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.

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.

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.

As natural products, non-ribosomal peptides (NRPs) exhibit biological activities with a broad spectrum, including anticancer, antibiotic and immunosuppression. Among U.S. Food and Drug Administration (FDA) approved drugs, fungal NRPs are a major category of pioneering pharmacological agents like immunosuppressive cyclosporine, antibacterial cephalosporin and antifungal echinocandins. Under the catalysis of complicated multimodular enzyme complexes known as non-ribosomal peptide synthetases (NRPSs), NRPs are synthesized with three core domains: adenylation (A), thiolation domain/peptidyl carrier protein (T/PCP) and condensation (C), which collectively form repetitive modules responsible for activating and incorporating specific amino acids or hydroxycarboxylic acid building blocks into the growing peptide chains. Beyond the core domains, optional domains are exemplified by epimerization (E), heterocyclization (Cy) and oxidation (Ox), facilitating the customization of the building blocks. These domains and the variability in the number of modules with NRPs significantly contribute to the structural diversity of the skeletons. Furthermore, post-modifications to the structural skeletons yield potent pharmacological groups for NRPs, contributing significantly to their structural diversity and biological activities, which not only provide opportunities for discovering naturally sourced and active NRPs, but also opens avenues for modifications to create non-natural NRPs via synthetic biological technology. To date, numerous strategies have been employed for developing NRPs, including heterologous expression, transcriptional factor activation, precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis and enzyme engineering. This review summarizes the progress in research on fungal NRPs, encompassing their bioactivities, biosynthetic pathways, enzymatic reaction mechanisms and metabolic engineering. A comprehensive understanding of fungal NRPs biosynthesis not only benefits for deciphering the corresponding enzymatic assembly mechanism, but also serves as a guidance for advancing novel fungal NRPs and their derivatives, thereby paving the way for developing potential drug candidates from NRPs.

Microorganisms have consistently been a crucial source for researchers to explore and develop new natural products. Currently, research methods involving gene editing tools for the discovery, biosynthesis, and metabolic engineering of natural products have garnered broad attention in this field. However, traditional methods for gene editing usually rely on the recombination ability of the host or introduced proteins. It’s difficult to establish a general platform for all bacteria mainly because of their complicated genetic background. This genetic diversity often causes laborious experimental operations with low efficiency. The CRISPR/Cas9 gene editing system, with its unique and flexible targeting advantages, overcomes common limitations such as sequence homology or site constraint in other gene editing methods and thus is more likely to function in diverse bacteria species. This simplifies experimental procedures, enhances work efficiency, and promotes the development of natural product research. This article introduces the applications of the CRISPR/Cas9 system for the discovery, biosynthesis, and metabolic engineering of natural products in microorganisms. It covers the development of the CRISPR/Cas9 system, cloning and genetic editing of natural product biosynthetic gene clusters, structural derivatization and metabolic engineering of natural products, and the activation of silenced natural product biosynthetic gene clusters. These aspects highlight the advantages of the CRISPR/Cas9 system in the research of natural products with microorganisms. Finally, solutions are proposed for addressing challenges that the CRISPR/Cas9 system currently faces in overcoming low recombination efficiency and host adaptability issues. Especially the CRISPR/Cas12a system which has broadened applications of the CRISRP/Cas9 system by preferring different PAM sites. In addition to functions that CRISPR/Cas9 system has realized, its potent multiple targeting ability further enhances the efficiency of target editing. It is believed that with the development of synthetic biology and information technology, an increasing number of genetic manipulation tools and methods related to the CRISPR/Cas9 system will be developed, continually driving progress in the research of natural products.

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.

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.

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.