The precise design and synthesis of carbohydrates with important biological functions and more complex structures is a frontier in synthetic biology. Recently, a novel strategy named Protein Glycan Coupling Technology (PGCT) based on bacterial oligosaccharyltransferases has been developed and widely used in the biosynthesis of bacterial glycoconjugate vaccines, which are one of achievements in modern medicine due to their effectiveness in fighting against infectious diseases. Herein, progress in developing key components for manufacturing glycoconjugate vaccines, such as oligosaccharyltransferases (PglL, PglS, PglB, and TfmP), carrier proteins (CRM197, diphtheria toxoid, recombinant Pseudomonas aeruginosa exotoxin A, and nanoparticles), polysaccharide biosynthesis gene circuits, and glyco-engineered strains is reviewed. Meanwhile, producing glycoconjugate vaccines through fermentation presents advantages in good product quality control for safety and efficacy, low production cost, and environmental-friendly manufacturing. PGCT has potentials to overcome some limitations of chemical conjugation production processes, such as complex purification and high cost, for competitiveness with existing chemical conjugates. As an emerging technology, more technological innovations are needed for PGCT. In the future, the directed evolution of oligosaccharyltransferases, the application of protein nanoparticle carriers, the combination rearrangement of glycosyltransferases, and the optimization of engineered bacterial strains with better metabolic pathways are expected to further promote the biosynthesis of conjugate vaccines. The next few years will be an important and exciting time for PGCT, as recent technological advances are being applied to the development of novel glycoconjugates, and ongoing large-scale clinic trials on the efficacy of glycoconjugate vaccines will also demonstrate the feasibility of this technology, making the future of PGCT vaccinology promising.

Glycoproteins with enveloped viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and human immunodeficiency virus (HIV), display a trimeric conformation. Different from the monomeric form, the trimeric proteins exhibit superior immunogenicity. Several trimerization motifs, such as Foldon derived from phage T4 fibritin, have been used to promote the formation of trimeric proteins with natural conformations. Although the Foldon-induced trimeric proteins are stable, their high immunogenicity limits applications in the development of vaccine antigens. In a previous study, we developed a recombinant human collagen type Ⅲ protein and determined its crystal structure, revealing a triple-helix conformation. However, the potential of this recombinant protein as a trimerization motif remained unknown. In this study, we demonstrated that the recombinant humanized type Ⅲ collagen (Rh3C) was able to act as a trimerization motif, facilitating the spontaneous trimer formation of the Rh3C-conjugated receptor-binding domain (RBD) within the spike (S) protein of SARS-CoV-2. This trimeric protein could induce a stronger SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a immune response, when compared with the monomeric RBD protein in the immunized mice. Notably, the Rh3C-RBD protein, when adjuvanted with the novel STING agonist CF501, also elicited significantly higher neutralizing antibody responses against both the pseudotyped SARS-CoV-2 (D614G) and its variant Omicron (BA.2.2) in the immunized mice. To showcase the broad applications of the Rh3C trimerization motif, we further demonstrated that the Rh3C-conjugated HA1 of the influenza virus could also elicit a stronger antibody response than free HA1. Considering the wide distribution of the Rh3C protein in human bodies, its use as a trimerization motif would not induce an immune response due to immune tolerance, thereby allowing the immune response to concentrate on targeted viral proteins. Therefore, this Rh3C-based trimerization motif holds great potential for the design and optimization of vaccines consisting of trimeric protein antigens.

