Terpenoids are a class of natural products with important physiological functions and significant biological activities that are widely found in nature and have a wide range of applications in the food, medical, and daily chemical industries. In the biosynthetic pathway of terpenoids, terpene synthases often determine the type and novelty of the terpene carbon skeleton, and tailoring enzymes, such as cytochrome P450 enzymes, can carry out a variety of post-modifications, ultimately resulting in terpenoids with a rich diversity of structures and functions. In recent years, with the development of genome-sequencing technology and synthetic biology, a large number of terpene biosynthetic enzymes of plant and microbial origin have been characterized, which, excitingly, include non-canonical terpene synthases that can also catalyze the generation of unique cyclized skeletons. Meanwhile, the use of combinatorial biosynthetic strategies has led to the creation of many novel and unnatural terpenoids, further enriching the kingdom of terpenoids. Here, we review the recent advances in non-canonical terpene cyclases and combinatorial biosynthetic pathways over the past five years, with a view to shedding light on the discovery and biosynthesis of novel terpenes in the future. Firstly, the newly discovered novel enzymes with terpene cyclization functions are reviewed, containing a new subclass of type Ⅰ terpene synthases, non-squalene triterpene synthases, UbiA-type terpene cyclases, cytochrome P450 oxygenases, methyltransferases, vanadium-dependent haloperoxidases, and haloacid dehalogenase, along with their sequences, functions, and possible cyclization mechanisms, which can contribute to our understanding of terpenoid biosynthetic enzymes and the discovery of novel terpenoids. This review then describes the combinatorial biosynthesis of non-canonical terpenoids. By combining terpene synthases with methyltransferases or natural/artificial cytochrome P450 oxygenases, a series of unnatural terpenoids containing non-canonical C₁₁ and C₁₆ backbones, or with unusual structural modifications, were produced. This could inspire the structural innovation studies of terpenoids in the future. The discovery of novel enzymes and the construction of novel combinatorial biosynthetic pathways will further broaden the structural diversity and chemical space of terpenoids, which is expected to provide more potential novel terpenoids for clinical drug development.

Photocatalysis has the advantages of mild reaction conditions, renewability, and strong reactivity, but the poor selectivity limits its further application in asymmetric synthesis. Enzymatic catalysis shows unique advantages of high selectivity and specificity, but it leads to some defects such as limited reaction types and relatively narrow substrate scope. Photoenzymatic catalysis combines the advantages of high reactivity of photocatalysis with high selectivity of enzymatic catalysis, providing a novel synthesis model, that is more in line with the requirements of modern green organic synthesis. The term “photoenzyme reactions” narrowly refers to the synergistic catalysis involving photoenzymes, which can be classified into the following four categories: natural photoenzymactic reactions, artificial photoenzymatic reactions, photo-biocatalysis cascade reactions, and photo-induced promiscuous enzymatic reactions. However, natural photoenzymes are rarely found in nature, the stringent substrate scope further hinders their application. Artificial photoenzymes integrate photosensitizers into the scaffold of natural enzymes, which have been well summarized in previous reviews. Photo-biocatalysis cascade reactions by combining photochemical steps and enzymatic steps can realize some complex organic synthesis processes. Since the first report on NAD(P)H-dependent KREDs-catalyzed enantioselective radical dehalogenation of lactones, photosensitive cofactor-dependent unnatural photoenzymatic catalysis demonstrated its great potential in the field of organic synthesis, and continues to thrive to date, which has addressed many problems difficult to be achieved in traditional organic synthesis. Since 2023, research into the promiscuity of photoenzyme catalysis has witnessed continuous breakthroughs, reporting diverse novel types of photoenzyme catalytic reactions and mechanisms. The precise control over stereoselectivity and even regioselectivity directly addresses the longstanding challenges in the field of organic synthesis. While there have been many publications summarizing the related research, yet rarely focused on this rapidly evolving field. In this review, we summarize the recent and representative reports of photo-induced promiscuous enzymatic reactions, and classify them according to asymmetric dehalogenation, hydrogenation, intramolecular cyclization, intermolecular C—C/C—N/C—S cross-coupling reactions through free radical pathways, etc. These reactions exhibit different mechanisms due to different enzymes and substrates. For example, in the process of redox initiation, there are two types: single-electron reduction initiation and single-electron oxidation initiation. In the radical termination process, single-electron reduction termination and single-electron oxidation termination may be used. The diversity of mechanisms also makes it possible to develop more photoenzyme-catalyzed promiscuous reactions. In the future, new photoenzymatic methods will be promoted by rapidly developing technologies such as genetic engineering, synthetic biology, enzyme engineering, flow chemistry, and artificial intelligence, and more efficient and highly selective new-to-nature reactions will emerge, significantly expanding the application range of photoenzyme catalysis in the field of green asymmetric synthesis.

