Huge challenges, such as food security, energy security, climate change, dual-carbon target, and so on, motivate human society to seek disruptive and innovative solutions. In vitro biotransformation (ivBT), bridging the gap between whole-cell-based fermentation and enzyme-based biocatalysis, is an emerging biomanufacturing platform designed for the production of biocommodities (e.g., synthetic starch, healthy sweeteners, organic acids, etc.) and bioenergy. In ivBT, in vitro synthetic enzymatic biosystem (ivSEB) is its high-efficiency biocatalyst. Based on the Chinese philosophy that “Tao is simple”, ivSEB is the in vitro reconstruction of artificial (non-natural) enzymatic pathways with a number of natural enzymes, artificial enzymes, and/or (biomimetic or natural) coenzymes, and/or artificial membrane, without living cell’s constraints, such as cell duplication, bioenergetics, basic metabolisms, regulation, and so on. ivBT enables it to surpass the limitations of whole-cell fermentation and has multiple advantages, such as theoretical product yield, at least 10-time volumetric productivity, tolerance to toxic substrate/product, and so on. This review defines the concept of ivBT, presents its design principles, distinguishes it from other seemingly-like concepts, such as cell-free protein synthesis and cascade enzyme biocatalysis, introduces several representative examples, and discusses its challenges and opportunities. The development of ivBT is based on the linear strategy of “Design-Build-GoNG-Optimization”, leading to super-biomanufacturing machines that can meet national needs, such as food security and new energy system. To address food security, we propose two out-of-the-box solutions: (1) in vitro biotransformation of cellulose to starch, possibly increasing the starch supply by a factor of 10; (2) artificial starch synthesis from CO₂ by combining ivBT and chemical catalysis. Furthermore, the revolutionary production of starch could open a door to the starch-based carbohydrate economy, wherein starch is a high-density hydrogen carrier, more than 2.5 times that of compressed hydrogen, and an ultra-high electricity storage compound, more than 10 times of lithium-ion battery. In a word, ivBT featuring ultra-high energy efficiency and potentially-low-cost production could become a third industrial biomanufacturing platform and help solve huge challenges.

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.

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.

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.

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.

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.

In order to achieve carbon neutrality and green economy, people use biorefinery technology to transform and utilize CO₂. Microbial electrosynthesis (MES) is an emerging technology that converts CO₂ into chemicals by electrically driven biocatalysts. Currently, the low efficiency of microbial carbon sequestration, an incomplete understanding of electron transfer mechanisms, low synthesis rate, and poor applicability of reactor components have been the limiting factors for the large-scale application of MES. In this paper, the mechanisms of electron supply in the MES system, including through electrodes and electron donors such as H₂, formic acid, CO, and other molecules, are systematically reviewed based on how cathodic microorganisms obtain electrons. It is an effective method to improve electron transport efficiency by modifying conductive nanowires of electroactive microorganisms and optimizing the expression of microbially associated hydrogenase, formate dehydrogenase and CO dehydrogenase using synthetic biology techniques. Additionally, cathode modification aimed at improving electron transfer rates between microbes and electrodes, enhancing the biocompatibility, and providing more reducing power can facilitate the generation of value-added products. In addition to enhancing the electron transfer efficiency of the cathode, the construction of a reactor with high efficiency of gas-liquid-solid mass transfer and electron transfer, the reduction of anode potential for water electrolysis, and the regulation of microbial activity are also important strategies to enhance MES performance. In the future, it is necessary to further elucidate the mechanism of microbial electron transport and strengthen the performance of MES by means of synthetic biological communities, and by designing a more efficient electrode interface that balances electron transfer rate, substrate mass transfer and biocompatibility. In terms of the scaling-up of reaction devices, electron transfer and gas mass transfer can be improved through the combination of various methods, and integrating product separation processes can promote the further development of the technology and provide new ideas for the realization of the “Carbon Peak and Carbon Neutrality” goal.

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.

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.