The central dogma of biology, which delineates the flow of genetic information from DNA to RNA to protein, along with the principles of cellular immunology, provides a foundational understanding for harnessing the power of synthetic biology to combat cancer. The application of synthetic biology in the design and production of novel tumor vaccines marks a pivotal advance in the field of cancer immunotherapy. This study delves into the cutting-edge development in the creation of therapeutic tumor vaccines, with a particular focus on two critical components: antigen selection and vaccine design. The request for more precise and effective tumor vaccines has garnered the attention of researchers globally. These vaccines are designed to target tumor-specific antigens or those related to tumor growth and survival pathways. Traditional approaches to antigen selection have typically involved targeting specific genes with tumors. However, the advent of high-throughput sequencing and mass spectrometry has revolutionized this process by enabling the screening of novel antigens, thereby enhancing the precision and immunogenicity of vaccines. In recent years, the landscape of tumor vaccines has been significantly broadened by the engineering of vaccines through various platforms. These include DNA-based vaccines, mRNA vaccines, viral or bacterial vector vaccines, and cell-based vaccines. These innovative approaches offer a stark contrast to traditional peptide vaccines, significantly amplifying the immune response against a variety of tumor types. The versatility of synthetic biology allows for the customization of vaccines to target a wide array of tumor antigens, thereby potentiating a more robust and targeted immune reaction. The progress made in synthetic biology is not only refining existing vaccine strategies but also accelerating the pace of experimental research in tumor vaccines. This rapid advancement holds the promise of continually improving the clinical therapeutic effects of these vaccines. As researchers continue to unravel the complexities of tumor immunology and synthetic biology techniques become more efficient, the intersection of these fields is expected to yield a new generation of tumor vaccines that are not only more effective but also safer and more accessible to patients. In conclusion, the integration of biological knowledge and technological innovation in synthetic biology is transforming the development of tumor vaccines. The focus on optimizing antigen selection and vaccine design is driving the creation of more potent and tailored immunotherapies. It is anticipated that synthetic biology will play an even greater role in enhancing the efficacy of tumor vaccines, offering cancer patients with hope in the ongoing battle against this devastating disease.

Recent outbreaks of infectious diseases, such as the middle east respiratory syndrome, Zika infection, Ebola hemorrhagic fever, and Coronavirus disease (COVID-19) pose significant challenges on the rapid development of efficacious vaccines. Virus-vectored vaccines, as an important new vaccine, can be administrated noninvasively through aerosol inhalation or oral administration, which could stimulate humoral, cellular, and mucosal immune responses without the need for adjuvants, showing good immunogenicity and safety in clinical trials or in emergency use. With the deeper understanding of the viral genome and structural proteins, synthetic biology has enabled the design and modification of viruses to produce recombinant viral vector-based vaccines with high titer, safety, and immunogenicity, and such research has significant implications for the vaccine development. This review highlights major strategies employed in the construction of virus-vectored vaccines, including the construction method of replication-competent or replication-defective viral vectors, and the development of viral vectors commonly used in producing the recombinant vaccines. Among these viral vectors, replication-deficient adenovirus-based vectors with gene deletion in the E1 and E3 regions are most mature for use. Currently, adenoviral vectors that have been used in the approved recombinant vaccines include Ad5, Ad26 and ChAdOx1. Vesicular stomatitis virus and flavivirus with small genomes are negative-sense and positive-sense single-stranded RNA viruses, respectively, which are easy to prepare and more suitable for being used in developing recombinant vaccines with small antigen proteins. Poxviruses and herpesviruses have large genomes for high packing capacity, but they are most difficult to be modified with synthetic biology methods. Different viral vectors need to be prepared using different strategies, and consequently vaccines developed with these vectors have different immune effects. The construction strategies of different viral vector vaccines introduced in this review will provide valuable theoretical reference for the research and development of novel viral vector vaccines. In the future, virus-vectored vaccines will be iteratively developed for higher safety, stronger protection, better compliance and lower production cost.

Human diseases, especially infectious diseases and cancers, pose unprecedented challenges to public health and the global economy, making the development of preventive and therapeutic vaccines a top priority for addressing these challenges. Among all vaccines, vector vaccines that activate T cell immune responses have significant advantages. This article reviews the immunological principles of vector vaccines, strategies for designing T cell vector vaccines, and their research advances. T cells, upon infection, can differentiate into various effector T cell subsets that play a crucial role in clearing pathogens. Research on the functions and mechanisms of effector T cells is essential for designing vaccines that can elicit T cell-mediated immunity. Currently, the development of vaccines for many viruses such as HIV and HCMV as well as cancers focuses on T cell-based vaccines. Various vectors, including viral vectors, bacterial vectors, and nucleic acid vectors, exhibit excellent performance on antigen delivery capability, immunogenicity, and protective efficacy. In addition, this article summarizes strategies for designing T-cell vector vaccines, including identifying appropriate antigen presentation pathways and vector delivery routes, ensuring biological safety, selecting suitable vaccine vectors, and evaluating the advantages and disadvantages of various vector vaccines. Notably, mRNA vaccines have played a crucial role in addressing the challenges posed by the COVID-19 pandemic. Technological advancements in vector vaccines are expected to accelerate the development of novel vaccines and enhance preparedness for emerging public health events. This review provides insights for the design of vector vaccines that are both safe and efficient. With advancements in vector vaccine technology and the progress of various interdisciplinary approaches, the next generation of vaccine development will continue to drive the evolution of vaccinology.