Steroids exhibit a range of biological activities and are commonly described as the ‘key to life’ in nature. Steroidal-based medications have emerged as the second largest pharmaceutical category following antibiotics, owing to their remarkable bioactivities such as anti-infective, anti-inflammatory, anti-allergic, and antitumor properties. This category encompasses more than 400 drug compounds, representing approximately 17% of FDA-approved medications. The synthesis of steroidal products continues to attract significant attention due to their diverse bioactivities and physicochemical characteristics in pharmaceutical applications. With the increasing demand for steroidal drugs and the fluctuating availability of sapogenin resources, the use of Mycobacteria to convert inexpensive phytosterols to produce key intermediates for steroid drugs has been established as the most mature and sustainable industrial route. However, the complex structure of steroids, particularly their highly oxygenated skeleton, poses challenges for the well-established semi-synthesis route of complex steroid medications. Recent strides in bioinformatics and genetics have significantly advanced the studies on synthesis of steroidal compounds. This review highlights recent advancements in the synthesis of high-value steroids, including the diverse steroid drug intermediate production via external steroidal modifying enzymes expression in engineered Mycobacteria, chemo-enzymatic synthesis of complex steroids, and yeast-based de novo synthesis. It specifically highlights the significant achievements in the chemo-enzymatic synthesis, which combines the precise site- and stereoselectivity of enzymatic transformations with the efficiency of chemosynthesis, enabling the concise synthesis of complex steroidal products. Recent advancements in chemoenzymatic strategies, especially those involving P450 hydroxylase, 3-sterone-Δ¹-dehydrogenase, reductase, and enzyme cascades, have significantly contributed to the efficient and straightforward synthesis of complex steroid medications. On this basis, the future research opportunities and challenges are also discussed, aiming to provide a reference for the efficient development of more value-added steroid compounds, including the development of new generation steroid intermediates, the discovery of novel steroid biocatalysts, and the establishment of steroid synthesis pathways in mycobacteria.

Glycosylation modifications, extensively present on the surfaces of eukaryotic proteins as a type of post-translational modification, hold significant physiological and pathological implications. The microscopic heterogeneity of natural glycoproteins has led to the emergence of the chemical synthesis of homogeneous glycoproteins with defined structures as a crucial frontier in exploring the structure-function relationships of glycosylation modifications. With the flourishing development of protein synthesis and glycoengineering technologies, various protein ligation and polysaccharide synthesis strategies have been developed, enabling the preparation of glycoproteins containing hundreds of amino acid residues. The development of glycoprotein synthesis strategies primarily revolves around chemical and enzymatic approaches for glycosidic bond formation, leading to effective synthesis schemes such as Lansbury’s aspartic acid acylation, chemical strategies based on glycosyl amino acid building blocks, and glycan remodeling strategies using endoglycosidases and glycosyltransferases. This review will discuss the chemical and enzymatic construction of glycosidic bonds, examining existing strategies for the total synthesis of glycoproteins and semi-synthetic approaches that combine with biological expression methods. It will introduce these strategies’ achievements in synthesizing complex homogeneous glycoproteins with different types of glycosylation modifications, such as those with multiple complex N-glycosylation modifications like HSV gD and those containing long hydrophobic segments like IL-2. Additionally, this review will highlight breakthroughs in understanding the structure-function relationships of glycosylation modifications in various physiological processes through these synthetic complex glycoproteins, including the relationship between glycan chain length and immunogenicity in antigenic glycoproteins, and the mechanisms by which O-GlcNAc regulates synaptic function in neurons. Finally, it will summarize the progress made in glycosidic bond construction, purification strategies, and protein solubility, and point out that further optimization of selectivity and synthetic yield remains a pressing issue in the field of glycoprotein synthesis. The wide application of glycoprotein synthesis technology in developing immunotherapies and understanding the molecular mechanisms of various diseases expands the development directions of synthetic science in the field of life and health, from understanding principles to developing products.

