Traditional Chinese medicine (TCM) is a treasure of Chinese civilization and also a good mine for drug development in China. Many TCM components come from rare biological species including plants, animals, and insects, making the preparation of these TCM pharmaceutical substances at large scales a bottleneck that substantially impedes TCM-based drug development. However, the rapid development of synthetic biology has provided a strategy for addressing this challenge. At present, significant progress has been made in the bio-production of individual TCM components, but the efficacy of TCM is mainly due to the synergistic effect of those ingredients, which are termed as pharmaceutical ingredient groups. Reports on constructing the bio-production platform of pharmaceutical ingredient groups are limited. Herein, we summarize research progress in the biogenic mechanism of important TCM pharmaceutical ingredient groups, such as volatile oils, saponins, flavonoids, lignans and alkaloids. Some individual components of pharmaceutical ingredient groups (e.g. ginsenosides) are synthesized by multiple branching pathways, which can be produced and formatted thereafter. On the other hand, some pharmaceutical ingredients such as sandalwood oil can be synthesized through single pathways/enzymatic reactions by engineering the key enzymes to optimize their ratio. We comment the strategy of combining enzyme engineering and metabolic engineering to optimize both the production of pharmaceutical ingredient groups and their ratio. At the end, we outline the prospect of synthetic biology research for producing pharmaceutical ingredient groups, including: (1) complete clarification of the biogenic mechanism of more complex pharmaceutical ingredient groups, (2) development of novel metabolic engineering approaches for breaking through homogenization of methodology, and (3) optimization of the catalytic characteristics of key synthetic enzymes by combining rational design and directed evolution.

Natural products play a crucial role as sources of therapeutic agents for human being and agricultural pesticides. With the development of sequencing technologies, genome mining employing various bioinformatic tools has become an important approach for discovering more natural products. Due to the large number of natural product biosynthetic gene clusters, screening those capable of generating the most potent bioactive molecules has gained significance. To avoid self-destruction, some bioactive molecule producers have evolved with self-resistance enzymes, which are slightly mutated versions of original enzymes, but not sensitive to the bioactive compounds. The presence of self-resistance enzymes in the biosynthetic gene cluster of natural products serves as an indicator for the biosynthesis of bioactive compounds. On the other hand, the biosynthetic gene clusters of natural products could be located using information with their structures and activities as probes, e.g. the accumulating knowledge on antibiotic resistance mechanisms has facilitated the discovery of new antibiotics. Moreover, dereplication of natural products with known resistance mechanisms has been achieved by using indicator strains expressing the resistance genes. While these approaches have successfully utilized self-resistance genes to connect molecules with their biological activities, a more impactful application is to accurately link biological activity with genomic information through target-guided mining of natural products. The concept is to use a self-resistance gene as a predictive tool to screen and identify biosynthetic gene clusters encoding compounds that inhibit specific targets. Recent breakthroughs in self-resistance gene identification have bridged the gap between activity-guided and genome-driven approaches for natural product discovery and functional assignment. This review summarizes progress in bioactive natural product discovery guided by self-resistance genes, as well as its applications, which include the following points: 1) locating biosynthetic gene clusters based on self-resistance genes, 2) predicting the targets of secondary metabolites through self-resistance genes, 3) rapid dereplication of bioactive compounds with self-resistance mechanisms, 4) genome mining of bioactive natural products guided by the target and the internal connection with self-resistance genes, and 5) the development of genome data mining tools directed by self-resistance genes.

