Increasing petroleum consumption and growing environmental concerns necessitate the sustainable production of chemicals and fuels from renewable resources. By utilizing renewable resources as raw materials and engineered microorganisms as the core tools, the bio-manufacturing of bio-based materials has become a hot research topic due to its green and low-carbon advantages. ω-Amino acids are a type of non-natural amino acids with amino and carboxyl groups located at the ends of the straight carbon chain. Self-cyclization of ω-amino acids produce lactams, which are the key monomers for the synthesis of polyamide materials, commonly known as nylon. Polyamide materials have wide applications and a huge global market over seven million tons per year. Nowadays, polyamide materials and their monomers are primarily produced through petrochemical routes with non-renewable resources. The research on biosynthesis of these materials and monomers is still in the early stages, but significant progress has been made in recent years. This review article systematically introduces the recent advances in the biosynthesis of ω-amino acids and lactams. To achieve the bio-manufacturing of bio-based polyamide materials, researchers have designed artificial biosynthetic pathways for ω-amino acids from renewable carbon sources such as glucose. The key enzymes for the cyclization of ω-amino acids to form lactams have been identified. By assembling the biosynthetic pathway in microbial chassis such as Escherichia coli and Corynebacterium glutamicum, production of ω-amino acids and lactams have been achieved. Furthermore, the metabolic flux was fine-tuned by regulating and optimizing the expression of key genes to improve the biosynthesis of ω-amino acids and lactams. Besides, biosensors of lactams have been developed to transfer the intracellular concentrations of lactams into easily detectable signals such as fluorescence. Such biosensors have been successfully used for high-throughput screening of ω-amino acid cyclization enzymes and dynamic regulation of biosynthetic pathway. These effects have resulted in the successful biosynthesis of C₄-C₆ ω-amino acids and lactams. Particularly, using glucose as a raw material, the production of valerolactam by fed-batch fermentation exceeded 70 g/L, with a productivity of about 1 g/(L·h), which approaches the level required for industrialization and commercialization. Finally, the review article discusses the current challenges faced in the biosynthesis of ω-amino acids and lactams, including the low yield of biosynthetic pathways, rate-limitations posed by key cyclization enzymes, and insufficient utilization of non-food carbon sources such as one-carbon compounds.

Biomimetic compartmentalization immobilization of multi-enzyme system is a frontier for in vitro synthetic biology, focusing on the spatial and temporal separation of reactions. Compared with simple co-immobilization, biomimetic compartmentalization immobilization can form substrate channels and promote the transmission of intermediates for sequential or coupling reaction. By controlling the relative positions of the enzymes on carriers, this method improves system stability, productivity, as well as purity of product. In this review, we summarized the recent advances of carriers for biomimetic compartmentalization immobilization of multi-enzyme systems, including metal-organic frameworks (MOFs), polymer vesicles and polymer capsules. Metal-organic frameworks (MOFs) are porous coordination materials which are composed of metal ions as nodes and organic linkers. MOFs possess unique characteristics including high porosity, large specific surface area and tunable structure, which are suitable for multi-enzyme systems. The strategies involving the hierarchically porous MOFs, MOF-on-MOF and multi-MOF combinations construct compartmentalized environments for efficient catalytic reactions in vitro. Polymer vesicles are hollow nanostructures composed of amphiphilic block copolymers. The membrane structure of polymer vesicles, similar to the natural phospholipid bilayers, has good mechanical stability and biocompatibility for protecting enzyme molecules, and provides unique microenvironment for sequential reactions. Multiple small vesicles were encapsulated into the larger vesicles to form a “vesicle-in-vesicle” by mimicking the structure of cellular organelles. Polymer capsules with a core-shell spherical nanostructure are formed by the templating method, and have structural stability and excellent shape controllability. Multilayered core-shell structures created by layer-by-layer self-assembly are applied for compartmentalized immobilization of multi-enzyme. In the future, the integration of microfluidic technologies with biomimetic compartmentalization immobilization of multi-enzyme is expected to provide highly efficient and stable multi-enzyme catalytic systems for in vitro synthetic biology and green biomanufacturing.