Stereochemical control is an important long-standing research topic in synthetic chemistry. Enzymes, as green and highly selective natural chiral catalysts, face constrains in synthetic applications due to their evolution-defined molecular structure and catalytic mechanism. Photocatalysis represents an important strategy to initiate free radical reactions by capturing photon energy to activate the chemical bonds of substrate molecules. As an emerging synthetic tool for asymmetric synthesis, photobiocatalysis merges the advantages of photochemistry and enzyme. Unfortunately, photoenzymes are rather rare in nature. Thus far photoenzymes identified are DNA photolyases, light-dependent protochlorophyllide reductases and blue light-responsive algal photodecarboxylases. Utilization of advanced molecular biotechnologies such as protein engineering and directed evolution under the guidance of chemical mechanisms of photocatalysis enables us to explore unknown photocatalytic functions of natural coenzymes, synergize photocatalysts and enzymes, and rationally design artificial photoenzyme with defined functions. The past few years have witnessed remarkable advances in these aspects, significantly surpassing the spectrum of substrates and reactions of enzyme catalysis, compensating for the scarcity of natural photo-enzymes and expanding the chemical boundaries and synthetic space of biocatalysis. This review summarizes the latest research progress in chemically-driven photoenzymatic asymmetric reactions. Based on their merging modes, the review categorizes the integration of light and enzyme into four classes: coupling of exogenous photocatalysts and native enzymes, photobiocatalysis driven by excitation of electron donor-acceptor complex, direct photoredox catalysis by coenzymes, and energy transfer photobiocatalysis. The chemical mechanism of bond activation by photocatalysis and synergistic control of stereoselectivity by enzyme in these photobiocatalytic systems are discussed in detail. In the end of this review, we also delineate the present challenges of asymmetric photobiocatalysis including the monotonicity of native photoactive cofactors and low catalytic efficiency for abiological reactions. This review also proposes future directions from the perspectives of new natural enzyme mining, expansion of artificial photoenzymes, enzyme de novo design, and whole-cell catalysis, which are anticipated to foster green bio-manufacturing of high-value functional molecules through the fusion of chemistry and biology and push forward the sustainable development of synthetic chemistry.

Organic acids, as important platform chemicals, have been widely used in food, pharmaceutical, chemical industries and agriculture. Currently, microbial production of organic acids relies primarily on sugars as feedstocks, which may suffer from the competition with food and arable lands. One carbon (C₁) molecules such as CO, CO₂, methane, methanol and formic acid are widespread and inexpensive, which are considered as ideal feedstocks for future bio-manufacturing. Bioconversion of C₁ feedstocks toward the production of organic acids helps mitigate greenhouse effect and realize carbon neutrality. Therefore C₁ sources have been regarded as raw materials of third generation biorefinery, and natural C₁ utilizing microbes attracted increasing attention. Although some microorganisms have native biosynthetic pathway of organic acids, the production efficiency is usually lower than expected. This review summarizes the recent progress on the biosynthesis of organic acids (3-hydroxypropionic acid, lactic acid and succinic acid) from C₁ feedstocks using synthetic biology methods. First, the native C₁ utilizing pathways are summarized, including CO₂, CO, methane, methanol and formic acid. Then the metabolic engineering strategies to improve organic acids production were systematically reviewed, including the optimization of rate-limiting enzymes expression, enhancement of the supply of precursor and cofactor, cofactor engineering, and inhibition of the product degradation. In addition, the challenges, solutions, and prospects of C₁ bioconversion to organic acids are also discussed, and coupling chemical catalysis and biological transformation may provide a promising industrial route for organic acids production. In particular, methanol is an ideal C₁ feedstock with many advantages like convenient storage and transportation, high liquid-to-liquid mass transfer efficiency, and it can also be massively produced from CO₂ by “liquid sunshine” technology. Therefore constructing high efficient methanol cell factory may enable organic acids production from CO₂, a carbon neutral production manner. This review may provide a guidance for C₁ biorefinery and industrial bioproduction of organic acids.

