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Table of Content

    28 February 2025, Volume 6 Issue 1
    Contents in Chinese and English
    2025, 6(1):  0. 
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    Perspective
    Exploring industrial biomanufacturing and the goal of “carbon neutrality”
    YING Hanjie, LIU Dong, WANG Zhenyu, SHEN Tao, ZHUANG Wei, ZHU Chenjie
    2025, 6(1):  1-7.  doi:10.12211/2096-8280.2023-075
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    Non-renewable resources, such as petrochemicals, have made great contributions to modern civilization. However, the extensive use of fossil fuels, which have been buried for hundreds of millions of years, has led to a substantial increase in carbon imbalance. The imbalance leads to severe ecological and environmental problems. Industrial biomanufacturing, often referred to as a “sunshine economy”, represents a novel sustainable production paradigm, utilizing renewable resources in a carbon-cycling mode. This paper discusses several ways in which biomanufacturing can support China’s “carbon peaking and carbon neutrality” goals from the perspectives of manufacturing feedstock, production mode and product usage. Biomanufacturing can reduce carbon emissions through feedstock substitution, technology iteration and product replacement. Utilization of straw biomass, producing non-food proteins and establishing a biobased industry landscape are important approaches for reducing carbon emissions in biomanufacturing. Efficient biomanufacturing of food and natural products can substantially improve production efficiency, conserve significant land resources, and thus provide land resources for “carbon replacement”. Optimizing agricultural products through biotechnology advancements and innovative product development is a crucial way to reduce pollution but also enhance the carbon sink capacity of the agricultural sector.

    Price to Cost-of-raw-materials Ratio (PC) of biomanufacturing: definition and application
    ZHANG Yi-Heng P. Job, CHEN Xuemei, SHI Ting
    2025, 6(1):  8-17.  doi:10.12211/2096-8280.2024-072
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    Biomanufacturing is one of the strategic emerging industries in China during the “14th Five-Year Plan” period. The author once proposed “Tao-Fa-Shu-Qi” for the industrial biomanufacturing and provided its philosophical guideline. Focusing on the “Fa” of biological manufacturing and further analyzing the concept of “Fa as rules” in biomanufacturing, the author first proposed the concept of “Price to Cost-of-raw-materials Ratio” (PC value), which is the ratio of product market price to cost with raw materials. Biomanufactured products can be categorized by PC value into high-value products, value-added products, biocommodity, and products for public good. The PC value is a key indicator for evaluating the technological capability and economic viability of biomanufactured products. It is simple, transparent, and publicly accessible, offering a new approach for categorizing biomanufactured products. This indicator aids in guiding new technologies towards pathways of efficiency enhancement and cost reduction, forecasting future manufacturing costs and market prices for bioproducts, and assessing the industrialization potential of emerging biotechnologies. This article focuses on the biomanufacturing of fructose syrup, fructose solution, crystalline fructose, allulose, myo-inositol, and tagatose as examples, analyzing pathways for developing new technologies and predicting their economic feasibility. The calculation and analysis of the PC value could provide a new methodological tool for the top-level strategic design of the future development of emerging biomanufacturing industries, and could effectively facilitate the high-quality development of the bioeconomy.