The natural products polyketides include over 10 000 molecules with a wide range of bioactivities and are among the most prominent classes of approved clinical agents. Usually, active lead compounds require structural modifications to improve their assimilation, distribution, metabolism, and excretion as well as to facilitate the drug development process. However, due to the large number of stereocenters and inert carbon atoms, it is challenging for chemical synthesis to accurately and efficiently derive polyketide scaffolds, making their biological synthesis for structural optimization of the polyketides a hot topic. In nature, the majority of polyketides are assembled from simple the building blocks acetate and propionate catalyzed by polyketide synthases, but a few polyketides with special building blocks provide inspiration for researchers to introduce unnatural building blocks selectively into the scaffolds of polyketides for their structure modifications. Polyketides can be built with predictable biosynthetic logic, each module of a modular polyketide synthase elongates the product backbone with two carbons by synergetic actions of its three essential domains: ketosynthase, acyltransferase and acyl carrier protein. The acyltransferase domain selects for and loads a carboxyacyl-Coenzyme A extender unit for the phosphopantetheinyl modification of the acyl carrier protein domain, whereas the ketosynthase domain then uses the extender unit to elongate the growing polyketide intermediate, before passing it to the following module. Given the hierarchical domain and module organization of the type Ⅰ modular PKSs that make these molecules, gene sequences and product structures are directly connected such that changes can be introduced site-selectively into the molecule by targeting building blocks and promiscuous acyltransferase domain with the corresponding domain. Besides, the biosynthesis of polyketide scaffolds depends on the assembly of a starter unit and variable extender units, therefore, introducing anticipated structures into the polyketides through incorporating the artificial extender units is considered as a powerful breakthrough for precise and effective modifications of the polyketides. This review summarizes three important enzymatic synthesis methods for unnatural polyketides extender units reported within the past decade. As results, a large number of unnatural extender units have been obtained through mining novel extender unit synthetase and exploring their substrates, or using enzyme engineering methods to modify the substrate spectrum. Also, this review comments on the cases of modifying polyketide structures using unnatural extender units to achieve the desired derivatives either through the natural synthetic pathway of polyketides or by utilizing modified synthetic pathways. Finally, we discuss some challenges existing in this research field and potential solutions for better applications of polyketides, including the compatibility issue of polyketides synthase with unnatural extender units, precursor supply for unnatural extender units, and etc. In recent years, interest and enthusiasm for the modifications of polyketides using unnatural extender moieties have increased dramatically, and our review draws a concise and clear map for the research of polyketide structure modifications by artificial extender units, with an expectation of laying a solid foundation for accelerating the development of polyketides drugs.

Actinomycetes, enriched with secondary metabolites, have emerged as a resource for drug discovery. These organisms predominantly harbor bioactive compounds such as polyketides, non-ribosomal peptides, aminoglycosides, and terpenes, with polyketides representing the most diverse class. Polyketides are divided into three major categories based on polyketide synthase: type Ⅰ, type Ⅱ, and type Ⅲ, in which type Ⅰ polyketides are most widely distributed and abundant, with macrocyclic lactone compounds serving as their archetypal representatives. Macrocyclic lactone compounds, frequently utilized as antibiotics, anti-cancer agents, immunosuppressants, and antiparasitic agents, hold immense biological significance. This review comments the biosynthetic process of macrolides, and strategies for biosynthesizing actinomycete polyketides are proposed, which encompass genome remodeling, regulatory pathway recombination, combinatorial metabolic engineering, and the modifications of polyketide structures. By knocking out competing gene clusters and superfluous genomic islands, augmenting the supply of precursors, and enhancing precursor supply and lipid stream processing, researchers can obtain genome-minimized and optimized industrial chassis, followed with manipulations such as promoter engineering, regulatory factor engineering, overexpression of the rate-limiting enzyme genes, enhanced substrate transport and tolerance, targeted modifications of the key enzymes, rational design of polyketides, etc. Furthermore, the optimized chassis and biosynthetic gene clusters are integrated to develop robust strains for multi-omics analyses and fermentation process optimization, which can be guided by rapidly developed synthetic biology enabling technologies and artificial intelligence, to develop a high-quality, efficient polyketides biosynthesis system. These advancements can offer robust technical support for the large-scale production of polyketides pharmaceuticals and their derivatives.

The (4+2)- and (2+2)-cycloadditions are important chemical reactions for constructing ring structures, with broad applications in the chemical synthesis and biosynthesis of complex natural products and chiral drugs. The discovery and development of enzymatic cycloaddition reactions, including both (4+2)- and (2+2)-cycloadditions, are currently hot topics in the field of chemical biology. Recently, several international and domestic research groups have successively reported multiple enzymatic (4+2)- and (2+2)-cycloadditions, revealing related protein structures and enzymatic mechanisms, designing new artificial cyclases, and developed different types of regio- and selective cycloaddition reactions through protein engineering. These studies provide a theoretical basis and successful examples for the design and optimization of novel cyclases using synthetic biology strategy, and will promote applications of the enzymatic reactions in organic synthesis.

