Skeletal muscle, one of the most abundant tissues in the human body, plays a crucial role in motor function, energy metabolism, immune regulation, and the aging process. The skeletal muscle tissue microenvironment is highly complex, involving a variety of cell types, a three-dimensional architecture, and specific mechanical properties. Replicating these intricate features in vitro to create a biomimetic skeletal muscle model has long posed significant challenges. The advent of organ-on-a-chip technology, which integrates microfluidics with 3D cell culture, offers a groundbreaking approach to faithfully replicate the key structural and functional characteristics of human skeletal muscle tissue. The organ-on-a-chip technology enables precise control over the microenvironment, facilitating the study of skeletal muscle development, disease progression, and drug screening in a highly controlled in vitro setting. The skeletal muscle-on-a-chip (SMoC) has been utilized to investigate a variety of muscle-related diseases, including Duchenne muscular dystrophy and amyotrophic lateral sclerosis, offering valuable insights into disease mechanisms and potential therapeutic strategies. Additionally, SMoC serves as a powerful tool for testing the efficacy and toxicity of new drugs, as well as exploring tissue repair and regeneration techniques. Recent advances in the design and fabrication of SMoCs have further enhanced their physiological relevance, including the incorporation of anisotropic scaffolds to guide muscle fiber alignment and the use of electrical and mechanical stimulation to mimic the native muscle environment. These improvements have led to more accurate disease models and more reliable drug testing platforms, making SMoC a versatile and promising tool in biomedical research. In the end, the prospects and challenges facing the future development of SMoC were discussed. Currently, SMoC still exhibit limitations in terms of cell sources and functionalities. However, the integration with emerging technologies such as gene editing and biosensing in the future could pave the way for significant advancements and breakthroughs. The development of SMoC is expected to further promote the process of translational medicine, with potential applications extending beyond basic research into clinical settings, where it could revolutionize personalized medicine, regenerative therapy and precision drug development.

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 and their derivatives are main sources for lead compounds in drug discovery and development. Canonical natural product discovery relies largely on biological activity-guided or chromatographic identification-oriented screening strategies, which have achieved great success so far. However, the limitations of these methods, such as time consumption, labor intensity, and the noises of abundant natural products, have constrained productivities in discovering novel active natural products for drug development and combating the rising threat of drug resistance. Modern biotechnology, particularly the development of DNA sequencing and computational technology, has made it possible to study the biosynthesis of natural products, enabling us to connect genetic sequences with natural product structures for predicting the potentials of natural products produced by specific biological species at the genetic level. Therefore, genome mining-directed discovery for natural products has emerged. In addition to mining methods dependent on the conservation of genes encoding core enzymes for natural product biosynthesis, recently developed activity-oriented and intelligence-assisted genome mining strategies provide more opportunities for discovering naturally medicinal products. This article reviews the history of genome mining, highlighting advances in related databases, tools, and algorithms, with a focus on recent cases and applications of classic genome mining as well as self-resistance mechanism, evolutionary theory and artificial intelligence guided mining in the discovery of naturally active products. Since genomic information contains enormous chemical potentials, the discovery of natural products with high throughput and efficiency can accelerate the development of new drugs, new chemicals and new catalysts.

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.

Natural products have long been considered as an important source for potential drugs. In history, natural products and their structural analogs have contributed substantially to the treatment of various diseases, especially cancers and infectious diseases. After a long history of applications, people have gradually begun to explore active ingredients in natural products that truly exert therapeutic effects, and discovered a series of functional compounds, such as morphine, quinine, ephedrine, etc. Over the past two hundred years, the discovery and research of natural products has undergone tremendous changes, from traditional identification and isolation methods to multidisciplinary approaches in the modern genomic era. Strategies for discovering natural products and tools for their prediction have been developed continuously. Although many novel and active natural products have been mined and discovered in the past two decades, considering the huge reserve of natural products in nature, a large number of genes or gene clusters encoding key enzymes for the biosynthesis of natural products have not yet been characterized, and both terrestrial and marine natural product resources are to be explored. Compared with traditional chemically synthesized molecules, natural products possess diverse skeletons for structural complexity, which have shown remarkable advantages in the discovery of new drugs. While there are still many challenges in discovering new drugs from natural products, such as the effective mining of molecules with new structural features, identification and isolation of functional natural products with trace abundance, derivatization of natural product analogs for exploring connections between their structures and activities, and the complete synthesis of complicated active natural products at large scales, etc., the emergence of novel analytical technologies and mining strategies is expected to substantially renovate natural product discovery. This review comments on the natural product drugs and semisynthetic drugs derived from natural products approved by the U.S. Food and Drug Administration within the past decade from January 2014 to October 2023, and provides an overview on the research progress on the biosynthesis of these natural products and their precursors. In addition, important progress in the biosynthesis of some drugs approved by FDA before is also briefly summarized. An in-depth understanding of the biosynthetic pathways and mechanisms underlying their efficacy is expected to provide valuable insights for the discovery and research of more new drugs in the future.

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.