    Invited Review
    Advances in microbial production of liquid biofuels
    GUO Shuyuan, ZHANG Qiannan, Gulikezi· MAIMAITIREXIATI, YANG Yiqun, YU Tao
    2025, 6(1):  18-44.  doi:10.12211/2096-8280.2023-040
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    With the socioeconomic development, the dependence of human beings on fossil fuels has led to their shortage and climate change. This has created an urgent need for alternatives that are renewable and environmentally friendly, and biofuels are one of them. Nowadays, widely recognized biofuels like fuel ethanol and biodiesel face challenges in terms of their production capacity due to limitation on raw materials such as grains and edible oils and high cost as well. Hence, the integration of metabolic engineering and synthetic biology has opened avenues for utilizing diverse substrates from other renewable sources, such as solar energy, light energy, electric energy, and waste biomass. Microbial cell factories, including microalgae, bacteria, and yeast, play a crucial role in synthesizing biofuels. The review comments on the evolution of the four generations of biofuels, encompassing fuel ethanol, biodiesel, bio-gasoline, jet and aviation fuels. We also discuss how microorganisms can be explored for producing the third- and fourth-generation biofuels from a variety of unconventional substrates such as carbon dioxide, methanol, and methane, multi-energy coupling to synthesize biofuels from lignocellulose by bacterial or yeast, CO2 conversion by microalgae or electrochemical-biological systems, the conversion of methanol and methane by methyltrophic microbes, and the application of synthetic biology. Furthermore, we overview biosynthetic pathways and engineering strategies for optimizing biofuels production. These strategies can convert raw materials to various fuel products, including fatty acids and esters, advanced alcohols and esters, isoprenoids, and polyketides. Finally, we highlight some challenges in biofuels production, including raw material supply and cost issue, low production yield, and limited product variety. Meanwhile, to address these challenges, we propose corresponding solutions. For example, by optimizing carbon fixation pathways, and converting carbon dioxide into low-carbon substrates like methanol, autotrophic microorganisms, methylotrophic microorganisms, and other cell factories can utilize carbon dioxide as the major raw material to synthesize various biofuels, which can benefit the application of biofuels and further promote their industrial production.

    Synthetic genetic circuit engineering: principles, advances and prospects
    GAO Ge, BIAN Qi, WANG Baojun
    2025, 6(1):  45-64.  doi:10.12211/2096-8280.2023-096
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    Synthetic genetic circuits are engineered gene networks comprised of redesigned genetic parts for interacting to perform customized functions in cells. With the rapid development of synthetic biology, synthetic genetic circuits have shown significant application potentials in many fields such as biomanufacturing, healthcare and environmental monitoring. However, the efforts to scale up genetic circuits are hindered by the limited number of orthogonal parts, the difficulty of functionally composing large-scale circuits, and the poor predictability of circuit behaviors. A longstanding goal of synthetic biology research is to engineer complex synthetic biological circuits, using modular genetic parts, as we do with electronic circuits. Synthetic biologists have developed various genetic toolboxes and functional assembly methods over the past few decades. Here we present an overview of the latest advances, challenges, and future prospects in genetic circuit engineering from four aspects corresponding to the four key engineering principles for circuit design, i.e. orthogonality, standardization, modularity, and automation. Firstly, the design and construction of orthogonal genetic part libraries are discussed in both prokaryotes and eukaryotes at the levels of DNA replication, transcription, and translation, respectively. Standardized characterization methods and the design of modular genetic parts are subsequently summarized. Furthermore, progress in developing modular genetic circuits are presented, providing new concepts and ways for engineering increasingly large and complex circuits. Finally, how to achieve automated design and building of genetic circuits are addressed from the advances in software, hardware and artificial intelligence, respectively, with an aim to replacing the presently time-consuming manual trial-and-error mode with the iterative "design-build-test-learn" cycle for improved efficiency and predictability of circuit design. The integration of these fundamental principles and the latest advances in information technology such as artificial intelligence and lab automation will accelerate the paradigm shift in genetic circuit engineering and synthetic biology research, making it feasible for designing synthetic lives to meet various customized needs.