One-carbon compounds are liquid or gaseous substances that can be naturally occurring or produced in industrial processes, offering the advantages of being abundant, cost-effective, and sustainable to produce. They are anticipated to serve as fundamental raw materials for the next phase of bio-manufacturing, encompassing easily transportable and storable liquid methanol, formic acid, and gaseous CO₂, CO, and CH₄. China is currently focusing on reducing carbon emissions and aims to progressively achieve the targets of carbon peak and carbon neutrality through diverse approaches. Amidst the flourishing landscape of bio-manufacturing, microorganisms are being genetically manipulated using synthetic biology techniques to efficiently harness one-carbon compounds for the creation of high-value products like lipids and single-cell protein. This initiative aims to reduce dependence on imported food and fossil resources, serving as a strategic measure to alleviate food and energy crises. This review presents a comprehensive overview of the most recent advancements in converting one-carbon compounds into valuable oils and single-cell proteins through the utilization of metabolic pathways, chassis genetic modification, and other methodologies involving methylotrophic microorganisms, acetogenic bacteria, yeast, and other microorganisms. It discusses pertinent studies on enhancing molecularly engineered strains through the fermentation process using one-carbon compounds and includes research cases focusing on the production of ultra-long-chain fatty acids. Furthermore, it collates industrial instances related to the conversion of one-carbon compounds from research institutions or companies. Lastly, by addressing the constraints in metabolic pathway design and genetic tools for utilizing one-carbon compound strains, as well as the energy conversion challenges between acetogenic bacteria and lipids-producing microorganisms, it offers foresight into the future opportunities and obstacles encountered in the bio-manufacturing of lipids and single-cell proteins. It suggests advancing inter-disciplinary, efficient systematic integration for fermentation within complex systemic bio-manufacturing processes, driving exploration on the biological conversion of one-carbon compounds, proposing novel solutions to current theoretical and practical challenges, and providing guidance for practical applications and industrial advancements.

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.

Hydrogenases are the most important enzymes in biological hydrogen production and hydrogen energy utilization. They are widely distributed, oxygen-sensitive, multiunit complexed metal enzymes. In vitro synthetic enzymatic biosystems (ivSEB) is a type of in vitro biotransformation (ivBT) technology, which is an emerging biomanufacturing powerhouse that combines microbial fermentation with enzymatic biocatalysis, allowing for novel and efficient hydrogen production, also breaking the Thauer limit and achieving a yield of hydrogen close to the theoretical value of chemistry (1 mole of glucose to produce 12 moles of hydrogen in maximum). It represents the future direction of biological hydrogen production. However, the recombinant expression of hydrogenase is the main bottleneck limiting the wide application of ivSEB for hydrogen production technology. Hydrogenases are widely distributed in all life domains, but are oxygen-sensitive and mostly consist of metalloproteins with multi-subunits, bearing [Fe] only, [NiFe] or [FeFe] dinuclear core in their catalytic center. Oxygen not only inhibits the activity of hydrogenase, but also affects the transcription of the enzyme-encoding gene and post-translational process of the enzymes. As a result, the levels of recombinant hydrogenase are usually low and the enzymatic activities are also incomparable to the native enzymes, often leading to high production costs due to the strict anaerobic purification procedures. In order to meet the requirements of industrial hydrogen production, hydrogenases must possess excellent catalytic properties, such as a high catalytic turnover number, great thermal stability, and the ability to tolerate trace amounts of oxygen. This review summarizes the studies on the structural and catalytic characterizations of hydrogenases, including their classification, oxygen resistance mechanisms, and progress in recombinant expression. Additionally, the evolution of natural electron transfer chains and the design of artificial routes, which can improve hydrogen production efficiency and reduce costs, are briefly discussed. The review also discussed the progress in the studies on the mechanisms of hydrogenases’ tolerance toward oxygen, the strategies for microbial expression of recombinant hydrogenases as well as the optimization of the artificial electron transfer chains adapted for the production of hydrogen using ivSEB, in expectations of promoting the applications of hydrogenases involved ivSEB, from renewable energy storage, anaerobic artificial respiration, to clean hydrogenation or dehydrogenation in biocatalysis.