Polyketides are a class of natural products isolated from a wide variety of species. In bacteria, diverse skeletons of polyketides lead to different biological functions, including anti-bacteria, anti-fungi, anti-tumor and immunomodulation. Polyketide synthases (PKSs) are responsible for the biosynthesis of polyketides through successive Claisen condensations of short-chain fatty acids. PKSs are classified into type Ⅰ, type Ⅱ and type Ⅲ, producing different polyketide scaffolds. Bacterial PKSs often hybridize with other biosynthetic enzymes to form PKS hybrids, such as PKS-NRPS or PKS-Ripps, exhibiting more complicated and unique structures. Additionally, different types of PKS can also form inter-PKS hybrids to generate different skeletons. In this review, we summarize recent advances in the structures and biosynthetic mechanisms of bacterial inter-PKS hybrids, including type Ⅰ PKS internal hybrids, type Ⅰ/Ⅱ PKS hybrids and type Ⅰ/Ⅲ PKS hybrids with the following context: (1) In atypical type Ⅰ PKSs, some modules may iteratively catalyze multiple rounds of carbon chain growth, resulting in iterative/non-iterative PKS hybrids; (2) trans-AT PKS and cis-AT PKS can also form PKS hybrids, and the synthesis of kirromycin is a representative example; (3) Type Ⅰ PKSs synthesize unique starter units for type Ⅱ PKSs to produce polyketide scaffolds with the alkyl groups; (4) Type Ⅲ PKSs can condense malonyl-CoA to form different aromatic acids through multiple tailoring steps, and the aromatic acids subsequently act as the starter unit or extender unit into the type Ⅰ PKS assembly line. By elucidating the biosynthetic gene clusters and biosynthetic pathways of inter-PKS hybrids, the reconstructions of inter-PKS hybrids for synthesizing pharmaceutically important analogues are possible. This review also comments the discovery of new inter-PKS hybrids and the engineering of their biosynthetic machineries, to gain more insights into their biosynthetic potential for the production of diverse molecules. By comparing the biosynthetic mechanisms of PKS and discussing the progress of engineering modifications, we prospect a variety of potential inter-PKS hybrid models, highlight the direction for the genome mining of novel polyketides, and provide insights for the engineering modifications of PKS. Through further in-depth and systematic studies on various inter-PKS hybrids in bacteria, it is expected to reveal more natural conundrums, generating a large number of new natural products through adaptive transformation for the research and development of microbial drugs.

The natural products terpenoids are widely distributed in animals (marine invertebrates), plants, microorganisms, with diverse molecular structures for bioactivities. A large number of terpenoids have been extracted directly from plants and microorganisms. However, traditional methods based on natural screening face challenges in discovering new terpenes due to the increasing number of known compounds at large quantities. The advent of next-generation sequencing and synthetic biology technologies marks the onset of the era of genome mining-driven natural product discovery, particularly in the exploration of new terpenoids. However, challenges persist in this regard, such as low efficiencies, interference of known compounds, and limited data throughput. In this review, we focus on recent advances in terpenoid discovery via microbial genome mining strategies, including the use of the precursor supplying microbial chassis (Escherichia coli, Saccharomyces cerevisiae, Aspergillus oryzae, Streptomyces albus, etc.), the microbial resources from extreme geographical environments, deep genome mining, and terpene mining platforms driven by artificial intelligence and automation techniques. To produce more terpenoids using heterologous hosts, multiple microbial chassis with enhanced precursor supply have been developed to improve their production yields and thus facilitate the discovery of structurally unique terpenoids. With the growing understanding of terpene biosynthesis machinery, the deep mining of terpenoid biosynthetic gene clusters and terpene synthases can effectively address issues related to repeated and irrelevant discoveries. Furthermore, the integration of artificial intelligence and automation platform with synthetic biology has ushered in the high-throughput intelligent discovery of terpenoids, which significantly improves the research and enables the discovery of numerous terpenoids with new structures. Finally, we address challenges and future directions for genome mining based terpenoid discovery. Driven by synthetic biology and artificial intelligence, a new chapter for the discovery of terpenoids and other natural products will open. We are looking forward to seeing more terpenoids to be developed as drugs and valuable chemicals in the future.

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.

Paclitaxel (Taxol) is a natural broad-spectrum anticancer drug, which is well-known for its potent anticancer activity. Its production mainly relies on the extraction and purification from the rare Taxus plant, followed by chemical semi-synthesis. The limited natural resource for paclitaxel imposes a significant constraint on its production capacity. In recent years, with the complete decoding of the Taxus genome and the rapid development of synthetic biology, constructing recombinant cells through synthetic biology techniques has emerged as an effective method to address this challenge. Since paclitaxel biosynthesis involves more than 20 steps of complicated enzymatic reactions and about half of them are P450 enzyme-mediated hydroxylation reactions, the complete elucidation of its biosynthetic pathway remains elusive. Meanwhile, the production of paclitaxel by engineered microbes is still at the initial stage, and there are numerous by-products, which seriously compromise the efficient synthesis of paclitaxel. Therefore, this article reviews research progress related to paclitaxel synthesis pathways, Taxus omics analyses, construction of chassis cells, synthesis of key precursors, modifications of crucial enzymes, and catalytic mechanisms underlying paclitaxel biosynthesis. Special attention is given to the recent breakthrough in elucidating the formation of oxetane ring and the discovery of Taxane 1-β- and 9-α-hydroxylases. Recent advances in the study of the catalytic mechanism of Taxadiene-5-α-hydroxylase and significant progress in engineering tobacco and yeast chassis will also be commented. Furthermore, challenges and future prospects involved in the paclitaxel synthetic biology research are discussed, such as the issues of low enzyme catalytic efficiency, significant product promiscuity, unknown specific reaction sequences, and the biosynthesis of critical paclitaxel intermediates, aiming to enhance the understandings of paclitaxel biosynthetic pathways and catalytic mechanisms for greener and more efficient production of paclitaxel.