DAI

2022, 3(4):  626-637.  doi:10.12211/2096-8280.2021-087

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The properties of natural living materials are tied with their biological function. For example, as bacteria grow under some conditions, they generate extracellular matrices composed of proteins and biopolymers to attain specific functions such as protection of bacteria from antibiotics and host defenses. In these dynamic processes, the spatial and temporal information required for the biofilm synthesis is encoded in the genome. The fast development of synthetic biology has greatly promoted the understanding of biology and broadened the application of engineered biological systems. Especially, the field of engineered living materials (ELMs) emerged at the intersection between the synthetic biology and the material science. In the last two decades, genetic engineering has applied living cells to express recombinant fusion proteins that can be purified and processed into protein-based materials. Also, metabolic engineering has applied living cells to synthesize small molecules that can serve as monomers for polymers and rubbers production. However, in these cases the cells only act as bio-factory and the properties of the final materials do not exploit the features of living biological systems. In contrast, ELMs are composed of living cells, which act as building blocks to modulate and direct the formation and function of the final materials. The resultant ELMs is programmable, self-regenerative and evolvable. The most pioneering efforts in ELMs development have focused on the engineering functional amyloids, which are secreted and assembled into nanofibrous structure on the cell surface. Especially, the curli of the E. coli was the very first engineered biofilm system. Since 2014, the related research about programming curli for ELMs assembly has accumulated rapidly. In this review, we discussed why and how this model system was chosen, engineered and developed, from the view of polymer physics and synthetic biology. We hope that our thoughts in this review would bring inspirations for more and further model system development in the ELMs.





Biosynthesis of high-performance protein materials and their applications

Jingjing LI, Chao MA, Fan WANG, Hongjie ZHANG, Kai LIU

2022, 3(4):  638-657.  doi:10.12211/2096-8280.2022-032

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Biomaterials, derived from high-performance structural proteins such as elastin, spidroin, and resilin, exhibit broad applications in high-tech fields from wearable devices, biomedicine to military scenarios. Advanced synthetic biology tools including genetic recombination, site-directed mutagenesis, and metabolic pathway optimization enable the generation of recombinant structural proteins with customized properties. Additionally, such recombinant proteins could further assemble into disordered aggregates or ordered hierarchies to fulfill multiple functionalities. In particular, two or more independently natural structure proteins have been connected to produce recombinant proteins as scaffolds with superior mechanical properties. The mechanical performance has surpassed chemically synthesized polymers. By harnessing the power of metabolic pathway optimization, engineered microbes could enable the large production of high-molecular-weight proteins as renewable materials. Furthermore, through the integration of synthetic biology and materials science, programmable materials with multiple composition and sophisticated spatial organization have been fabricated, with diverse functionalities including self-repair, information storage, impact resistance, wound healing, etc. The protein-based materials established a new niche for current material systems. Despite recent advances, there are still huge challenges to be addressed in rational design, scalable production, and systematic integration for recombinant proteins. Synthetic biology, coupling with advanced techniques in materials science, provides a feasible route to tackle the aforementioned challenges and promote the innovation in bioinspired protein materials. In this review, we outline key advances in the design and fabrication of biosynthetic protein-based materials. First, we highlight the achievements in protein design, structural reconstruction and programmable assembly of novel biomaterials. Next, we discuss the developments of typical advanced protein materials, including biofibers and bioadhesives. Finally, we envision the ideal biosynthetic platforms, which would enable the rational de novo design and mass production for protein-based materials in the future.