The development of cancer vaccines is confronted with significant challenges. Synthetic biology emerges as a potent tool for addressing these challenges, due to its ability to modify and engineer microbes capable of adapting to and colonizing on tumor tissues to change the immunosuppressive tumor microenvironments, augment antigen presentations, and stimulate both innate and adaptive immune responses against tumors in situ. This review comments on several pivotal applications of synthetic biology in engineering bacterial and viral vectored cancer vaccines. We start with discussion on methods to mitigate the pathogenicity of bacterial or viral vectors, including the removal, deactivation, or modification of their virulent genes. Furthermore, we address strategies for enhancing their tropism and fitness within tumor tissues, such as the alteration of their cellular entry proteins or the implementation of environmentally controlled gene expression systems. Approaches to minimize their systemic toxicity are also described. To fully harness the potential of tumor microenvironment modifications induced by microbial replication, we underscore studies employing synthetic biology methods, which involve the introduction of foreign genes into the microbial genomes, thereby enabling the production of agents like cytokines, chemokines, or monoclonal antibodies to enhance the recruitment and activation of innate and adaptive cells, promote immunogenic cell death, and augment the presentation of tumor-associated antigens. We also delve into the applications of synthetic biology for the introduction of tumor antigens to the vectors, discussing various loading methods, locations, and releasing mechanisms to generate an optimized tumor-specific immune response. At the end, we highlight substantial challenges that arise in the development of microbial vectored cancer vaccines, including safety considerations, intricate interactions between anti-vector and anti-tumor immunity, and the inherent complexity of tumor biology, and propose strategies for addressing these obstacles. In conclusion, this review emphasizes the crucial role of synthetic biology in the engineering of microbes, which is instrumental in advancing the development of cancer vaccines.

Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, it has evolved into different lineages, including Alpha, Beta, Delta, and Omicron. The development of broad-spectrum vaccines has become a necessity for preventing the highly mutated respiratory viruses. Traditional vaccine antigens, originating from prototype strains, cannot cover rapid mutations with these viruses, leading to breakthrough infections. With the development of synthetic biology, new technologies such as multivalent coupling of antigens, reconstructed dominate antigen modules, engineering design of conserved epitopes, epitope display, and computation-guided reconstruction have enabled redesigning antigens to achieve stronger immunogenicity with broader spectrum. The technology of synthetic biology is also applicable in the vaccine production process, such as antigen expression in nanoparticles, viral vectors, nucleic acids, and subunits. This article reviews the applications of synthetic biology technology in developing broad-spectrum vaccines in recent years, particularly for the broad-spectrum SARS-CoV-2 vaccines, and summarizes how to display common antigens and cross-antigenic sites by the reverse vaccinology for the activation of broad-spectrum immune responses against different mutant strains, achieving broad-spectrum vaccine protection effects through “remaining constant in response to ever-changing”. The article also provides a comprehensive comparison of the strengths and limitations of different broad-spectrum vaccine design strategies and discusses challenges to applying synthetic biology in the development of vaccines, offering valuable insights for universal against highly mutation viruses.

With the research progress and clinical application of immune checkpoint inhibitors and chimeric antigen receptor T-cell therapies, immunotherapy has substantially changed the treating modalities for various tumors. Tumor neoantigen vaccines, as a promising immunotherapy method, aim to trigger a novel T cell response against neoantigens. Neoantigens, with their high specificity, can induce and expand the tumor-specific T cell receptor repertoire, which were discovered through the second-generation sequencing of DNA extracted from both the patient’s tumor and non-tumor tissue samples. The sequences and HLA types are then analyzed for alignment to pinpoint tumor-specific mutations. To validate the significance of these mutations, RNA sequencing data are integrated with the results. Subsequently, bioinformatics platforms are employed for the prediction and analysis of neoantigens encoded by mutated genes and HLA types, enabling the identification of potential immunogenic neoantigens. Finally, the immunogenicity of these neoantigens is assessed through techniques such as ELISPOT and tetramer assays. Tumor vaccines can be categorized as peptide-based, DNA-based, RNA-based, and DC-based products. Viruses, lipid nanoparticles, and nano delivery systems can activate antigen-presenting cells, enhancing their ability to recognize and present tumor-associated antigens, thus promoting the activation of CD8⁺ T cells. Neoantigen vaccines can be administered through various routes, including subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal injection, intravenous injection, or intralymphatic injection. Preliminary clinical studies have shown that neoantigen tumor vaccines have demonstrated evidence of strong tumor-specific immunogenicity and antitumor activity. In this review, we summarize in detail the source, prediction, and identification of tumor neoantigens, as well as the classification and immunization scheme of neoantigen vaccines. In addition, we highlight strategies for optimizing tumor neoantigen vaccines, including prediction algorithms, expressing multiple epitope structures, increasing immunogenicity, administration methods and delivery systems, and combining adjuvants and various treatments, providing new insights for the development of personalized immunotherapy.