Microbial natural products (NPs) are a major source for mining small molecule drugs, which have been widely used in medicine, agriculture, and other fields. Growing antimicrobial resistance and other public health problems necessitate the rapid discovery of microbial NPs with novel structures and bioactivities. With rapid advances in high-throughput screening and low-cost DNA sequencing technologies, highly diverse biosynthetic gene clusters (BGCs) have been detected in bacteria and fungi, but characterized compounds are limited, representing the tip of an iceberg, and much more novel small molecules are awaiting for being discovered. Although various strategies have been developed for NP discovery, effectively linking the biosynthetic pathways to their encoded products remains a challenge. Recently, (meta)genomic library construction strategies have shown advantages in elucidating NP biosynthetic pathways more efficiently, and significantly accelerated the discovery of novel NPs by combining with high-efficient targeted BGC screening approaches. In this review, we summarize three strategies for discovering microbial NPs based on (meta)genomic library construction and targeted BGC screening. We also discuss the cloning vectors including Cosmid/Fosmid, BAC/PAC and FAC/YAC, and comment strategies for library construction and targeted BGC screening, such as LEXAS and CONKAT-Seq. Furthermore, we compare strengths, limitations, and applicability of different libraries. At the end, we prospect the future developments of these strategies for the high-throughput discovery of microbial NPs.

A Public Health Emergency of International Concern (PHEIC) is defined by the World Health Organization (WHO) as “an extraordinary event which is determined to constitute a public health risk to other states through the international spread of disease and potentially requires a coordinated international response”. To date, WHO has declared seven PHEIC events, including the H1N1 influenza, Ebola, poliomyelitis, Zika, COVID-19 and mpox. Vaccination remains as an effective method in preventing infectious diseases. The International Health Regulations (IHR) Emergency Committee's recommendations for preventing or reducing the international spread of disease and avoiding unnecessary interference with international traffic include an emphases on the development of diagnostics and therapeutics for diseases, as well as the vaccine development. The mRNA vaccine represents a platform technology for the development of next-generation vaccines, and possesses distinct advantages, such as a shortened development cycle, scalable and cost-effective production, as well as enhanced amplification capacity, highlighting its potential in rapid responding to emerging and re-emerging infectious diseases. In recent decades, the development of mRNA synthesis technology and nucleic acid delivery system has facilitated the rapid development of mRNA vaccines and their clinical applications. Here, we overview the development of mRNA vaccines in response to the past PHEICs, and discuss challenges and trends in this regard. Currently, COVID-19 mRNA vaccines have been authorized for human use, while multiple mRNA vaccines against influenza, Zika, mpox and Ebola have been evaluated in clinical or pre-clinical studies. Despite their proven efficacy, there is still room for further improvement of the mRNA vaccines. The mRNA design, optimization, delivery, formulation, manufacturing, storage, and transportation can be further improved by integrating synthetic biology, biochemistry, artificial intelligence, and other multidisciplinary technologies. Although the emergence of the next PHEIC cannot be predicted with certainty, we are optimistic that the mRNA vaccine technology will play a pivotal role in preventing pandemics in the future.

In recent years, bacterial infections have emerged as the second leading cause of death globally, posing a serious threat to public health and demanding prioritized intervention from the healthcare community worldwide. While antibiotics have conventionally been used as the primary strategy to combat bacterial infections, their efficacy is increasingly compromised due to the emergence of drug-resistant bacteria, especially multi-drug-resistant and even pan-drug-resistant superbacteria. Vaccines are thus considered as one of the most scientific, economical, safe, and effective means to prevent infectious diseases and improve public health, which are estimated to save 2 to 3 million lives annually, and can serve as a critical tool in the battle against antimicrobial resistance. However, the complexity of bacterial structure and pathogenic mechanism has hindered the development of vaccines. Challenges include screening and rationally design of effective antigens, ensuring compatibility of various antigen combinations, establishing animal models for preclinical evaluation, and defining reliable endpoints for clinical efficacy assessment. As a result, only a small number of bacteria vaccines have been successfully developed so far, and none of them has been licensed to combat the most prevalent drug-resistant infections, such as Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Synthetic biology is a brand-new multidisciplinary focusing on repurposing natural biological systems and inventing innovative biological tools, technologies, devices, and systems for practical applications, and its concepts, principles and technologies have been extensively employed to facilitate vaccine development, including rational design, screening, and optimization of antigen, carrier, adjuvant and delivery system as well as the modulation of bacterial pathogenicity and immune responses. Herein, we outline the current status of the development of bacterial vaccines and the advancement of clinical trials for drug-resistant bacterial vaccines. Then, we summarize the application of synthetic biology technology in the development of major bacterial vaccines. Finally, we prospect the potential of synthetic biology in creating novel bacterial vaccines. Researchers have access to a greater variety of design possibilities for bacterial vaccines through synthetic biology. To maximize these benefits, we should employ synthetic biology and related technologies more efficiently in developing bacterial vaccines. Meanwhile, we should develop a scientific, reasonable, effective, and feasible management system, as well as regulatory measures, to expedite the development of efficient bacterial vaccines, therefore addressing the problem of antibiotic resistance to protect human health.