    Applications of protein engineering in pharmaceutical industry
    WEN Yanhua, LIU Hedong, CAO Chunlai, WU Ruibo
    2025, 6(1):  65-86.  doi:10.12211/2096-8280.2024-061
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    Protein engineering performs specific designs and modifications on proteins through directed evolution, semi-rational or rational design, computer-assisted design, and so on. The engineered proteins, with improved properties, have significant applications in food, medicine, fuel, and material industries. For the chemical and pharmaceutical industry, engineered enzymes can serve as efficient biocatalysts for the synthesis of active pharmaceutical ingredients (API) and their intermediates, aligning with the concepts and principles of green chemistry and manufacturing. For the biopharmaceutical industry, the engineering of peptide or protein modifying enzymes can boost the efficiency in preparing drug candidates, while engineered diagnostic enzymes can make detection more accurate and sensitive. Moreover, protein engineering can improve the bioactivities of biological drugs such as therapeutic enzymes and antibodies, increase stability, and mitigate immunogenic response for their safety and efficacy. Here, we review the tremendous progress in protein engineering, elucidate its importance in the research and development of chemically derived drugs and biologics, and provide examples of its applications. These examples encompass the discovery of enzymes or antibodies, the process of protein engineering, and the subsequent economic advantages. We aim to showcase the practical implementation of protein engineering in the pharmaceutical industry and facilitate technology transfer, thereby fostering seamless integration between research, development, and industrial production. Furthermore, we discuss challenges such as cost-effectiveness and market changes in the synthesis of API, and multi-target optimization, long cycle and high risk in the discovery and development of biopharmaceuticals. Finally, we look forward to the prospects of protein engineering in pharmaceutical industry. In the future, automated pipelines consisting artificial intelligence and self-driving laboratories will accelerate the design-build-test-learn cycle, leading to rapid progress in molecular design and discovery.

    Research on the application of optogenetic tools in learning and memory
    ZHENG Yikun, ZHENG Jie, HU Guopeng
    2025, 6(1):  87-104.  doi:10.12211/2096-8280.2024-042
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    Optogenetics represents an advanced technology that facilitates precise control of gene expression and neuronal activity in living cells through light. Introduced by neuroscientist K. Deisseroth in 2005, this methodology has transformed neuroscience research, empowering researchers to modulate excitable tissues and neural circuits with exceptional spatiotemporal accuracy. Optogenetics necessitates the expression of light-sensitive proteins, including channelrhodopsins, halorhodopsins, and various microbial opsins, within specific cells. Employing viral vectors and tissue-specific promoters, these proteins ensure targeted expression. Exposure to designated wavelengths of light permits these proteins to activate or inhibit cellular activity, thereby modulating neuronal behavior. The implementation of optogenetics has significantly enhanced comprehension of learning, memory, and neural plasticity. This technology enables the examination of the molecular dynamics associated with synaptic plasticity, long-term potentiation (LTP), and long-term depression (LTD), which are pivotal for memory. Real-time manipulating of specific neuronal populations can elucidate the intricate neural circuits involved in these phenomena. Additionally, optogenetics has facilitated the exploration of potential therapeutic approaches for neurological conditions such as Alzheimer’s disease by meticulously controlling memory-associated circuits. The utility of optogenetics transcends fundamental research, yielding promising prospects in addiction to studies and motor function enhancement. By modulating distinct neural circuits, it is possible to alter addiction-related behaviors and augment motor functions. Furthermore, the amalgamation of optogenetics with cutting-edge technologies like artificial intelligence and deep learning is anticipated to refine stimulation protocols, resulting in more precise and efficacious experimental outcomes. Notwithstanding its transformative capacity, the clinical application of optogenetics encounters significant obstacles, including the requisites for safe and effective gene delivery systems and the formulation of light-sensitive proteins with optimal characteristics for applications in human beings. Future investigations should concentrate on surmounting these hurdles while expanding the applications of optogenetics in neuroscience and related fields. The integration of optogenetics with multidisciplinary approaches is poised to unveil new realms in brain research, yielding profound insights into mechanisms governing memory, learning, and neural plasticity.

