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Significant research progress in synthetic biology DING Mingzhu, LI Bingzhi, WANG Ying, XIE Zexiong, LIU Duo, YUAN Yingjin Synthetic Biology Journal    2020, 1 (1): 7-28.   DOI: 10.12211/2096-8280.2020-057 Abstract （8086）   HTML （1484）    PDF（pc） （3953KB）（10987）       Knowledge map    Save As an emerging, interdisciplinary field, synthetic biology has made great advances in many directions due to wide acceptance of its core principles and rapid progress in DNA synthesis. In this paper, recent development in gene circuits, genome design and synthesis, cell factories, and synthetic microbial consortia is reviewed. The complexity of artificial gene circuits that can be designed and constructed is gradually increasing with more refined control. Synthetic genomes are routinely assembled, expanding from prokaryotes (Mycoplasma to Escherichia coli) to eukaryotes (Saccharomyces cerevisiae), and improved capacity in genome design promotes the research of biological evolution. Metabolic pathways of ever-increasing lengths are constructed based on modularization and orthogonality principles to produce molecules of complex structures, and fundamental rewiring of cellular metabolism is performed for enhanced robustness and compatibility. The design and construction of synthetic microbial consortia have been expanded from two-species systems to multi-species systems, so that more sophisticated functions can be achieved. At the end of this paper, new research directions resulted from the interdisciplinary integration of synthetic biology and other disciplines are discussed. Table and Figures | Reference | Related Articles | Metrics

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Microbial promoter engineering strategies in synthetic biology YU Huimin, ZHENG Yukun, DU Yan, WANG Miaomiao, LIANG Youxiang Synthetic Biology Journal    2021, 2 (4): 598-611.   DOI: 10.12211/2096-8280.2020-092 Abstract （6323）   HTML （689）    PDF（pc） （1858KB）（7866）       Knowledge map    Save Synthetic biology is of vital importance to the green biomanufacturing industry and sustainable development strategies of our country. Promoter is the core-component of synthetic biology, playing a significant role in highly efficient and fine-tuning expression and regulation of target genes at the transcriptional level. Herein we summarized and discussed the key progress and future frontiers of microbial promoter engineering, particularly for prokaryotic microorganisms. Firstly, we introduced the basic DNA sequence characteristics of promoters and the regular mechanism for promoter recognition and transcription-initiation by RNA polymerase sigma factors. Inducible mechanisms for both negative and positive regulation were particularly highlighted with the typical lac operator of Escherichia coli as an example. Then, effective strategies for obtaining improved-promoters were summarized, which were roughly divided into two categories: endogenous promoter mutation and heterologous promoter replacement. For the endogenous promoter mutation, the following strategies, e.g. point mutation toward sigma factor consensus sequence, coupling optimization of -35 and -10 regions with RBS sequence, random mutation or saturation mutagenesis of UP element or spacer sequences accompanying with promoter library construction and high-throughput screening were emphasized. For the heterologous promoter replacement, strategies such as substituting the native promoter into stronger ones from other microorganisms, introducing phage-source chimeric promoters, tuning the constitutive promoter into inducible pattern and integrating positive regulator(s), were mainly discussed. We further sorted out the representative inducers for inducible promoters reported so far, including both chemical molecules and physical signals. Progress in constitutive promoters of non-model and model microbial organisms were simply summarized as well. Next, arising from the breakthrough development of dynamic metabolic regulation and artificial intelligence (AI), we proposed that the innovative research on identification and evolution of new and unique promoters with dynamic-response features and AI de novo design for promoters with novel/superior functions will be the new frontiers of promoter engineering. Finally, we analyzed the challenging scientific issues in the microbial promoter engineering, from the viewpoint of both basic research and large-scale applications; and further discussed the research priority coupling with the vigorous development of synthetic biology. Table and Figures | Reference | Related Articles | Metrics

