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.

Natural products with the bisintercalator family are a group of C2-symmetric cyclic non-ribosomal peptides produced by actinobacteria, possessing potent antimicrobe, antitumor and other bioactivities. Bisintercalators can be divided into two groups based on the size of their macrocycles: the minor and major scaffold types with eight and ten amino acid residues, respectively. Structure diversity with bisintercalators arises from variations in aromatic heterocycles, amino acid residue identities and quantities, and post-assembly line modifications. The major scaffold type bisintercalators harbor two structurally rigid six-membered nitrogen heterocycle-containing amino acids, which can further undergo oxidative and acylation tailorings. The minor scaffold type bisintercalators seemingly derive their rigidity from disulfide or thioacetal bridges formed by sulfydryls of two cysteines, and the thioacetal bridges allow variable S-alkyl elongation and conversion of S-alkyl sulfur into sulfoxide moiety. In addition, bisintercalators also exhibit differences in other amino acid identities, which further contribute to their diverse activities, including antimicrobial, antitumor, antifungal, anti-malarial, or antiviral effects. The chemical synthesis of these nonribosomal peptides is complex due to their intricate architectures, making microbial fermentation a more efficient production method. On the other hand, structural optimization can be achieved for bisintercalators through combinatorial and precursor-guided biosynthesis. Therefore, understanding the biosynthetic pathways of bisintercalators is crucial for yield enhancement via the pathway-specific regulation and also offering biocatalytic parts for structural modifications. This knowledge will facilitate future discovery and drug development for this promising natural product family.

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.

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.

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.

Filamentous fungi, which present distinct morphology and cell structure, play a critical role in human health as well as industrial and agricultural production. However, the unique characteristics of filamentous fungi make them difficult to be manipulated with traditional genetic engineering methods. Thus, the development of an efficient gene editing system is essential for exploring biological resources and understanding metabolic processes in filamentous fungi. The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) system promotes more efficient and effective gene editing in different species, and brings a revolutionary breakthrough in fungal fundamental research and applications. In this review, we first briefly introduce the history, working mechanism, and classifications of the CRISPR/Cas mediated gene editing system. Next, we comment the functional components of CRISPR/Cas9 such as selective marker, Cas9 and gRNA and the delivery methods of these components in various filamentous fungi. Furthermore, we systematically discuss the applications of CRISPR related technologies, including CRISPR/Cas12, base-editor, CRISPRa, CRISPRi and CRISPR mediated epigenetic regulation, in the genetic engineering of filamentous fungi, particularly in marine-derived filamentous fungi. Finally, we address challenges with relative low gene editing efficiency and off-targets effects in engineering filamentous fungi, and highlight the potential solutions for developing novel CRISPR/Cas-based gene editing systems. This review can provide guidance for developing an efficient gene editing platform in filamentous fungi and pave the way for further exploration of the secondary metabolites and establishment of robust fungal cell factories.

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.

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.

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.

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.