    Progress in the miniaturization of CRISPR-Cas systems
    DONG Ying, MA Mengdan, HUANG Weiren
    2025, 6(1):  105-117.  doi:10.12211/2096-8280.2023-068
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    The CRISPR-Cas gene editing technology has revolutionized the fields of biology, medicine, agronomy, etc. due to its simplicity and efficiency. Laboratory-developed tools, such as the widely recognized CRISPR-Cas9, have played a pivotal role in addressing a multitude of genetic diseases. By harnessing the targeted nucleic acid capabilities of the CRISPR-Cas system, researchers have successfully integrated various functionalities into Cas proteins, including fluorescent markers, transcriptional regulatory proteins, and base editing components. This has unlocked new possibilities, including chromosome imaging, transcriptional regulation, and precise base editing. Currently, Cas nucleases with large molecular weights, often exceeding 1000 amino acids, are commonly used. However, adeno-associated virus (AAV) vectors, which are extensively employed in gene therapy, have limited capacity to accommodate additional functional components beyond the coding sequences of CRISPR nucleases and guide RNAs (gRNAs). This limitation severely constrains their utilization in gene therapy and other applications. As a result, a significant focus of research has been placed on the miniaturization of CRISPR tools, making them compact enough to align with current delivery methods. Compact Cas protein variants within CRISPR-Cas systems hold the potential to create and deliver genome editing and regulatory tools into human cells using AAV. Hence, the development of miniaturized CRISPR-Cas systems presents a crucial avenue for addressing this technical challenge. This article provides a comprehensive review of research progress in miniaturizing key proteins within two classes of Cas systems: Cas9 and Cas12 for targeting DNA, and Cas13 for targeting RNA. This review encompasses the screening of novel Cas proteins, the reduction of protein structural domains, and the modification of guide RNAs, all with the intention of presenting innovative ideas for the further advancement of compact, precise gene editing, and regulatory tools. The miniaturization of CRISPR-Cas systems is a critical step toward unlocking their full potential in various fields, including biomedicine, agriculture, and basic research. As researchers continue to explore and refine these compact gene editing and regulatory tools, we can expect significant advancement in understanding and manipulating genetic information. This ongoing progress promises to have a profound impact on the future of science and technology. At present, the limitations of the miniaturized CRISPR-Cas system are mainly with the size of protein molecular weight and the efficiency and specificity of gene editing. If we can solve these problems and obtain a smaller structure in future research, not only can we optimize the transmission of the system in the body, but also develop high-efficiency and low-damage treatment methods for clinic applications.

    Research progress in the production of α-arbutin through biosynthesis
    ZHONG Quanzhou, SHAN Yiyi, PEI Qingyun, JIN Yanyun, WANG Yihan, MENG Luyuan, WANG Xinyun, ZHANG Yuxin, LIU Kunyuan, WANG Huizhong, FENG Shangguo
    2025, 6(1):  118-135.  doi:10.12211/2096-8280.2024-054
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    Arbutins are a kind of natural glycoside compounds found widely in nature. α-arbutin, one of its isomers, has received increasing market attention due to its efficient and safe whitening effect and other excellent pharmacological effects. Studies have revealed that the production methods of α-arbutin mainly fall into three categories: plant extraction, chemical synthesis, and biosynthesis. For the plant extraction, raw materials are widely available, and the process is simple, but the yield fails to meet the requirement for large scale production and applications. The chemical synthesis has a higher yield but with harsh reaction conditions, and thus is not environmentally friendly. Through research has found that the biosynthesis of α-arbutin has higher yield, safer environment, more competitive cost and other advantages compared with the natural extraction and chemical synthesis as well, making it the mainstream production method. This article discusses the advantages and disadvantages of different synthetic methods and studies on the seven enzymes commonly used in the biosynthesis of α-arbutin including α-amylase, sucrose phosphorylase, cyclodextrin glycosyltransferase, α-glucosylase, dextransucrase, amylosucrase, and sucrose isomerase. These enzymes use different sugar donors and catalyze the transglycosylation reaction with hydroquinone as the receptor substrate to synthesize α-arbutin. Additionally, we provide a comprehensive review on research progress in the whole-cell catalysis and microbial fermentation to produce α-arbutin, and potentials for its industrial production are assessed. Furthermore, we highlight challenges that exist in the biosynthesis of α-arbutin, such as the oxidation of hydroquinone during synthesis that increases cell toxicity and reduces the yield, the low utilization rate of glucose and the generation of other glycoside products, and the poor performance of experimental strains, and corresponding solutions are proposed. Finally, future directions for α-arbutin synthesis are prospected, with the aim of providing new ideas for achieving more efficient and lower-cost production of α-arbutin and enhancing its applications in the fields of cosmetics and medicines.