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Computational design and directed evolution strategies for optimizing protein stability RUAN Qingyun, HUANG Xin, MENG Zijun, QUAN Shu Synthetic Biology Journal    2023, 4 (1): 5-29.   DOI: 10.12211/2096-8280.2022-038 Abstract （3820）   HTML （438）    PDF（pc） （2169KB）（7004）       Knowledge map    Save Most natural proteins tend to be marginally stable, which allows them to gain flexibility for biological functions. However, marginal stability is often associated with protein misfolding and aggregation under stress conditions, presenting a challenge for protein research and applications such as proteins as biocatalysts and therapeutic agents. In addition, protein instability has been increasingly recognized as one of the major factors causing human diseases. For example, the formation of toxic protein aggregates is the hallmark of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Therefore, optimizing protein folding and maintaining protein homeostasis in cells are long-standing goals for the scientific community. Confronting these challenges, various methods have been developed to stabilize proteins. In this review, we classify and summarize various techniques for engineering protein stability, with a focus on strategies for optimizing protein sequences or cellular folding environments. We first outline the principles of protein folding, and describe factors that affect protein stability. Then, we describe two main approaches for protein stability engineering, namely, computational design and directed evolution. Computational design can be further classified into structure-based, phylogeny-based, folding energy calculation-based and artificial intelligence-assisted methods. We present the principles of several methods under each category, and also introduce easily accessible web-based tools. For directed evolution approaches, we focus on library-based, high-throughput screening or selection techniques, including cellular or cell-free display and stability biosensors, which link protein stability to easily detectable phenotypes. We not only introduce the applications of these techniques in protein sequence optimization, but also highlight their roles in identifying novel folding factors, including molecular chaperones, chemical chaperones, and inhibitors of protein aggregation. Moreover, we demonstrate the applications of protein stability engineering in biomedicine and pharmacotherapeutics, including identifying small molecules to stabilize disease-related, aggregation-prone proteins, obtaining conformation-fixed and stable antigens for vaccine development, and targeting protein stability as a means to control protein homeostasis. Finally, we look forward to the trends and prospects of protein stabilization technologies, and believe that protein stability engineering will lead to a better understanding of protein folding processes to facilitate the development of precision medicine. Table and Figures | Reference | Related Articles | Metrics

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Recent development of directed evolution in protein engineering QI Yanping, ZHU Jin, ZHANG Kai, LIU Tong, WANG Yajie Synthetic Biology Journal    2022, 3 (6): 1081-1108.   DOI: 10.12211/2096-8280.2022-025 Abstract （4406）   HTML （400）    PDF（pc） （3627KB）（6336）       Knowledge map    Save Directed evolution aims to accelerate the natural evolution process in vitro or in vivo through iterative cycles of genetic diversification and screening or selection. It has been one of the most solid and widely used tools in protein engineering. This review outlines the representative methods developed in the past 10 years that increase the throughput of directed evolution, including in vitro and in vivo gene diversification methods, high-throughput selection and screening methods, continuous evolution strategies, automation-assisted evolution strategies, and AI-assisted protein engineering. To illustrate the significant applications of directed evolution in protein engineering, this review subsequently discusses some remarkable cases to show how directed evolution was used to improve various properties of enzymes, such as the tolerance to elevated temperature or organic solvent, the activities on non-native substrates, and chemo-, regio-, stereo-, and enantio-selectivities. In addition, directed evolution has also been widely used to expand the biocatalytic repertories by engineering enzymes with abiotic activities. In addition to the native enzymes, directed evolution has also been used to engineer de novo designed enzymes and artificial metalloenzymes with activities comparable to or exceeding the ones of the native enzymes. Finally, this review has pointed out that further improving the efficiency and effectiveness of directed evolution remains challenging. Some advanced continuous evolution and high throughput screening strategies have been succesfully demonstrated in improving the throughput of directed evolutions extensively, but they have been limited to engineering certain protein targets. To resolve those issues, continuously improved computational modeling tools and machine learning strategies can assist us to create a smaller but more accurate library to enhance the probabilities of discovering variants with improved properties. Additionally, laboratorial automation platforms coupled with advanced screening and selection techniques also have great potential to extensively explore the protein fitness landscape by evolving multiple targets continuously in a high throughput manner. Table and Figures | Reference | Related Articles | Metrics

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Rewiring and application of Yarrowia lipolytica chassis cell SUN Meili, WANG Kaifeng, LU Ran, JI Xiaojun Synthetic Biology Journal    2023, 4 (4): 779-807.   DOI: 10.12211/2096-8280.2022-060 Abstract （2103）   HTML （254）    PDF（pc） （2749KB）（6112）       Knowledge map    Save 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. Table and Figures | Reference | Related Articles | Metrics

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Application of dynamic regulation strategies in metabolic engineering Yu Zheng, SHEN Xiaolin, Sun Xinxiao, Wang Jia, Yuan Qipeng Synthetic Biology Journal    2020, 1 (4): 440-453.   DOI: 10.12211/2096-8280.2020-029 Abstract （3842）   HTML （302）    PDF（pc） （2215KB）（5829）       Knowledge map    Save As a sustainable biochemical reactor, microbial cell factories are widely used in the production of value-added compounds such as natural products, pharmaceuticals and nutraceuticals. In order to make microbial cell factories produce target compounds with high titer, productivity and yield, metabolic engineering strategies are employed to rationally modify and regulate their metabolism. However, knockout and overexpression of genes inevitably bring stresses such as redox imbalance and toxic intermediate accumulation. While dynamic control strategy has been proven as a promising tool to address these challenges by balancing carbon flux and energy generation and dissipation for cell growth and generation of target compounds. As a result, many dynamic regulation elements and gene circuits have been developed and widely used in metabolic engineering so far. In this review, we summarize four types of attractive dynamic regulation systems based on metabolite-response, quorum sensing-response, environmental parameter-response and protein level regulation, with a focus on the construction method of various regulatory elements and their applications in metabolic engineering. In addition, challenges faced by different dynamic control strategies in industrial applications are analyzed. At the same time, we prospect the application potentials of some strategies such as high-throughput screening, protein engineering, computer simulation and mathematical model analysis coupled with gene control elements in solving the problems of narrow response threshold and limited control range of dynamic regulation tools. With the further development of synthetic biology and metabolic engineering, we believe that dynamic control strategy will be widely used for the construction of microbial cell factories in the near future. Table and Figures | Reference | Related Articles | Metrics

