Table of Content

    31 August 2023, Volume 4 Issue 4
    Invited Review
    Advances and applications of evolutionary analysis and big-data guided bioinformatics in natural product research
    Fanzhong ZHANG, Changjun XIANG, Lihan ZHANG
    2023, 4(4):  629-650.  doi:10.12211/2096-8280.2022-073
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    Nature has invented a myriad of natural products through billions of years of evolution. Natural products own unique structural features selected by evolutionary pressure and serve as a treasure trove for drug discovery. The rapid growth of microbial genomic data now provides new opportunities for evolutionary and big data analysis of biosynthetic gene clusters, which not only gives us a clearer picture about the global landscape of natural products, but also enables us to reveal the evolutionary trajectory of natural products. Such holistic understanding of natural products can facilitate the phylogeny-guided genome mining, allow better functional prediction of biosynthetic enzymes, and even open the door to biosynthetic redesign to create non-natural molecules by evolution-guided engineering. The core essence of evolutionary and large-scale bioinformatics lies in that it visualizes the entire sequence space and their distribution of a particular analyte family. Therefore, big data-driven bioinformatics has the potential to answer some challenging questions such as "How many natural products remain to be discovered?", and "How long can natural products discovery be sustainable?" This review summarizes recent advances in the application of evolution and big data-guided bioinformatics for natural products research from several perspectives including: ① natural product discovery; ② functional and structural prediction of biosynthetic enzymes and their products; ③ bioengineering, with the emphasis on the assembly line enzymes such as polyketide synthases and non-ribosomal peptide synthetases. Due to the modular domain architecture they have, the assembly line enzymes have been the main targets for genome mining. The phylogenetic analysis of their domains has shown to be a powerful and effective way to predict their enzymatic function and substrate specificity. Recently, the evolutionary mechanism of the assembly line enzymes has been investigated, and several evolution-guided engineering strategies were shown to have much higher efficiency for the assembly line reprogramming, providing a potential breakthrough for the bioproduction of complex polyketides and peptides. Non-modular enzymes are also discussed with selected representative examples. Finally, we present current challenges and future prospects of big data-driven natural products research. {L-End}

    Recent advances in photoenzymatic synthesis
    Yang MING, Bin CHEN, Xiaoqiang HUANG
    2023, 4(4):  651-675.  doi:10.12211/2096-8280.2022-056
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    Biocatalysis has the advantages in terms of sustainability, efficiency, selectivities and evolvability, thus it plays a more and more important role in green and sustainable synthesis, both in industrial production and academic research. However, compared with the well-known privileged chemocatalysts, enzymes suffer from the relatively limited types of reactions it can catalyze, which is unable to meet the future needs of green biomanufacturing. On the other hand, photocatalysis has emerged as one of the most effective strategies for the generation of reactive chemical intermediates under mild conditions, thereby providing a fertile testing ground for inventing new chemistry. However, the light-generated organic intermediates, including radicals, radical ions, ions, as well as excited states, are highly reactive resulting in the difficulties of controlling the chemo- and stereo-selectivities. The integration of biocatalysis and photocatalysis created a cross-disciplinary area, namely photoenzymatic catalysis, which can not only provide a new solution to stereochemical control of photochemical transformations with the exquisite and tunable active site of enzymes, but also open a new avenue to expand the reactivity of enzymes with visible-light-excitation. In addition, photoenzymatic catalysis inherits the inherent advantages of biocatalysis and photocatalysis, such as mild reaction conditions, representing green and sustainable synthetic methods. We have witnessed the booming development of photoenzymatic catalysis during the past several years. In this review paper, the recent advances in this field are highlighted. According to the cooperative modes of photocatalysis and enzymes, this paper is divided into following four parts: photoredox-enabled cofactor regeneration systems, cascade/cooperative reactions combining enzymes with photocatalysts, unnatural transformations with photoactivable oxidoreductase, and artificial photoenzymes. In this paper, we summarize the representative works and emphasize on the catalytic mechanisms of photoenzymatic transformations as well as the strategies for realizing abiological transformations. At the end of this review, by analyzing the challenges of photoenzymatic synthesis, the future directions are prospected. We hope that this review can inspire the discovery of more novel photoenzymatic systems and ultimately spur the applications of photoenzymes in industrial productions of high value-added enantiopure chiral products. {L-End}