    Efficient biosynthesis of glucoraphanin in Brassicaceae crops by genetic engineering
    LIU Xiaoyue, WANG Pandi, WU Gang, LIU Fang
    2025, 6(1):  136-156.  doi:10.12211/2096-8280.2024-031
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    Glucoraphanin (GRA), a secondary metabolite of plants, is a glucosinolate (GSL) derived from methionine. It is relatively stable in nature, and both GRA and its degradation product sulforaphane (SFN) play important roles in anticancer, neuroprotection, and other broad biological functions and health-benefits, and in particular, SFN has been reported as the best natural product for anticancer. In this article, we review the physicochemical properties, sources, biological functions, synthetic pathways, current production status of GRA, and discuss the potential strategy for the efficient biological synthesis of GRA in the future. The synthesis pathway of GRA involves three stages: side chain elongation, core structure information, and side chain modification. GRA can be converted into SFN and other active compounds by plant myrosinase (MYR) and intestinal microorganisms. Brassicaceae crops such as broccoli have high levels of GRA, and are currently the main source of GRA. However, the cultivation cycle of GRA-rich plants is long, and its extraction yield is low. Therefore, the development of economical and renewable new resources of GRA will greatly advance its applications. With the elucidation of the biosynthesis and regulation pathways of GRA, its genetic engineering-assisted efficient biological synthesis shows great potential, suggesting that the possibility for developing strategies with the manipulation of multiple genes for regulated expression at different dimensions to synthesize GRA more efficiently compared to the current mainstream strategy through manipulating single genes. This review focuses on the genetic engineering-assisted efficient biosynthesis of GRA in Brassicaceae crops, systematically outlining potential genes for engineering at each stage of GRA synthesis and highlights chassis crop species from the perspective of enrichment organs, aiming to providing ideas and strategies for the future regulation of GRA biosynthesis in plants through transgenic technology and molecular breeding for large-scale sustainable production of GRA.

    Key technologies for DNA storage: encoding, error correction, random access, and security
    XU Huaisheng, SHI Xiaolong, LIU Xiaoguang, XU Miaomiao
    2025, 6(1):  157-176.  doi:10.12211/2096-8280.2024-066
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    DNA information storage is a new technology that uses DNA molecules as data carriers. It encodes information for synthesizing DNA with a specific sequence and reads out data through sequencing technology. Compared with traditional magnetic, optical, and electronic storage media, DNA storage has significant advantages in data density, retention duration, energy efficiency, and security, since it is not easily affected by electromagnetic interference. With the rapid increase in the total amount of global data, DNA storage has gradually become a research hotspot with its efficient storage capacity, low maintenance cost, and unique chemical property for synthesizing easily. However, DNA storage technology is still in its early stages of development and there are still many technical bottlenecks to be addressed. For example, an important advantage of DNA storage is its ultra-high storage density and long-term stability. However, achieving these goals require overcoming many technical challenges, such as reducing the error rate for synthesis and improving the encoding efficiency. Understanding existing key technologies, such as DNA encoding, error correction, random access, and DNA information encryption, can help identify and address those shortcomings, thereby promoting further technological innovation and development in DNA storage. Encoding strategy is one of the core aspects of DNA storage technology, directly determining data storage efficiency, reading accuracy, and error correction capability. To achieve efficient and stable DNA information storage, it is essential to develop more advanced encoding algorithms to enhance storage density, reduce synthesis and sequencing error rates, and ensure data accuracy and integrity. Moreover, the information security of DNA storage is becoming increasingly important, particularly in terms of data and privacy protection. As a potential data carrier, DNA storage needs to address challenges related to data encryption, information security, and tamper-proof to ensure data confidentiality and integrity. Therefore, integrating modern cryptographic techniques with DNA storage to establish a secure and reliable information storage system has become a key research focus in this field. This article first introduces the basic process of DNA storage, and then reviews the key technologies involved in DNA information storage, especially the research progress of encoding strategies, error correction technology, random access and DNA information encryption. In addition, the current development status and main challenges of DNA storage technology are also discussed. For example, the scale of DNA data storage in the laboratory is small, and the operation time for synthesis is long. Moreover, most DNA storage steps rely on experimenters, making it difficult to automate the information storage and reading process. With the advancement of synthetic biology and encoding and decoding methods, we believe that these bottlenecks will be solved in the near future, and promote the transformation of technology from laboratory research to practical applications.