Application of cell-free synthesis strategy in biomaterial research

Botao JI, Zhigang QIAN, Xiaoxia XIA

2022, 3(4):  658-675.  doi:10.12211/2096-8280.2021-069

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As various issues such as global warming, environmental pollution and the exhaustion of fossil resources become increasingly severe, biomaterials have attracted increasing attention in the fields of biotechnology and biomedical engineering due to their many advantages such as renewable, degradable nature, and good biocompatibility. Based on the “bottom-up” design concept, cell-free expression system as a useful supplement to the cell synthesis system, greatly accelerates the development of biomaterials and expands their application range, becoming a frontier of the materials synthetic biology. This review first introduces the difference between cell-free expression system and conventional cell expression system, and emphasizes the unique advantages of cell-free expression system. Next, this review overviews the application of cell-free strategies in sustainable production, functionalization and innovative design of biomaterials, as well as promotion of new types by accelerating the Design-Build-Test (DBT) cycle. All of these examples fully demonstrate the advantages of cell-free systems in the design and synthesis of novel biomaterials. Although the use of cell-free expression systems for biomaterial synthesis encounters challenges such as cost, weak post-translational modification, and urgent need for cross-field cooperation, we still believe that in the near future, with the development of synthetic biology and the deepening of interdisciplinary research, cell free strategy will provide a faster, cheaper and more friendly way to produce new biomaterials, thereby protecting the earth environment on which we depend. In addition, the continuous development of smart materials that are combined with cell-free strategies, such as virus detection biosensors, will protect the health of human beings around the world to a great extent, and even save thousands of lives. All in all, the cell-free expression system will definitely provide a powerful boost to the research and industrialization of new biomaterials, and make a non-negligible contribution to the joint solution of global environmental problems and the protection of human health.





Cultured meat from biomaterials: challenges and prospects

Can ZHANG, Liyang SHI, Jianwu DAI

2022, 3(4):  676-689.  doi:10.12211/2096-8280.2022-020

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Cultured meat technology aims to manufacture meat by exvivo culture of animal cells, which has emerged as a promising alternative for livestock meat production. Raising animals for meat production have led to some negative effects like global public health, environmental pollution, energy consumption, and animal welfare concerns. Compared to conventional animal agriculture, cultured meat is more sustainable, environmentally friendly, nutrition precision, and it can lessen the animal welfare concerns in the future. Although cultured meat is considered to supple or even replace conventional meat, it is still in its early stages and faces many challenges which need to be resolved before being on the market. Muscle is a well-organized but also intrinsically complex tissue. Benefiting from the extensive research on tissue engineering, it has already been successfully used for generation of bio-artificial muscles to repair muscle injury, but it has never been used for generation of meat. To fully mimic conventional meat's texture, flavor and nutritional properties, applications of tissue engineering technology and biomaterials are required. This review details the benefits and challenges of cultured meat with regard to isolation and purification of stem cells, long-term and large-scale culture of stem cells in an undifferentiated state, and efficient myogenic differentiation of stem cells. Given the vital important role that biomaterials play in fabricating muscle fibers, this review provides in-depth analysis of highlighting promising scaffold materials that can be applied to develop cultivated meat. Especially combining some precursors into edible synthetic scaffolds could create functional scaffolds that promote stem cell multilineage differentiation and thereby improve the flavor of the final meat product. Concluding remarks and future perspectives are also included for future research into scaffolds capable of supporting the growth of high-quality meat while minimizing production costs. In general, with increasing demand of meat and further development of technologies, cultured meat may ultimately act as a more healthy and sustainable choice.





Genetically encoded click chemistry, an enabling tool for materials synthetic biology