    Application of phage therapy in the treatment of intracellular pathogens
    Kai WANG, Wan ZHANG, Yunhai HUANG, Lixin ZHANG, Chunbo LOU
    2023, 4(4):  676-689.  doi:10.12211/2096-8280.2023-002
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    Intracellular pathogens are a class of pathogens that can invade eukaryotic cells and survive in cells. After entering the host cell, they can regulate the intracellular environment so as to facilitate their own reproduction and spread, while the host's cell membrane and other structures will protect intracellular pathogens from being attacked by antibiotics, resulting in treatment failure. the increasingly serious drug resistance of intracellular pathogens makes the problem more difficult. It is necessary to explore bacteriostatic methods other than antibiotics, and phage therapy is a good choice. Bacteriophages have been used to treat bacterial infections as early as their discovery because they can effectively kill extracellular pathogens. However, phage therapy to deal with intracellular pathogens is still in the exploratory stage. In this review, we introduce the strategies of intracellular pathogens invading and settling in eukaryotic cells, as well as the mechanism of their resistance to antibiotics. It shows the unique bactericidal mechanism of phage therapy and its outstanding advantages, and the great potential of phage therapy in dealing with intracellular pathogens, especially drug-resistant pathogens. Of course, the application of phage therapy in the treatment of intracellular pathogens still faces many challenges, such as the fact that bacteriophages can not easily pass through the eukaryotic cell membrane to contact with intracellular pathogens. Finally, we discussed the possible development direction of bacteriophage therapy in the treatment of intracellular drug-resistant pathogens. We believe that first of all, we must adopt the "Trojan horse" strategy,cell-penetrating peptides modification or nanomaterial modification to make it possible for bacteriophages to enter eukaryotic cells efficiently. On this basis, the bacteriophage itself can be modified to obtain a recombinant bacteriophage with stronger bactericidal efficacy, and the bacteriophage resources, including mild bacteriophage, should be further explored. {L-End}

    Current developments in the use of engineered bacteria for cancer therapy
    Jiawen CHEN, Jiandong HUANG, Haitao SUN
    2023, 4(4):  690-702.  doi:10.12211/2096-8280.2022-062
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    Bacterial therapy has a long history, which originated at the end of the 19th century when bacteria were used for the first time to treat cancer. William B. Coley, a bone sarcoma surgeon, used heat-killed Streptococcus pyogenes to treat patients with unresectable sarcomas and found that the cancer had disappeared spontaneously. Over the next 40 years, he injected more than 1000 cancer patients with heat-killed bacteria Streptococcus pyogenes and Serratia marcescens, which are now known as Coley's toxins. However, owing to criticism from the medical community, rare reproducibility of the results and the emergence of radiotherapy and chemotherapy, bacterial therapy gradually disappeared from medical practice. With the development of immunology and synthetic biology, bacterial therapy has once again gained importance. Over the past 20 years, bacterial therapy has become a major focus area for research, and several types of bacteria have been used in clinical and preclinical studies. Therefore, bacterial therapy is considered an innovative and ideal strategy to treat cancer. In this review, we summarized the recent progress in the treatment of tumours with genetically engineered bacteria. Various bacteria include Salmonella, Escherichia coli, Bifidobacterium and Streptococcus pyogenes, which can accumulate, especially in tumours, and suppress their growth. However, in order to improve bacterial tumour-targeting ability and reduce bacterial virulence, various bacterial species have been genetically engineered through molecular biology techniques, thus forming different genetically engineered strains by using Clostridium novyi-NT, Listeria monocytogenes and Salmonella typhimurium. These genetically engineered bacteria can be selectively colonized in tumours and can inhibit cancer growth. In addition, they can also be used as live delivery carriers for cancer treatment, which can overcome the limitations of conventional antitumour therapies, such as high toxicity to normal tissue cells and the inability to treat deep tumour tissues. Potential targets including cytokines, cytotoxic agents, regulatory factors, prodrug-converting enzymes and small interfering RNAs (siRNAs) can be delivered via genetically engineered bacteria to treat cancer. However, various problems remain to be overcome before bacterial therapy can be used for clinical cancer treatment. The balance between bacterial virulence and anti-tumor ability is a key point, and more sophisticated genetic circuits need to be designed to modify the bacteria. While reducing the virulence of the bacteria, it is also important to enhance the ability of the bacteria to target to the tumor tissue to reduce the impact on other normal tissues. Genetic instability of bacteria is also a potential problem, as mutations may produce ineffective or deleterious phenotypes. However, with the development of synthetic biology, the above problems would be solved in the future, and bacterial therapy will be an approach with great potential for tumor treatment. {L-End}

