1,3-Propanediol (PDO) is an important chemical extensively used in material science and the cosmetics industry. The biomanufacturing of PDO offers numerous advantages, such as the renewability of raw materials and environmental friendliness. Among various microorganisms, Clostridium pasteurianum emerges as an ideal choice for industrial PDO production due to its safety, non-pathogenic nature, rapid glycerol metabolism, fast growth rate, independence from expensive culture medium components, and its inherent efficient metabolic pathway for PDO production. This review begins by introducing the current state and challenges of PDO biomanufacturing, followed by an in-depth discussion of the methods for producing PDO using C. pasteurianum. Special attention is paid to the glycerol metabolism mechanism, strategies for glycerol fermentation, and the design of the fermentation process. Notably, our research group has identified C. pasteurianum mutant strains and developed robust processes that have largely addressed the organism’s traditional sensitivities to environmental conditions, especially regarding iron concentration and impurities of raw glycerol. In an electricity-aided fermentation process, PDO concentration as high as 120.6 g/L was achieved, with a productivity of 4.8 g/(L·h) and a yield reaching the theoretical maximum. We further discuss the natural limitations of genetic engineering in C. pasteurianum, exploring strategies based on rational genomic modification and directed evolution. Finally, the development of efficient downstream processing technologies is emphasized as crucial for realizing the cost-effective microbial production of PDO from renewable resources, since the industrial application of PDO requires a very high purity (>99.9%). The discussion on PDO downstream processing mainly focuses on evaporation, distillation, and extraction-based purification techniques. Through a comprehensive coverage of metabolic engineering, strain evolution, fermentation optimization, and product separation technologies, this review discusses about the characteristics and advantages of PDO production from C. pasteurianum, highlighting key considerations for advancing this microorganism as a new industrial chassis.

Chitin, a linear homo-polysaccharides composed of N-acetylglucosamine (GlcNAc) through β-1,4-glycosidic bonds, is the richest nitrogen containing biomass resource on earth, with an annual production of 10 billion tonnes. Chitin is widely distributed in nature, mainly found in the shells of shrimps and crabs, the exoskeletons of insects, and the cell walls of fungi. Due to its abundance and renewablity, especially the presence of the valuable nitrogen element, chitin receives widespread attention. However, the abundant hydrogen bonds in the structure of chitin and its huge molecular weight make it highly crystalline and insoluble in water, which leads to challenges in its degradation and high-value utilization. Thus, chitin resource is often discarded as wastes or buried, leading to serious environment issues and wasted resources. Conversion of abundant chitin resources into high value-added chemicals has both environmental and economic significance. Nowadays, the utilization of chitin resources is mainly done by efficient, low-cost chemical method, but causing huge environmental pollution. Compared with chemical method, the biological method shows great potential in the context of green and sustainable development due to the advantages of environmentally friendly process and mild reaction conditions. In this review, the sources and classifications, catalytic mechanisms and properties of key enzymes for chitin degradation are introduced. Secondly, the current status of chitin biodegradation to monosaccharides (GlcNAc and glucosamine) and oligosaccharides (N-acetyl chitooligosaccharides and chitooligosaccharides), and further bio-converted into nitrogen-containing chemicals are reviewed. Although many studies on enzymes involved in chitin degradation and conversion have been carried out with certain achievements, the diversity and complexity of these enzymes, coupled with the low activity and secretory nature and other factors, have hindered the real industrial chitin degradation and conversion. Consequently, the challenges in biodegradation and high-value conversion process of chitin such as low activity of enzyme, poor efficiency and high cost are highlighted. Finally, the important role of rapidly developing synthetic biology technologies in chitin utilization is envisaged, which will aid the efficient bio-refining of chitinous resources.