Nanoparticle vaccines have been established firmly as a cornerstone of modern immunization strategies, with a compelling history that trace their pioneering use in human being back to 1981. Within the past four decades, these vaccines have not only demonstrated their efficacy, but have also been developed as powerful tools in fighting against a range of infectious diseases, most notably hepatitis B virus (HBV) and human papillomavirus (HPV). Their success can be attributed to their exceptional immunogenicity and impeccable safety as well, making them invaluable in curbing the spread of viruses and safeguarding the health and well-being of human being. The global outbreaks of the COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has made vaccination into the forefront of public health priorities. This unprecedent challenge has accelerated the progress of various vaccine technologies, with nanoparticle vaccines attracting considerable attention. However, due to their relatively empirical design approaches and complicated manufacturing processes, progress in the clinical trials of SARS-CoV-2 nanoparticle vaccines has not been highlighted particularly. Therefore, the imperative for developing nanoparticle vaccines is to figure out their rational design, requiring groundbreaking advancement in novel technologies and theories. In this endeavor, synthetic biotechnology has emerged as an indispensable tool, driving the technological innovations of the production of nanoparticle vaccines. This article begins with an overview of technological advancements in the development of nanoparticle vaccines, encompassing progress from self-assembled nanoparticles to assist-assembled nanoparticles, and ultimately to antigen-display on formed nanoparticles. Furthermore, discoveries in understanding the unique roles of nanoparticle vaccines in enhancing antigen immunogenicity are updated, particularly in the function of nanoparticles with novel antigen presentation pathways. Finally, a comprehensive summary of the clinical trials of nanoparticle vaccines on fighting the COVID-19 pandemic is presented. In conclusion, we firmly believe that nanoparticle vaccines, bolstered by the scaffolding of synthetic biotechnology, are poised to emerge as steadfast guardians in the global battle against emerging and highly infectious diseases, and ongoing progress in this regard not only holds great promise, but also has potentials to revolutionize contagious disease prevention and control on a global scale.

Influenza viruses are highly variable and transmissible, and their infections can cause infectious respiratory diseases, such as seasonal influenza outbreaks around the world, one of the most serious public health problems at present, which can be prevented by influenza vaccination. The genome sequences, protein structures and functions of influenza viruses, as well as their packaging mechanisms are relatively clear. they are also important models, which can be used for developing conditional control genetic elements and the construction of intelligent responsive viruses. With the development of reverse genetics and synthetic biology technology, influenza viruses that are genetically engineered can better control virus replication to improve the safety of vaccines, and induce strong immune responses in human being, which have attracted wide attention in tumor immunotherapy. Several studies using simple or modified influenza viruses for treating liver cancer, melanoma, or lung cancer have found breakthroughs. In this paper, three novel strategies for attenuating influenza viruses, namely, proteolytic targeted chimeric virus, conditionally replicating influenza-attenuated live virus and highly interferon-sensitive virus, are described. The oncolytic effects of influenza viruses encoding premature stop codon chimeric antigen peptide, influenza viruses recombining with PD-L1 or CTLA4 immune checkpoint and influenza viruses expressing GM-CSF with truncated NS1 fragment on melanoma and hepatocellular carcinoma are reviewed, respectively, which suggest that the influenza viruses can be used as a live attenuated vaccine and a potential carrier for oncolytic viruses, and future researchers can be focused on constructing influenza viruses with more innovative strategies and different viruses to build a live attenuated vaccine and oncolytic viruses, in order to obtain high safety and more clinical curative treatment, improving the life quality of the patients.