Qikun YI, Chenbo SUN, Zhongguang YANG, Ri WANG, Songzi KOU, Zhaoxia LI, Fei SUN

2022, 3(4):  690-708.  doi:10.12211/2096-8280.2021-076

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Precise control over the structure and function of a material constitutes a fundamental challenge facing materials science. Due to the diverse specific interactions among biomolecules, direct assembly of biomolecules or even living cells into higher-order structures may provide a solution to this. In recent years, genetically encoded click chemistries (GECCs)-a collection of new protein chemistries that are inspired by the spontaneous isopeptide bond formation within naturally occurring microbial proteins-have gained traction among materials scientists. They are able to covalently assemble functional protein molecules directly into advanced architectures, with efficiency and selectivity rivaling traditional click reactions, while conferring genetic programmability on the resulting materials. These tools have facilitated the integration of protein engineering, materials science, and synthetic biology, and thus have opened enormous opportunities for the cross-disciplinary research. Here, we provide a concise review and discussion over the recent developments and applications of these protein chemistries, and the trends thereof. We compare the GECCs of different generations in reactivities, examine the synthesis of various uncommon protein molecule enabled by protein topology engineering, glance over the entirely protein-based hydrogels with dynamically tunable properties or underwater adhesiveness assembled via GECCs, glimpse into the design of subunit vaccines, especially those against COVID-19, and further explore the modulation of genetically engineered living materials by GECCs, featuring self-production, micropatterning, and self-assembly. Despite the tangible potential held by these molecular tools, unceasing protein engineering efforts remain necessary to further optimize and expand the arsenal of GECCs. As their chemical reactivities are fully encoded within the information (i.e., the amino acid sequence), GECCs may also serve as a source of inspiration to develop new chemistries for other synthetic information polymers in a broader term. Together, this emerging GECC toolbox has greatly empowered materials synthetic biology and will continue to provide solutions and inspirations for new biotechnologies.





Biosynthesis of antimicrobial peptides and its medical application

Daixu WEI, Hailun GONG, Xuwei ZHANG

2022, 3(4):  709-727.  doi:10.12211/2096-8280.2022-001

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Due to their broad-spectrum antibacterial activity and low incidence of drug resistance, natural antimicrobial peptides have become a potential alternative to antibiotics. In addition to being able to control pathogenic bacteria and fungi, antimicrobial peptides also have many other biological effects, such as anticancer, antiviral, antiparasitic and immunomodulatory activity, exhibiting broad biomedical application prospects. This review introduces the distribution and mechanisms of antimicrobial peptides, and summarizes the biosynthesis methods of antimicrobial peptides. We further compare and analyze the advantages and disadvantages of various antimicrobial peptide biosynthesis approaches relying on microbial expression systems and introduce new interdisciplinary peptide-design strategies based on synthetic biology. In addition, we also briefly summarize the applications of antimicrobial peptides. The application prospects of antimicrobial peptides can be classified into seven medical fields, including antiinflammatory drugs, antiviral drugs, antiparasitic drugs, anticancer drugs, medical tissue engineering, drug delivery systems, skin care and cosmetology. Furthermore, we also identify potential problems such as low expression yield, difficulty in extraction, high process cost, poor stability and insufficient biosafety of existing antimicrobial peptides. To solve these issues, computational prediction and directed gene editing technology can be used to create new antimicrobial peptides with improved antibacterial properties and reduced toxicity. It is also important to improve the industrial infrastructure of antibacterial peptide biosynthesis and develop strategies for rapid recovery of high-purity antibacterial peptides. Antimicrobial peptides can also be combined with existing antibiotics to prevent bacterial resistance to traditional antibiotics. Finally, antimicrobial peptides can be combined with new biomaterials to reduce their toxicity to tissues and organs in vivo.





Biosynthesis of elastin-like polypeptides and their applications in drug delivery