The development of environmentally benign, biodegradable materials is considered an important way to address “white pollution”. Importantly, organic acid is one of the crucial monomers for preparing biodegradable materials. In recent years, the synthesis of organic acids through green and efficient methods has attracted much attention. As the most promising carbon source recognized for renewability and affordability, lignocellulose is considered a promising carbon source for the biochemical industry. Converting lignocellulose into organic acid is critical to preparing biodegradable materials and achieving carbon neutrality, which meets the requirements of the green and sustainable development strategy. Hence, researchers are focusing their investigations on the lignocellulose biorefinery. To date, innovations in synthetic biology have significantly advanced organic acid manufacturing. For example, the yield of succinic acid has exceeded 150 g/L, which facilitates the formation and development of the bio-based biodegradable materials industry. In this paper, various lignocellulose pretreatment technologies were reviewed, including physical, chemical, biological, physicochemical, and other emerging pretreatment methods. To realize the goal of efficient utilization of lignocellulose, the refining processes of lignocellulose were also reviewed, including detoxification of inhibitors, reductive catalytic fractionation, consolidated bioprocessing, and other methods. After the pretreatment and refining process, lignocellulose is transformed to sugars and aromatic compounds, which can be utilized for producing various organic acid compounds, such as succinic acid, 3-hydroxypropionic acid, cis,cis-muconic acid, 2,5-furandicarboxylic acid, 2-pyrone-4,6-dicarboxylic acid. Next, using the optimization of production of these organic acid compounds as examples, several synthetic biology strategies were summarized, including constructing biosynthetic pathways, optimizing regulatory elements, enlarging the substrates spectrum, and other strategies for improving cell production capacity. Finally, the development trends of the biodegradable materials industry are summarized and prospected. The development of emerging pretreatment and consolidated bioprocessing to facilitate the efficiency of lignocellulose utilization were discussed. Improving the robustness of microbial cell factories and designing the systematic lignocellulose conversion pathways could further optimize the performances of organic acid synthesis. The insights given in this review could facilitate further development on the industrial production of biodegradable materials, towards addressing the global energy crisis and “white pollution”.

Lignin is a major component of lignocellulose, accounting for 15%-30% on a dry weight basis, with an annual yield estimated to be 20 billion tonnes. Lignin is a heterogenous aromatic polymer of phenylpropanoids linked by various C—C and C—O bonds. It is an integral component of the secondary cell wall from terrestrial plants, providing plants with rigidness and fending off microbial pathogens. The abundance and renewability of lignin has recently attracted ample interest in valorizing this readily available polymer. However, the complex nature of lignin presents a significant challenge for lignin breakdown and utilization, and at present the majority of lignin is simply burned as a fuel. Among the different methods, biological utilization of lignin has emerged as a highly attractive approach, since it proceeds under mild conditions and is generally considered environmentally friendly, especially considering that environmental sustainability is trending worldwide. This review comprises three major sections. First, we will summarize key enzymes that nature has created to break down lignin, including laccase, manganese peroxidase, lignin peroxidase, dye-decolorizing peroxidase, and versatile peroxidase etc. Relevant enzymes and their catalytic mechanisms will also be briefly discussed. Second, we will review key reactions in priming and processing lignin derived aromatics before they enter microbial metabolic pathways: O-demethylation, hydroxylation, decarboxylation, and ring opening, as well as representative enzymes involved and their catalytic mechanisms. Finally, we will present engineering efforts toward biological valorization of lignin and lignin derived aromatics, which is largely driven by synthetic biology approaches. Biological valorization of lignin is undoubtedly a field full of potential, however its realization still faces several major hurdles, such as low conversion efficiency and long processing time. Nevertheless, as synthetic biology is developing rapidly, harnessing the power of genetic and metabolic engineering to improve the efficiency of lignin breakdown and utilization, microbial tolerance to toxic aromatics, and redox balance will certainly be a promising path forward, paving the way for industrial application in the near future.

Phosphinothricin (PPT) is one of the top three herbicides, known for its broad spectrum, high herbicidal activity, and non-selectivity, with a highly optimistic market prospect. However, PPT exists in two enantiomers (D-PPT and L-PPT), with the herbicidal activity primarily stemming from L-PPT. Therefore, efficient synthesis of L-PPT with high optical purity is crucial. Pesticide manufacturing enterprises have attempted to develop chemical synthesis methods for L-PPT using approaches such as racemic compound splitting, asymmetric synthesis, natural amino acid chiral source method, and chiral auxiliary induction. However, due to challenges such as low stereo-selectivity, low product yield, and high production costs, large-scale production has not been achieved. Under the guidance of Academician Yin-Chu Shen, the “Father of Biopesticides in China”, our research group has conducted scientific research and industrial practice on the biosynthesis of L-PPT for over 20 years. In cooperation with multiple enterprises, we have developed more than ten process routes and technologies. Among them, five routes (racemic mixture derivatization-resolution route, racemic PPT-chiral separation route, generic compound cyanation followed by hydrolysis route, de novo synthesis from common chemicals route, and synthesis of homoserine followed by chemical synthesis) are discussed in detail in this review. Each route’s reconstruction, establishment of bioinorganic amine technology, creation of biocatalysts, high-density fermentation for enzyme production, product separation and purification, and reaction equipment are included. Notably, we developed the BioHPP^®, a biomanufacturing technology for the synthesis of highly optically pure L-PPT. Based on this technology, a ten-thousand-ton digital and intelligent production line for L-PPT was established. Utilizing smart sensors and actuators, real-time data collection, transmission, analysis, and feedback adjustment were achieved at over a thousand control points. This led to fully automated parameter collection and control, increasing production efficiency by 50% and reducing labor intensity by more than 70%, thereby realizing the bio-intelligent manufacturing of ten thousand tons of L-PPT. Based on the long-term accumulation of our research efforts, we summarize and analyze the mainstream production processes of D,L-PPT, detailing on the principles and methods of biomanufacturing technology and synthetic biology to construct the key synthesis system for L-PPT. We also compare the characteristics and key points of industrialization implementation of these routes in terms of substrate synthesis and selection, types of biocatalysts, use of amino donors, and separation and purification. It can be foreseeable that, with the aid of synthetic biology technology, an increasing number of high-optical-purity chiral pesticides will be produced on a large scale through biomanufacturing in the future.

