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.

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.

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.

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.

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.

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.

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.

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.

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.

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”.