Hydrogenases catalyze the reversible conversion of hydrogen gas into protons and electrons which is promising for industrial application. However, free hydrogenases face challenges such as oxygen sensitivity and low electron transfer rates. This review summarized the immobilization of hydrogenases by carbon materials, metals, semiconductors, polymers and metal-organic-frameworks (MOFs). Carbon materials provide the advantages of low cost and large specific surface areas, while they tend to agglomerate. Hydrogenases are immobilizated on carbon materials through adsorption, usually involving electrostatic interactions and hydrophobic interactions, and are used in bioelectrocatalysis, biofuel cells and bioreactors. Metals and semiconductors, known for high conductivity and excellent reactive activity, are expensive and less stable. Through adsorption involving electrostatic interaction and hydrophobic interaction, immobilization of hydrogenases on metals and semiconductors are normally applied in bioelectrocatalysis, biofuel cells and photoelectrocatalysis. Polymers have good biocompatibility and mechanical strength but low conductivity. Immobilization of hydrogenases on polymers can improve the stability and oxygen tolerance of hydrogenases. Immobilization on polymers is realized through adsorption and entrapment, involving hydrogen bonds, hydrophobic interactions and π-π interactions, and is often used in bioelectrocatalysis and photoelectrocatalysis. MOFs are designable and have high specific surface areas, which provide wide choices for hydrogenases immobilization. However, MOFs tend to collapse in harsh conditions. Immobilization on MOFs through adsorption and entrapment involves coordinate bonds, hydrophobic interaction, and π-π interaction. Furthermore, the prospect of immobilization of hydrogenases by novel hybrid materials was proposed which can expand the applications of immobilized hydrogenases. Immobilization of hydrogenases facilitates the stability of hydrogenases, which can be applied in efficient production and application of hydrogen, as well as biological asymmetric hydrogenation for chiral medicine preparation. Immobilization of hydrogenases provide alternative options for transforming energy structures, realizing green manufacturing and solving environmental problems.

The traditional chemical manufacturing based on petroleum as raw material has had profound impacts in the development of modern society. However, it also has many drawbacks, such as environmental pollution and lack of sustainability. In contrast, biomanufacture with microorganisms as industrial chassis is gradually becoming a hot spot in industrial production due to its advantages of environmental friendliness and sustainability. Nonetheless, the limitations of traditional industrial biotechnology, including susceptibility to microbial contamination, complex fermentation processes, and difficulties in achieving continuous fermentations, have hindered the competitiveness of their products in terms of production costs compared to chemical industries To address these challenges, “Next Generation Industrial Biotechnology” (NGIB) with extremophiles as non-conventional chassis, has been undergoing continuous development with increasing global attentions.The basis of NGIB is extremophiles, such as halophiles, acidophiles, and thermophiles, known for their ability to thrive in extreme environments. Through molecular engineering of extremophiles, especially Halomonas spp., the recombinants can utilize various inexpensive carbon sources for continuous open fermentation, leading to the production of diverse high-value products with reduced cost. This review defines and summarizes the characteristics of extremophiles, highlighting their ability to grow rapidly in extreme environments like high salt, high temperature, and extreme pH. Subsequently, the review summarizes current genetic engineering approaches for extremophiles, such as promoter engineering, CRISPR-based gene editing, community fate strategy, and stable plasmid vectors. Additionally, metabolic engineering methods such as precursor supplementation, pathway disruption, byproduct reduction, and enhanced transport are discussed, along with various products including PHA, proteins, amino acids, and small molecule derivatives. The review also identifies challenges in extremophile engineering, such as the lack of suitable plasmid vectors, low plasmid transformation efficiency, lack of efficient gene editing tools, and long growth and fermentation cycle, but proposes corresponding solutions. Finally, the review proposes leveraging the characteristics of different types of extremophiles to produce advantageous products, thereby driving the development of next generation industrial biotechnology based on various extremphiles, and achieving green, environmentally friendly, and sustainable biomanufacturing.

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.

