Organoid technology, which leverages the cultivation of three-dimensional (3D) miniature organ models from stem cells in vitro to simulate the structure and function of human organs, has emerged as a cornerstone in biomedical fields such as disease modeling and drug screening. Despite its significant contributions, the rapid advancement of this frontier technology also raises profound ethical challenges, necessitating a robust framework for its normalization and legalization. This paper firstly provides a comprehensive analysis of the development of organoid technology, elucidating its distinctive characteristics and substantial value in biomedical applications. Currently, the technology is advancing towards the construction of more complex organ systems, achieving higher fidelity, and integrating more intricately with other cutting-edge technologies. This evolution enhances the capability of organoids to mimic human physiology accurately, thereby improving the predictive accuracy of medical research and pharmaceutical developments. The paper examines the primary ethical challenges raised by organoid technology, including the informed consent of donors, privacy protection, and equitable access to this technology. These issues are pivotal in maintaining public trust and compliance with ethical norms. Additionally, it explores the ethical implications of modeling sensitive organs such as the brain and embryos, raising questions about the moral status of such models and potential psychological impacts on society. It also reviews how various countries and international organizations respond to these ethical controversies. Major developed countries and international bodies have adopted a variety of governance measures, primarily in the form of ethical guidelines or standards, which provide a flexible yet sometimes insufficient framework for addressing rapidly evolving technologies. In contrast, China lacks specific legislative policies regarding organoid technology, indicating a need for tailored governance strategies that align with both international standards and local ethical considerations. In order to bridge these gaps, this paper attempts to construct a “moral assessment scale” framework. This tool is designed to quantify ethical considerations and guide decision-making in organoid research and application. The paper advocates for strengthened interdisciplinary cooperation and the improvement of the informed consent process, essential for ethical compliance. Moreover, establishing an international unified ethical code could facilitate global cooperation and harmonize standards across borders. Supporting ethical research and fostering public discussions are also crucial for the responsible development of organoid technologies. By thoroughly examining the ethical challenges and proposing actionable solutions, this paper aims to foster a balance between scientific innovation and ethical responsibility. This balanced approach will not only advance the field but also ensure that it develops in a manner respecting human dignity and societal values, ultimately better serving human health and well-being.

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.

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.

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 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.

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.

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.

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.

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 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.