Zhaoying YANG, Fan ZHANG, Jianwen GUO, Weiping GAO

2022, 3(4):  728-747.  doi:10.12211/2096-8280.2021-094

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Elastin-like polypeptides (ELPs) are artificially synthetic peptide polymers inspired by human elastin. ELPs are composed of repeat units of a Val-Pro-Gly-X-Gly, where X can be any amino acid except proline, and they can exhibit different biological functions along with X residue changes. ELPs are thermally responsive and demonstrate lower critical solution temperature phase behavior. They are soluble at temperatures below a characteristic transition temperature (T_t) and reversibly phase separate into an insoluble, coacervate phase above the T_t. Moreover, the phase behavior is retained when the ELP is either genetically fused to peptides or covalently conjugated to small molecules, and this phase behavior can be adjusted through changing X residue and chain length of ELPs. As ELPs are typically produced from synthetic genes, the structure and function of ELPs can be accurately regulated through genetic engineering. The amino acids or peptides with reactive side chains can be incorporated into ELPs through recombination synthesis as well. This precision control over ELP is unmatched by synthetic polymers. Based on these properties, ELPs can be engineered to assemble into unique architecture and used as soluble macromolecular carriers, therapeutic drug depots, hyperthermia-targeted drug carriers and self-assembled micelles. Lastly, as ELPs are derived from natural protein sequences, they show desirable biological properties including excellent biocompatibility, low immunogenicity, and non-toxic effects. Due to these attributes, ELPs have been widely used in biomedical fields including protein expression and purification, in vitro diagnosis, drug delivery and tissue engineering. By focusing primarily on applications of ELPs in drug delivery, this review introduces the design principles, physicochemical properties, biosynthetic methods of ELPs and ELP conjugates, as well as exemplifies representative applications of ELPs in drug delivery, as extending the half-life of drugs, tumor targeted delivery, local delivery, hyperthermia-targeted delivery and sustained released of the drugs in vivo. Challenges and problems faced in this emerging field are discussed at the end of this review.





Low-carbon biomanufacturing of polyhydroxyalkanoates: analysis and application based on carbon conversion rate

Qian WANG, Qingsheng QI

2022, 3(4):  748-762.  doi:10.12211/2096-8280.2021-088

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With the increasing environmental pollution and the policy introduction of global ban on free plastic bags, the bio-manufacturing of bio-based degradable plastics represented by polyhydroxyalkanoates (PHAs) is one of promising ways to respond this challenge. PHAs are polyesters of hydroxyalkanoates synthesized by many bacteria and haloarchaea as carbon and energy storage materials. There are more than 150 types of polyhydroxyalkanoate monomers reported, resulting in a variety of PHAs with diverse properties. However, how to ensure low-cost, green, low-carbon and sustainable production of PHAs is still facing huge challenges. By using synthetic biology approaches, it is an important solution to construct microbial cell factories that synthesize PHAs and efficiently convert cheap carbon sources and renewable raw materials into various types of PHAs with excellent mechanical properties. This paper summarizes the biosynthetic pathways of various PHA monomers and analyzes the theoretical carbon conversion rate of various monomer synthesis pathways. It is proposed that the synthesis of PHA monomers with high carbon conversion rate is preferred to increase the yield of copolymer monomers. At the same time, it summarizes the current research progress in the creation of low-carbon biosynthesis pathways and the use of one-carbon compounds as carbon sources to synthesize PHAs. These are all effective ways to achieve low-carbon biosynthesis of degradable plastics. Finally, we summarized and prospected the development trend of synthetic biology in the field of low-carbon biomanufacturing of PHAs. In the future, with the integration and development of synthetic biology and new technologies, more low-cost and high-value-added PHAs can be obtained through green bio-manufacturing, thereby promoting the industrialization of bio-based plastics and better serving the global green and low-carbon society.





Research progress of modern biotechnology-promoted green degradation of polyethylene terephthalate in plastics