    Application and prospect of CRISPR-Cas9 system in tumor biology
    Mengdan MA, Mengyu SHANG, Yuchen LIU
    2023, 4(4):  703-719.  doi:10.12211/2096-8280.2022-054
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    The RNA-directed CRISPR-Cas nuclease system was first discovered in bacteria as part of the adaptive immune system, and its ability to modify genetic components has led to a variety of practical applications, such as base editing, insertion or deletion of long segments, transcriptional regulation, and epigenetic modification. Because CRISPR-Cas gene editing tool is not only powerful, but also highly specific and efficient, it can accurately and rapidly screen the whole genome and facilitate gene therapy for specific diseases, so that it has been widely used in related research on the treatment of human diseases. The occurrence of tumor is the result of malignant degeneration of normal cells caused by the combined action of multiple factors, multiple stages and multiple mechanisms. CRISPR-Cas gene editing technology can accurately change genetic information at the DNA level and simulate the corresponding malignant characteristics of cells caused by the change of genetic information, thus becoming a molecular mechanism to explore the occurrence, development and metastasis of tumors. To investigate signaling pathways related to drug resistance in tumors and develop potential approaches to gene and cell therapy for tumor therapy. From early acquisition of oncogene mutations to metastatic colonization of distant tissues and the development of drug resistance, this continuous process leaves clear phylogenetic signatures at each step. Mapping detailed cancer cell lineages using the CRISPR gene-editing tool can reveal the dynamic processes behind the development and progression of cancer metastases, which will help track tumor development patterns. Despite its rapid development, CRISPR-Cas system still has limitations in its delivery efficiency, safety, and off-target effects in tumor therapy. The progress of CRISPR-Cas9 as a cancer research tool is reviewed. The research progress of this technique in establishing tumor model, studying the mechanism of tumor development, diagnosis, treatment and lineage tracking of tumor development was summarized. The development prospects of this technology in the new hot areas of cancer research and precision medicine in clinical medicine were discussed, and the technical challenges and future development directions were pointed out. {L-End}

    Base editing technology and its application in microbial synthetic biology
    Yannan WANG, Yuhui SUN
    2023, 4(4):  720-737.  doi:10.12211/2096-8280.2022-053
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    The discovery and development of the CRISPR/Cas system have a revolutionary influence on life sciences. A series of tools derived from the CRISPR/Cas system have brought great convenience to research in the field of life sciences. The base editors developed based on the CRISPR/Cas system are gene editing tools that can achieve base conversions and transversion on target. The base editors are constructed by fusing cytosine or adenosine deaminase, and other functional elements to Cas proteins with abolished double strand DNA cleavage activity to convert cytidine or adenine into other bases at genome on-target sites guided by sgRNAs. Base editors have shown great potential in biology, medicine and related fields. Although they have already been continuously optimized, there are still problems affecting further application of base editors. In this review, we briefly describe the development of DNA base editors. Furthermore, we introduce in detail the problems of the limited editing range of base editors as well as the corresponding optimization strategies by increasing the target sites recognized by the locator moiety and expanding or narrowing the editing window of the effector moiety. At the same time, we introduce several off-target editing detection methods specially developed for base editing. Based on usual and developed detection methods, multiple and frequent off-target editing caused by base editors were found at both DNA and RNA levels. We also introduced various effective optimization strategies to improve the editing specificity of the base editors in every respect. Most of these strategies are based on protein modification, but also on optimization of sgRNA and spatio-temporal regulation of base editing systems. These measures greatly enrich the application scenarios of the base editors. Then, we discuss the progress on applying base editors to the field of microbial synthetic biology, including revealing the metabolic pathway and synthesis mechanism of natural products as well as improving the production of target compounds in multiple species. Finally, we envisage the promising development of base editing in synthetic biology in the future. {L-End}

    Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi
    Jicong LIN, Gen ZOU, Hongmin LIU, Yongjun WEI
    2023, 4(4):  738-755.  doi:10.12211/2096-8280.2022-076
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    Filamentous fungi are the producers of antibiotics, pigments, enzymes, hormones, and other natural products, which are widely applied in the industries of medicine, chemical engineering, agriculture, and basic biology studies. The genetic background of filamentous fungi is complex, and molecular biology studies of filamentous fungi are difficult. Genome editing can cut specific sites of the genomic double-stranded DNA, to finish the insertion, deletion, or replacement of genomic information in vivo based on non-homologous end joining repair or homologous recombination repair. CRISPR system is the most widely used genome editing technology, which has been applied in genetic breeding, metabolic engineering, and the production of valuable natural products with filamentous fungi. The secondary metabolites of filamentous fungi, the gene editing principles, biopart design and expression, and delivery strategy of the CRISPR/Cas system were introduced, and the application of CRISPR/Cas system of filamentous fungi was summarized. Possible solutions to solve the problems of off-target effect and low conversion rates of gene editing were discussed. Besides, the application of the CRISPR/Cas system in the characterization of fungal gene function, natural product biosynthetic pathway recovery and rebuilding, construction of efficient filamentous fungi chassis cells, and natural product biosynthesis were also discussed. {L-End}

    Progress in metabolic engineering of microorganisms for the utilization of formate
    Zhenzhen CHENG, Jian ZHANG, Cong GAO, Liming LIU, Xiulai CHEN
    2023, 4(4):  756-778.  doi:10.12211/2096-8280.2023-032
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    One carbon resources are expected to become the next generation raw materials for the production of high value-added chemicals to achieve the recycling and utilization of carbon resources and promote the development of green industries. To achieve this aim, microbial utilization of formate produced from various one carbon resources, is one of the important strategies to build a green and sustainable platform for one-carbon biomanufacturing. However, there are many problems in the process of microbial utilization of formate, such as the low efficiency of formate utilization, the slow growth rate of formate-utilizing microorganisms, and the low yield of target metabolites and so on. To solve these problems, in this paper we systematically summarize and analyze the research progress in metabolic engineering of microorganisms for the utilization of formate from following three aspects: formate-utilizing microorganisms, metabolic pathways and metabolic engineering strategies. For formate-utilizing microorganisms, we summarize the metabolic characteristics and applicative advantages of natural formate-utilizing microorganisms, as well as the potential and advantages of model microorganisms for the application of metabolic engineering. For formate-utilizing metabolic pathways, we review the key steps, energy/reducing power consumption and characteristics of natural formate-utilizing pathways, reconstruction and optimization of formate-utilizing pathways and artificial formate-utilizing pathways, and then discuss the potential of metabolic engineering modification of these pathways. For formate-utilizing metabolic engineering strategies, we describe the useful strategies to improve the metabolic efficiency of formate assimilation pathways, such as optimizing the expression level of pathway genes, engineering key pathway enzymes, blocking the competitive pathways, reconstructing cofactor regeneration system, and modular pathway engineering, and then summarize the key approaches to improve the cell growth of formate-utilizing microorganisms, such as adaptive laboratory evolution, enhancing the cell growth of formatotrophs and improving the ability of microorganisms to synergistically utilize formate. Finally, we prospect the developmental direction of microbial utilization of formate from three aspects of formate-utilizing microorganisms, metabolic pathways and metabolic engineering strategies, which would lay the foundation for the development of formate bio-economy. {L-End}