In agroecosystems, microorganisms have rich, diverse, and complex ecological functions, so-called emergent properties, which refer to novel characteristics that emerge in complicated systems as their complexity increases. Interestingly, although emergent functions originate from the joint action of multiple species, the number of species required for triggering such a phenomenon is not so large, typically less than ten. This not only provides the possibility of using synthetic bacterial communities (SynComs) to explore the generation of emergent functions, but also makes it possible to use SynComs to modify the symbiotic microbiota of plant hosts. Seed microbiome, which consists of the earliest microbial residents of a plant, has much simpler community structures compared with either rhizosphere or phyllosphere microbiome. Considering the priority effect, however, it is believed that the seed microbiome plays an important role in the evolution and assembly of plant symbiotic microbial communities, which is currently overlooked. Under particular conditions, even if the members of the seed microbiome have become rare species or disappeared in the later stage, they may still affect the development of plant symbiotic microbiome with legacy effect. Notably, limited by the dry and oligotrophic microhabitat conditions, biofilms should be the main morphology for the microbes existing on the surface of and inside seeds. Applying functional synthetic microbial communities as the seed coatings or biofilms may be the most effective intervention strategy for plant microbiome manipulations, as it targets the most critical period of the early development of plant-associated microbiota. In the context of smart agriculture, the integration of seed chip technologies and drone intelligent platforms could facilitate the high-throughput field characterization and application of SynComs, enabling the discovery of functional SynComs with specific emergent properties that work with plant seeds. Therefore, the application of synthetic bacterial biofilms, or coatings, provides a feasible approach, and is expected to bring breakthrough for the development of microbe-crop breeding technology.

Astaxanthin is a value-added terpene with strong antioxidant activity as well as other physiological functions, such as anti-cancer, enhancing immunity, eye protection, and cardio-cerebrovascular protection. Natural astaxanthin mainly comes from algae and aquatic crustaceans such as lobster shell. Astaxanthin presents with stereoisomerism and geometric isomerism, which have different biological activities and applications. Currently, astaxanthin in the market is obtained primarily through natural extraction from Haematococcus pluvialis or Xanthophyllomyces dendrorhous and chemical synthesis as well. While H. pluvialis has a long growth cycle and high light demand, leading to low biomass productivity and extraction rate for high production cost of astaxanthin, X. dendrorhous has a low astaxanthin yield and is easy to degenerate, making them challenging for the large-scale commercial production. The chemical synthesis of astaxanthin involves multiple reactions with complicated processes, producing mixed isomers and various byproducts, which consequently compromises its antioxidant capacity. Moreover, the assimilation and utilization of chemically synthesized astaxanthin in vivo is poor compared to its natural product, making it not suitable for being used by human being. With the continuous development of synthetic biology, microbial fermentation has been developed as an effective way for the commercial production of astaxanthin to better meet consumer demand. At present, astaxanthin-producing microorganisms include bacteria, fungi, and algae. This review introduces astaxanthin's structure, properties, production methods, and processes for its extraction and purification, with an emphasis on natural and engineered biosynthetic pathways. The latest progress in the production of astaxanthin by different microorganisms such as H. pluvialis, Yarrowia lipolytica and Escherichia coli is summarized, along with strategies for increasing astaxanthin production through genetic engineering and fermentation process optimization. Future metabolic engineering strategies are proposed, such as over-expression of astaxanthin synthesis genes, promoters with higher substitution intensity, subcellular localization, metabolic pathway optimization, etc, to increase astaxanthin yield for wide usage in food, medical, cosmetic and feed industries.