Lei LI, Xin GAO, Hongbin QI, Chao LI, Fuping LU, Shuhong MAO, Huimin QIN

2022, 3(4):  763-780.  doi:10.12211/2096-8280.2021-089

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Polyethylene terephthalate (PET) has been widely used in the food packaging and clothing industries due to its good durability, high plasticity, and safety. However, PET is difficult to be degraded by microorganisms or enzymes due to its high hydrophobicity and high crystallinity, which has led to severe environmental and social problems globally. Some of the high-quality PET can be reused in food packaging, but the majority of waste PET is downgraded through conventional mechanical recycling ways and cannot be recycled in a green and efficient manner. The proportion of recycled plastics that relies on recycling and green degradation is decreasing year by year, and only a small percentage of plastic can be recycled, accounting for 17.6% of annual plastic use. Thus, exploring a safe and efficient biodegradation for plastic to solve "white pollution" has become a major research issue that needs to be pursued urgently. Herein, this paper reviews the current status of research on PET biodegradation, focusing on the mining methods of microbes and genes encoding novel enzyme based on macrogenomics and proteomics. The characteristics and mechanism of plastic-degrading enzymes from different sources were elucidated through an analysis of the crystal structures. Importantly, the activity and degradation efficiency of the plastic-degrading enzymes were improved by directed evolution and smart-computing strategy. This provides insights into the structural modification of plastic-degrading enzymes and introduces some frontier research fields. The idea of "two-direction modification" is proposed to improve the degradability for PET raw materials using modifying degradation enzymes. Furthermore, new enzymes mining and modification for plastic degradation, the development of multi-enzyme catalytic system and the improvement of sustainable performance of plastic will significantly stimulate the new ideas for exploring efficient biodegradation of PET in the future.





Effects of mechanical signals on stem cell fate determination

Chengzhi SONG, Yang SUN, Yi CAO

2022, 3(4):  781-794.  doi:10.12211/2096-8280.2021-066

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Because of their outstanding self-renewing potential and pluripotency, stem cells are believed to have a broad range of applications in various fields such as tissue engineering, drug discovery, gene therapy, and cell therapy. While traditional studies in the field of stem cell fate determination mainly focused on bio-chemical factors, mechanical signals have come into sight of the science community as a crucial role in this area as well. A clearer understanding of the effect of mechanical forces and the mechanotransduction pathways in stem cells are surely helpful for their biomedical and clinic applications. Using synthetic hydrogels with well-defined rigidities as the substrates, the mechano-sensing mechanism and the lineage specification of stem cells have been extensively studied. Recent studies indicate that beyond the effect of substrate rigidity, other mechanical/physical properties, such as stress relaxation, degradation, and porosities, are also critical to stem cell self-renewal and differentiation. However, it remains challenging to control these mechanical parameters of synthetic hydrogels precisely and independently. The advance in synthetic biology may provide novel synthetic protein hydrogels with controllable mechanical responses for the study of force sensing mechanisms and lineage specification of stem cells. In this review, we focus on the impact of mechanical signals on stem cell differentiation and the underlying molecular mechanism. The mechanical signals could be passed down to the nucleus either through a direct mechanical connection of ECM (extracellular matrix)-integrin-FA (focal adhesion)-cytoskeleton or through some signaling molecules. Meanwhile, we introduce some commonly used synthetic hydrogels systems that have been widely used as model systems for stem cell studies. We also highlight different mechanical responses of stem cells cultured in 2D and 3D. It is believed that precise characterization of the cellular behaviors and the mechanical signaling pathways are crucial and can be realized by constructing more specialized hydrogels using advanced synthetic biology tools.





Research progress on the construction of three-dimensional porous structure of bone tissue repair scaffolds based on silk fibroin materials

Yunfei SHAO, Hui WANG, Yiran ZHU, Shuchun WANG, Yulin JIANG, Jianchen HU, Jing WANG, Keqin ZHANG