    Rewiring and application of Yarrowia lipolytica chassis cell
    Meili SUN, Kaifeng WANG, Ran LU, Xiaojun JI
    2023, 4(4):  779-807.  doi:10.12211/2096-8280.2022-060
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    Engineering microbial chassis cells to efficiently synthesize high value-added products has received increasing attention. This biomanufacturing mode based on excellent performance microbial chassis cells has become the research frontier in the field of synthetic biology. Yarrowia lipolytica, an unconventional oleaginous yeast, is emerging as one of the popular microbial chassis cells in the field of advanced and green biomanufacturing. This is due to its unique physiological and biochemical characteristics, such as the inherent mevalonate pathway, adequate acetyl-CoA supply, broad substrate spectrum, and high tolerance to multiple extreme environments. These characteristics make Y. lipolytica a superior chassis candidate for the advanced and green biomanufacturing. In recent years, the researches and applications on the rewiring of Y. lipolytica chassis cell for biomanufacturing have gradually increased, which promoted the further upgrading of Y. lipolytica chassis cells. This review firstly describes the development of the genetic elements for rewiring Y. lipolytica chassis cell, including promoters, terminators, and selecting markers. Then, this review summarizes the expression modes and integration methods for endogenous and heterogenous genes, including gene expression based on episomal plasmid, genomic integration based on homologous recombination (HR) and non-homologous end joining (NHEJ). This review further summarizes the research progress of various synthetic biology tools developed for Y. lipolytica, including various gene overexpression methods, biosensor-based dynamic regulation strategies, CRISPR/Cas-based gene expression regulation methods, and the emerging strategies such as genome-scale metabolic modelling, genome-wide mutational screening, etc. This review also introduces the achievements of rewiring Y. lipolytica chassis cell for the synthesis of different high value-added products, including proteins, organic acids, terpenes, functional sugars and sugar alcohols, fatty acids and their derivatives, flavonoids and polyketides, and amino acid derivatives. In addition, the prospects of Y. lipolytica chassis cell-based biomanufacturing are discussed in light of the current progresses, challenges, and trends in this field. Finally, guidelines for building next-generation Y. lipolytica chassis cell for production of the aforementioned products are also emphasized. {L-End}

    Research progress on recombinant collagen expression system
    Jiahao PAN, Weisong PAN, Jian QIU, Donling XIE, Qi ZOU, Chuan WU
    2023, 4(4):  808-823.  doi:10.12211/2096-8280.2023-020
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    Collagen is the most abundant protein in mammals, and its production has been widely used in biomedicine, cosmetics, leather, biotechnology, etc. At present, collagen is generally divided into animal collagen and recombinant collagen. Although animal collagen is the main source of collagen, most of it comes from animal carcasses, and its collagen has been cross-linked and embedded in native tissues, which is more demanding on extraction and purification technology. In addition, pathogen contamination and allergy risks have become unavoidable problems for animal collagen. Recombinant collagen is a protein obtained by using human collagen cDNA fragments as the backbone gene, cloning the gene to the selected expression vector and converting it into an expression cell, and finally achieved by purification technology. Due to its single molecule, clear structure and easy control, recombinant collagen is the best alternative to replace animal collagen in biomedicine and tissue engineering. In this paper, the structure, category, biosynthesis mechanism and market scale of collagen are briefly described. Emphasis is placed on the construction strategies, advantages and limitations of different expression systems of recombinant collagen, including prokaryotic, yeast, plant, baculovirus and mammalian or human cell expression systems. Prokaryotes and yeast have a short cycle of producing recombinant collagen, but do not have a triple helix structure. The plant expression system produces recombinant collagen with a moderate cycle and a certain triple helix structure. The baculovirus-insect expression system and the mammalian expression system have a long cycle of recombinant collagen production and a complete triple helix structure. The practical application of recombinant collagen in ophthalmology, cartilage engineering, skin treatment and other biological medicine is described. Currently, the most commercially valuable use of collagen is subcutaneous injection of soluble protein to repair damaged skin. At the same time, collagen, as the main component of animal skin, can cross-link collagen in raw hides through chemical processes such as tanning, so that collagen becomes harder, more durable, and corrosion-resistant leather. By designing collagen scaffolds that are familiar with the natural cytoplasmic matrix environment, it can effectively reveal the pathogenesis of cell behavior and disease etiology. It is expected to provide suggestions on the research of recombinant collagen and future industrial development. {L-End}