Although bioactive natural products have played significant roles in pharmaceutical research, their application potential is still limited by low isolated yields and structural modification challenges. To overcome these obstacles, developing environmentally friendly and highly efficient synthetic strategies offers exceptional approaches to obtain complex bioactive natural products and their analogs. Driven by advancements in microbial genetics and enzyme engineering, chemoenzymatic strategies, which merge enzymatic and synthetic transformations, are steadily emerging as potent tools in the synthesis of bioactive natural products, pharmaceutical components and other valuable molecules. These fashionable strategies offer not only advantages of chemical synthesis, such as simplicity, flexibility and scalability, but also those of biosynthesis, including environmental friendliness, high selectivity and efficiency. This will establish a linkage into the next-generation synthesis which is expected to break the boundary between chemistry and biology. Versatile cytochrome monooxygenases, P450s, can achieve inert C—H bond selective oxidation in mild and green conditions, a classically challenging organic transformation, providing novel retrosynthetic plans for complex natural products and becoming one of the hotspots in synthetic science. This review summarizes the recent applications of chemoenzymatic synthesis of natural products using P450-catalyzed site-selective oxidations as critical steps to improve the synthetic efficiency and avoid unnecessary functional group transformations and protection/deprotection steps, categorizing the case studies by structure features, such as steroids, terpenoids, and other types of natural products. At the end of this review, the current challenges in this field, such as heavily relying on the native activities of enzymes, are also analyzed and discussed, along with emerging research directions and technologies in new enzyme mining and enzyme engineering that may provide solutions to these challenges in the future. With constantly cross fusion of biosynthesis, chemical synthesis, synthetic biology, protein engineering, machine learning and other research field, P450-catalyzed site-selective oxidations will be becoming routine tools for synthetic chemists.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of peptide natural products that often contain noncanonical amino acids and structural motifs with promising potential as drug leads. One unique structural unit found in RiPPs is the C-terminal S-[(Z)-2-aminoethenyl]-D-cysteine (AviCys) or (2S,3S)-S-[(Z)-2-aminoethenyl]-3-methyl-D-cysteine (AviMeCys). Avi(Me)Cys-containing RiPPs usually exhibit potent antimicrobial or anticancer activities, which strictly require the presence of the C-terminal AviCys motifs. Despite the potential of Avi(Me)Cys-containing RiPPs as drug leads, lack of synthetic methods and biosynthetic systems to access these type of cyclic peptides impede the application of Avi(Me)Cys-containing peptides in medicinal chemistry. In this review, we summarize the current understanding of the biosynthesis of Avi(Me)Cys-containing peptides and the progress made in the development of chemical methods to synthesize Avi(Me)Cys motifs and derivatives. This review contains two following major sections: ① The biosynthetic process of Avi(Me)Cys motifs in the different families of Avi(Me)Cys-containing RiPPs, including lanthipeptides, lipolanthines, linaridins and thioamitides, are introduced with three essential enzymatic steps: first, a cysteine decarboxylase oxidatively decarboxylated the C-terminal cysteine, generating a highly reactive enethiol; subsequently, distinct enzymes catalyze the dehydration of a serine/threonine (Ser/Thr) residue or the dethiolation of a Cys residue in the precursor peptide by incorporating a dehydroalanine (Dha) or dehydrobutyrine (Dhb) residue; finally, a putative cyclase catalyzes the Michael-type addition between the enethiol group and a Dha/Dhb residue to yield the Avi(Me)Cys motif. Detailed enzymatic investigation of these biosynthetic steps are introduced. ② The chemical synthesis of the Avi(Me)Cys building block and their analogues via diverse synthetic methodology, including the radical thiol-yne coupling, the oxidative decarboxylation/decarbonylation, and the condensation of amides with acetals. Overall, further elucidation of the complete biosynthetic pathway for Avi(Me)Cys motifs in related RiPPs, along with advancements in the chemical synthesis of Avi(Me)Cys-containing natural product peptides, will facilitate the effective utilization of these bioactive peptide natural products.