    Legal issues for the storage of DNA data
    JIAO Hongtao, QI Meng, SHAO Bin, JIANG Jinsong
    2025, 6(1):  177-189.  doi:10.12211/2096-8280.2024-033
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    DNA data storage is a key for the development and application of synthetic biology. With the advent of high density storage technology for long period and more stable and secure data storage, the shortage of storage capacity caused by the explosive growth of data can be addressed. Thus, exploring the legal issues for the storage of DNA data is more important than ever for ethics with the emerging science and technology. At the human rights level, human dignity and privacy need to be protected, and the data gap between human beings available and unavailable to the data to be minimized, through the development of complete policy and legal systems for DNA data storage. At the security level, it is suggested to strengthen the security governance of DNA data storage technology by combining information security and biosafety issue. At the level of intellectual property, it is suggested to improve patent protection, and optimize the legal environment of intellectual property, so as to promote the technological innovation and application of DNA data storage technology.

    Two hypothesises for the origins of organisms from the synthetic biology perspective
    LI Jiyuan, WU Guosheng
    2025, 6(1):  190-202.  doi:10.12211/2096-8280.2024-035
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    The inquiry into the essence of organisms has long been a thriving topic in biology and philosophy. Hypothesises are commonly employed in biological research to understand lives. These hypothesises can be grouped into two categories: ① the machine hypothesis, likening the components and organizational structure of organisms to the operation of machines, and ② the autopoietic hypothesis, likening organisms to complex systems with purposeful and unique attributes. Both play an epistemic role in various fields of biology, serving as theoretical hypotheses, heuristic tools, and means of scientific communication. The machine hypothesis, for instance, has been influential in areas such as molecular biology and systems biology, where organisms are viewed as intricate machines made up of interacting components. The autopoietic hypothesis, on the other hand, has been more prominent in theoretical biology and philosophy of biology, highlighting the self-organizing and self-producing nature of living systems. The development of synthetic biology, which aims to redesigning and constructing biological systems from scratch, has challenged the traditional dichotomy between natural and artificial entities. Both the machine and autopoietic hypothesises are reflected in the advancement of synthetic biology, as researchers attempt to engineer living systems using principles and methods adapted from various disciplines, including engineering, computer science, and materials science. While the hypothesises serve epistemic purposes, their usage also raises some controversies, particularly in the context of synthetic biology. The conflation of ontology and epistemology, where hypothesises are mistaken for literal descriptions of reality, can lead to ethical concerns. For example, the machine hypothesis may suggest that organisms are merely complicated machines to be manipulated, potentially diminishing their intrinsic value and ethical status. This article examines the origin and clarification of these two hypothesises, their applications in synthetic biology, and addresses the potential confusions and ethical implications arising from their usage. It advocates for a cautious approach to the usage of the epistemological hypothesis, considering both its epistemic impact and ethical consequence. As synthetic biology continues to advance, it is crucial to maintain a critical and nuanced understanding of hypothesises employed, recognizing their heuristic value while also acknowledging their limitations and potential pitfalls. The discussion of hypothesises for organism origins in the context of synthetic biology highlights the importance of interdisciplinary collaboration and dialogue between scientists, philosophers, and ethicists. By examining the philosophical and ethical issues of hypothesises, we can better navigate the complex and rapidly evolving landscape of synthetic biology, ensuring that our scientific endeavors are guided by a deep appreciation for the intricate and multifaceted nature of lives.