Biomanufacturing is a green production that applies such bio-organisms as plants, animals, microorganisms, enzymes as well as in vitro synthetic enzymatic biosystems, to process and/or synthesize numerous value-added compounds, which would change the world′s future of industrial manufacturing in the energy, agricultural, chemical, and pharmaceutical industries. The competition of biomanufacturing is a key part of the battlefield of science and technology. Here we attempt to apply the ancient Chinese philosophy to provide enlightenment to the future development of industrial biomanufacturing. The ancient Chinese philosophy of “Tao-Fa-Shu-Qi” encompasses four key elements: “Tao is a way or direction, Fa is rules, Shu is techniques, and Qi is tools for accomplishing goals”. First, we define and explain the “Tao and Fa” of industrial biomanufacturing analyzes. Second, we analyze the limits and restriction set by Fa. Third, we expound this philosophy of “Tao and Fa” and how it guides way or choice of biomanufacturing type for the desired products. Based on “Tao-Fa-Shu-Qi”, we also present some predictions that a few hot products cannot be manufactured economically by seemingly-promising new techniques based on the limits and restriction of Fa. We take Amyris, a pioneering American company in synthetic biology as an example to analyze and discuss the important roles of “Tao and Fa” in the selection of biomanufactured products, far more important than “Shu and Qi”. Amyris’ failure was destined at its beginning because it went a wrong way (Tao) and ignored basic laws (Fa), although it exhibited advanced abilities of technologies and tools (“Shu and Qi”). Also, we briefly discuss opportunities and challenges of ensuring food security of China by using two disruptive technologies-making synthetic starch from lignocellulosic biomass and carbon dioxide catalyzed by in vitro synthetic enzymatic biosystems. In a word, the ancient Chinese philosophy “the way is simple, from top to down, the way guides techniques and tools” would provide top-level design methodology, identify the future research and development priorities in industrial biomanufacturing, and help effectively solve the major challenges, such as food security, dual carbon goals, and sustainable development.

As a class of enzymes widely distributed in nature, peroxidases are involved in important life processes such as innate immunity and epidemic prevention of organisms, anti-oxidative stress of plant microorganisms, fungal lignin degradation, plant cell wall metabolism and wound healing. With the rapid development of DNA sequencing, gene editing, recombinant protein expression and high-throughput screening technologies, more and more peroxidases have been discovered, characterized and recombinantly expressed. These peroxidases, characterized by their species diversity, abundant quantity, and excellent catalytic performance, have attracted extensive attention in many fields of application research. In recent years, remarkable progress has been made in the recombinant expression of peroxidases, further promoting their development in the field of applied research. Additionally, as we deepen our understanding of the catalytic properties of peroxidases, new opportunities have emerged for their application in the field of biosynthesis. Their high catalytic activity allows for rapid oxidation reactions under mild conditions and enables the construction of multi-enzyme cascade systems in conjunction with other enzymes, thereby facilitating the efficient synthesis of complex compounds. This paper provides a brief overview of peroxidases from the perspective of systematic evolutionary classification and function. It systematically reviews recent progress in the recombinant expression of peroxidases in Escherichia coli, yeast, and fungi, as well as their application achievements in environmental remediation and compound detection. The focus is on the latest research advances in the application of peroxidases for the biosynthesis of high-value-added compounds. The paper also discusses the current issues in this field, such as substrate and product non-specificity and the cytotoxicity of the cofactor H₂O₂. Peroxidases have enormous potential for applications in medical diagnostics, environmental protection, and biosynthesis. However, current technologies and applications still face several challenges, such as the stability and activity of peroxidases in complex environments, high production costs of enzyme preparations, and poor specificity. In the future, by integrating the latest advances in protein engineering, synthetic biology, and immobilization technology, these challenges can be effectively solved, promoting the widespread application of peroxidases across various fields.