Over the past century, the scientific foundation of embryonic development has primarily relied upon meticulous examination of developmental processes in model organisms. However, investigating the development of mammals has presented numerous challenges, including interspecies disparities, ethical considerations, and technical constraints. With the rapid advancement of stem cell technology, researchers have endeavored to overcome these obstacles by harnessing the potential of stem cells to generate sophisticated invitro embryo models. The rapid advancement of stem cell technology has revolutionized our approach to study embryonic development. While the ability of current embryo models to fully simulate the authentic developmental process is yet to be verified, they undeniably present new possibilities for developmental biology research. This review primarily focuses on mouse and human, summarizing the types of stem cells used in constructing embryo models and elucidating the roles and importance of different stem cells in simulating developmental processes. This review systematically presents and dissects crucial events and spatiotemporal dynamics in the embryonic development of both mice and humans across various stages. We thoroughly discuss the remarkable milestones achieved by existing embryo models, explore methods for evaluating the biomimicry of these models, and highlight the crucial role of bioengineering methods in embryo model development. The pivotal role of bioengineering in advancing embryonic model development is underscored, emphasizing its indispensable contribution to providing the requisite technical scaffolding for the realization of instruct multicellular induced self-organization with high-level spatiotemporal orders. Additionally, we provide perspectives for the optimization and progressive refinement of embryo models, so as to improve their relevance and applicability. In summary, engineered advances in stem cell-based synthetic development could not only improve our understanding of the inherent complexities of embryos, but also hold the potential for applications in disease research, drug screening, reproductive medicine, toxicological assessments, and other related fields, thereby opening new avenues for both fundamental and translational research.

As the global issue of infertility continues to escalate, particularly with the increasing incidence of male infertility, research in testicular organoids offers new hope and strategies in this field. This review comprehensively discusses the application of testicular organoids in simulating the natural sperm-producing environment, delving into the mechanisms of spermatogenesis, and addressing challenges in male reproductive health. Firstly, we introduce the cellular composition, physiological functions, and the complete process of spermatogenesis within the testicular organ, emphasizing the crucial role of the testicular somatic cell microenvironment in normal testicular development and sperm production. Subsequently, we provide a comprehensive review of the construction of in vitro spermatogenesis systems and the associated research progress through techniques such as testicular tissue culture and reconstruction of testicular organoids in vivo. Moreover, testicular organoids, as a system mimicking spermatogenesis environments in vitro, exhibit significant potential in exploring molecular mechanisms, drug screening and toxicity assessment, as well as preserving and restoring male fertility. Finally, we discuss the limitations of current research in the field of testicular organoids and future research directions. Challenges include accurately simulating the physiological processes of the testis in vitro and improving the quality of sperm obtained in vitro for clinical applications. Future research directions involve delving into the complex interactions between germ cells and somatic cells, aiming to better simulate the testicular microenvironment in vitro, and striving towards safe and effective translation of these research findings into clinical applications for treating male infertility. Additionally, we should ensure that the genetic stability and functionality of germ cells cultured in vitro meet the requirements for clinical applications, and pay attention to the relevant ethical issues. Despite the complexity of the testicular microenvironment and the challenges in fully replicating human spermatogenesis in vitro, the ongoing development in the field of testicular organoids holds promise for providing novel solutions in clinical reproductive medicine and male health research.