    Research Article
    Development of CRISPRa for metabolic engineering applications in cyanobacteria
    Tiantian WANG, Hong ZHU, Chen YANG
    2023, 4(4):  824-839.  doi:10.12211/2096-8280.2022-077
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    Cyanobacteria can be used as a model for photosynthesis research and as a chassis for the production of fuels and chemicals from light energy and CO2. However, the genetic tools of cyanobacteria are still relatively limited. Development of efficient tools for programming gene expression is important for cyanobacterial systems and synthetic biology. Here, we developed a CRISPR transcriptional activation system (CRISPRa) for programming heterologous gene expression in a model cyanobacterium Synechococcus elongatus PCC 7942. Among the transcriptional activators tested, endogenous RNA polymerase ω-subunit RpoZ resulted in optimal performance and was chosen for subsequent studies. We established CRISPRa by fusing dCas9 that lost DNA cleavage activity with RpoZ, and expressed single guide RNAs (sgRNAs) under a strongpromoter. We further improved heterologous reporter gene expression by deleting the rpoZ gene in S. elongatus PCC 7942, enhancing the expression of dCas9-RpoZ fusion, and optimizing the sgRNA targeting sites. Using this optimized CRISPRa system, we engineered S. elongatus PCC 7942 for improved production of isopentenol, an ideal biofuel candidate. Furthermore, we demonstrated that this system was able to simultaneously activate multiple genes of the biosynthetic pathway and repress a gene of the competing pathway, thereby increasing the isopentenol production by 17 times. Thus, this CRISPRa system could serve as a powerful tool for the construction of photoautotrophic cell factories. {L-End}

    Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases
    Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI
    2023, 4(4):  840-851.  doi:10.12211/2096-8280.2022-048
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    Restriction endonucleases are a large family of endonucleases characterized with high specificity of DNA sequence recognition and cleavage catalysis, and are vital tools extensively applied in biology studies. Restriction endonucleases are usually produced through heterologous co-expression with the methylases to protect the hosts from cytotoxicity. The recently developed cell-free protein synthesis (CFPS) technology is attractive as the advantages of easy manipulation, high efficiency and lower cytotoxicity. In this study, we applied CFPS technology to produce restriction endonucleases. We first constructed the linear DNA template (LDT) of restriction endonucleases and conducted protein expression in E. coli-based cell-free reactions without methylases protection for 6 hours. Then, restriction endonucleases were isolated through two steps purification of affinity chromatography and gel chromatography. By using this strategy, three restriction endonucleases EcoRⅠ, BamHⅠ,and BsaⅠ were successfully synthesized and the specific DNA cleavage activities were tested (EcoRⅠ 3.7 × 105 ~ 3.7 × 106 U/mg, BamHⅠ8.3 × 102 ~ 4.1 × 103 U/mg, and BsaⅠ 4.4 × 105 ~ 4.4 × 106 U/mg). Furthermore, we developed a real-time catalytic activity detection method, which will facilitate the study on catalytic mechanism and screening of restriction endonucleases. Our CFPS-based restriction endonucleases production system established in this study exhibits advantageous properties such as time-efficient (1~2 days for the overall process), high protein yield (32.5~130 mg/L cell-free reaction), and high catalytic activity (1.3 × 105 ~ 5.7 × 108 U/L cell-free reaction), which will provide new insights to the discovery, engineering, and production of restriction endonucleases in the future. {L-End}