DNA-Encoded Library (DEL) technology, as an emerging means of small molecule drug screening, has become an important and indispensable technology platform for new drug discovery and development. The technology incorporates many advantages from combinatorial chemistry, molecular biology, and chemical bioinformatics, which greatly improve the efficiency of compound library synthesis and screening. Meanwhile, driven by the development of nucleic acid-compatible chemical reactions and high-throughput sequencing technology, DEL technology has made remarkable progress and gradually become a fast, economical, and efficient high-throughput screening platform, and has been more and more widely used in seedling compounds screening by universities, research institutes, and large pharmaceutical companies. The success of a DEL screening relies heavily on the chemical space and structural diversity of the compound libraries, both of which are directly affected by the number of chemical reactions compatible with nucleic acids. Therefore, developing the on-DNA chemical reactions to continuously enrich the chemical toolbox for DEL synthesis and thus enhance the structural diversity and drug potential of the molecules in the libraries has been the focus in this field. In recent years, the number of on-DNA chemical reactions has increased significantly, greatly broadening the scope of chemical reactions available for DEL construction. Meanwhile, a series of novel reaction methods, such as photocatalysis, electrocatalysis, and biosynthesis, have also emerged in the application of on-DNA chemical reactions and further expanded the field that on-DNA chemical reactions can reach. In this paper, we systematically review the metal-catalyzed on-DNA chemical reactions in recent years, including C(sp²)—C(sp²) bond-formation reactions, C(sp³)—C(sp³) bond-formation reactions, C(sp²)—C(sp³) bond-formation reactions, and C(sp²)—X bond-formation reactions; the synthesis of on-DNA privileged heterocycles with single-ring, fused-ring, and spirocyclic rings by using target-oriented synthetic and diversity-oriented synthetic strategies; the research progress of photocatalytic and enzyme-catalyzed on-DNA chemical reactions. However, the current developments in on-DNA reactions also have limitations, such as compatibility with nucleic acids and substrate suitability. In the future, it is important to exploit more robust on-DNA reactions that can proceed under mild conditions, new types of on-DNA reactions, and the combination of high-throughput screening and computer-assisted on-DNA reactions.

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.

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.

Natural product is an important source of small-molecule drugs and probes, but its synthesis is challenging and has attracted lasting attention in the field of organic chemistry. With the continuous advancement of chromatographic techniques for separation and spectroscopic methods for structural analysis, the pace of discovering tiny bioactive natural products is accelerating, concomitantly leading to an increase in the diversity and complexity of the newly identified structures. However, to meet the demand of the quantity for the study of their structure-activity relationships, target identification, in vivo activity evaluation, etc., growing challenges in the requirement for the synthetic efficiency, economy, and scalability of natural products are emerging. Synthetic practices in a chemoenzymatic way have provided multi-dimensional visions for natural product research, which emerged as a hot research topic in recent years. On the one hand, enzymatic catalysis has provided highly efficient and selective synthetic methodologies that would complement traditional synthetic methods. On the other hand, the introduction of enzyme-catalyzed reactions would bring a new mode of strategic design for synthesis, enabling the rapid and diverse synthesis of natural products with high efficiency. In this context, how to integrate the enzyme-catalyzed reactions into the synthesis of natural products is the key to a successful chemoenzymatic synthesis. We herein summarized three roles played by the applications of enzyme-catalyzed reactions in the current practices of chemoenzymatic synthesis of natural products. ①The involvement of biocatalysis would introduce a chiral center or a key functional group into the starting material, or supply complex synthetic precursors (e.g., polysubstituted (hetero)aromatics, chiral pools, etc.) via in vitro enzyme-catalyzed reactions or fermentation, hence advancing the starting line of synthesis; ②Late-stage enzyme-catalyzed chemo-, regio-, and stereoselective modifications of substrates with heavily substituted functional groups or inert positions of complex skeletons; ③The strategic application of enzymatic catalysis as a key carbon-carbon bond-forming step in the construction of the skeleton of natural product. Finally, we have also discussed the current challenges and future trends of the chemoenzymatic synthesis of natural products in three facets, including the design of synthetic strategy, the development of synthetic methods, as well as persons involved in the research. Thus, the integration of interdisciplinary methods and technologies, including chemical synthesis and biocatalysis, would invigorate the synthesis of natural products.

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.