2022, 3(4):  795-809.  doi:10.12211/2096-8280.2022-026

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Bone defects caused by trauma, tumors or congenital diseases seriously affect the physical and mental health of human beings. Autologous bone and allogeneic bone transplantation are the gold standard in clinical treatment, but they have certain limitations due to their limited sources, risks of immune response and infection. In recent years, with the improvement of medical technology and cognitive level, the treatment concept of bone tissue defect has gradually changed from tissue transplantation to tissue regeneration mode. Three-dimensional (3D) porous scaffolds play a key role in bone tissue engineering research, serving as biological carriers for seed cells to survive before forming tissues, providing a space for tissue regeneration. The ideal bone tissue scaffold should have good biocompatibility, a porous structure which is conducive to the growth and differentiation of bone cells, suitable mechanical properties, and matching degradation properties. Successful 3D scaffold material design requires understanding the composition and structure of natural bone tissue, selecting appropriate biomaterials, and controllably constructing 3D porous structures at multi-scales through certain fabrication techniques. Natural bone tissue is a composite material with a hierarchical porous structure, which is composed of dense cortical bone in the outer layer and porous cancellous bone in the inner layer. From the perspective of bionics, the regulation of porous scaffolds at multiple scales is an important link in mimicking the hierarchical structure of bone tissue. Silk fibroin (SF), as a natural protein material with good biocompatibility and biodegradability, excellent mechanical properties and easy processing, has become an excellent candidate for the construction of 3D porous scaffolds. This paper summarizes and discusses the research progress of the construction of 3D porous scaffolds based on SF biomaterials. First of all, the characteristics of the multi-layered porous structure of natural bone tissue are summarized; secondly, the composition and structural characteristics of SF materials, as well as their excellent biocompatibility, mechanical properties and biodegradability are described; subsequently, the typical fabrication techniques of SF-based 3D porous scaffolds for bone tissue repair are introduced, including freeze-drying, particle leaching, 3D bioprinting, and composite manufacturing technology, and their ability to control the porous structure of 3D scaffolds, the effects of porous structure on cell growth behavior and bone tissue regeneration are also discussed; finally, the design and fabrication challenges and development prospects of SF-constructed bone tissue repair scaffolds are prosposed, emphasizing that synthetic biology could provide a powerful tool for solving the problems existing in the application of SF-based porous scaffolds in the field of bone tissue engineering.



Research Article



Coated probiotic-based drug carriers for oral delivery of tumor antigens

Sisi LIN, Chao PAN, Yifan ZHANG, Jinyao LIU

2022, 3(4):  810-820.  doi:10.12211/2096-8280.2022-010

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Cancer vaccines, as a form of cancer immunotherapy, trigger host anti-tumor immunity by delivering tumor antigens. Oral administration has the advantages of convenience, simplicity and high efficacy. However, oral tumor vaccines have been rarely reported and existing forms are solely suitable for gastrointestinal cancer. The situation is associated with lack of advanced oral delivery systems. In this study, yeast cell membrane-coated probiotics are developed as drug carriers for oral delivery of tumor antigens. Yeast cell membranes play a dual role in protecting probiotics and antigens and facilitating targeted delivery of tumor antigens to the gut lymphatic system, while probiotics serve as both immune adjuvant and antigen carrier. As a model antigen, ovalbumin (OVA) is loaded onto probiotic surface by complexing with polyethyleneimine and hyaluronic acid via electrostatic interaction. Subsequently, antigen-loaded probiotics are individually camouflaged with yeast membranes by physical extrusion through a porous membrane. As reflected by similar growth to uncoated probiotics, both the coating and preparation procedures have limited influences on bacterial viability. Experimental results show that, compared to unmodified bacteria, yeast cell membrane coated probiotics retain their ability to grow and divide after incubation in simulated gastrointestinal environments and exhibit an improved bioavailability following oral ingestion. The presence of embedded β-glucan on yeast membranes promotes the phagocytosis by microfold cells that locate in intestinal epithelium. After oral gavage, the coating facilitates the accumulation of wrapped probiotics in Peyer's patches and then in mesenteric lymph nodes. Furthermore, the maturation of dendritic cells and corresponding antigen presentation are enhanced by coated probiotics, which induce an OVA-specific immune response as manifested by an upgraded serum level of OVA-IgG in mice after oral administration. These results demonstrate that yeast cell membrane-coated probiotics show enhanced tolerance against insults in the gastrointestinal environment and improved delivery of antigens into the gut lymphatic system, hence activating anti-tumor immunity.