    Philosophical reflections on synthetic biology in the Anthropocene
    TANG Xinghua, LU Qianneng, HU Yilin
    2025, 6(1):  203-212.  doi:10.12211/2096-8280.2024-039
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    Synthetic biology, as a science transforming life science in the 21st century, is interdisciplinary in nature, breaking boundaries, emphasizing human roles, and shaping our way of living through technology, presenting common challenges in the Anthropocene era we live in. As a technology of the Anthropocene, synthetic biology blurs the line between nature and artificiality, merging the two and demonstrating the profound impact of technology on life itself. Synthetic life not only obscures the boundary between nature and artificiality but also transcends traditional disciplinary divisions, becoming a research object across various fields, thereby promoting interdisciplinary collaboration and integration. In this process, the openness of synthetic life and the generative nature of synthetic biology determine its future-oriented characteristics, altering the direction of technological research in the Anthropocene. Finally, the “big questions” and “small questions” that the philosophy of technology concerns about are unified in synthetic biology. Synthetic biology encompasses both general issues and its specific developments and applications. In summary, philosophical reflections on synthetic biology as a technological platform in the Anthropocene contribute to a deeper understanding of synthetic biology within a new theoretical framework.

    Research Article
    Effect of terminators on the downstream transcript unit with gene expression in Escherichiacoli
    REN Jiawei, ZHANG Jinpeng, XU Guoqiang, ZHANG Xiaomei, XU Zhenghong, ZHANG Xiaojuan
    2025, 6(1):  213-227.  doi:10.12211/2096-8280.2024-046
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    During gene transcription, RNA polymerase initiates the process by recognizing the promoter sequence, and terminates it upon recognizing the terminator sequence located at the 3′-UTR, leading to dissociation of the transcription complex. Therefore, promoters and terminators within the transcription unit play the role of initiating and terminating transcription, respectively. For downstream transcription units, in addition to the direct effect of terminating transcript read-through, the dissociation of the RNA polymerase from the terminator may affect the binding of the promoter to RNA polymerase in the subsequent transcription unit, thus indirectly altering the expression of the downstream transcription unit. This interplay between terminators and promoters across transcription units remains poorly understood, therefore, elucidating the impact of terminators on the transcriptional strength of downstream transcription units is of great significance for the precise regulation of gene expression and the development of efficient terminators. In this study, a library containing 405 different combinatorial elements (terminator-spacer-promoter) was constructed by combining nine terminators, five spacer sequences, and nine promoters using one-pot assembly technology. All combinations in the library were sequenced and analyzed in terms of fluorescence intensity based on the FlowSeq technology to establish the correlations between combinatorial sequences and downstream gene expression. The results showed that combinations of weak terminators, short spacers, and strong terminators were more favorable to enhance the expression of downstream genes, while combinations of strong terminators, long spacers, and weak terminators reduced the expression of downstream genes. Quantitative analysis of transcription revealed that weak terminators not only enhanced downstream leakage transcription (21~70-fold enhancement), but also facilitated downstream promoters to re-recruit RNA polymerase for re-promoted transcription (2~3-fold enhancement). This study has elucidated the effect and mechanism of terminators on the regulation of gene expression in the downstream transcription units, providing a design framework for the construction of gene circuits using terminators.