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From chemical synthesis to biosynthesis: trends toward total synthesis of natural products ZHANG Faguang, QU Ge, SUN Zhoutong, MA Jun′an Synthetic Biology Journal    2021, 2 (5): 674-696.   DOI: 10.12211/2096-8280.2021-039 Abstract （7828）   HTML （642）    PDF（pc） （6155KB）（5498）       Knowledge map    Save The complexity and diversity of natural products have made them a rich source for drug and agrochemical discovery. To overcome the supplying limitation of natural resources, tremendous effort has been made by the academic and industrial communities during the past two centuries for the total artificial synthesis of natural products. In this regard, total chemical synthesis has achieved significant progress, and numerous highly complex natural products have been synthesized through different chemical processes. Despite these great achievements in total chemical synthesis, there are still many challenges including expensive chemical reagents, harsh reaction conditions, difficult control on stereoselectivity, long synthetic route, and low product yield. Notably, the development of synthetic biology has allowed more and more natural products to be produced through biological cell factories, which provides a new and complementary strategy for the synthesis of natural products at a large scale. This review critically comments on the representative advances in total chemical synthesis of natural products (Section 1), and then highlight major progress and trends in the biosynthesis of pharmaceutically important natural products (Sections 2 and 3). In Section 2.1, we selected the production of penicillin, erythromycin, and avermectin as examples to analyze the modification and optimization of natural product biosynthetic pathways. The discovery and utilization of secondary metabolites from microorganisms has been a continuous driving force in the field of natural products. Notably, significant progress has been made in the total biosynthesis of natural products from secondary metabolism via the genetic manipulation of microbial cells. In Section 2.2, we selected Vitamin B12 and Tropane alkaloids as examples to demonstrate the use of heterologous expression and biological production for natural product synthesis. In recent years, on the basis of analyzing the structure of natural products in animals, plants, and microorganisms, great advances have emerged in exploring their biochemical reaction mechanisms and synthetic routes. More importantly, expressing and regulating the relative genes in heterologous microbial cells have enabled the complete biosynthesis of many natural products. Furthermore, in Section 3, human insulin, artemisinin, saframycin, azaphilone, kainic acid, and podophyllotoxin were selected as examples to showcase the power of merging chemical and biological processes for the total synthesis of natural products. Although there are still many challenges in the total synthesis of new and complex natural products, biosynthesis will ultimately play a significant role in the construction of natural molecules and their relative analogues. By taking advantage of the merits with organic chemistry, synthetic biology, and artificial intelligence, the development of highly efficient and automatic biosynthesis could be a trend in this field. Table and Figures | Reference | Related Articles | Metrics

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Applications and prospects of genome mining in the discovery of natural products YANG Qian, CHENG Botao, TANG Zhijun, LIU Wen Synthetic Biology Journal    2021, 2 (5): 697-715.   DOI: 10.12211/2096-8280.2021-012 Abstract （4510）   HTML （437）    PDF（pc） （6343KB）（5208）       Knowledge map    Save Natural products have been an abundant source of leader compounds for new drugs, but traditional isolation and analysis technologies to obtain novel natural products cannot satisfy the requirement for drug discovery. Genomic data have been utilized for identifying potential drug targets, or exploring biosynthesis pathways for natural products that were neglected before. Genome sequencing has unveiled a plethora of undeveloped chemical diversity in microorganisms and plants. From genome sequences, a large amount of information is available, from functional enzymes to conserved patterns/signatures, even potential structures and features that can be interpreted to hunt for new biocatalysts. With the advent of the genomic era, the computational mining of genomes has become an important part in the discovery of novel natural products as drug leads. Meanwhile, the development of high-throughput sequencing and the establishment of DNA database, genome mining methods and tools have contributed to the discovery and characterization of these natural products. In spite of the diversity of natural products, the biosynthetic rules and thus the biosynthetic machineries for many of these compounds are often remarkably conserved, which is highlighted in the high amino acid sequence similarity of the core biosynthetic enzymes, such as polyketides synthases (PKS), non-ribosomally peptides synthetases (NRPS), and many others. Besides, most of natural products are considered to be produced by the host to kill or limit the growth of competitors through the inhibition or inactivation of essential housekeeping enzymes. Therefore, accumulating knowledge on the self-resistance mechanisms, for instance, mining for SRE (self-resistance enzyme), have promoted research on natural products. Moreover, a phylogeny-guided mining approach provides a method to quickly screen a large number of microbial genomes or metagenomes to detect new biosynthetic gene clusters of interest, and many web tools and databases have been developed and utilized by researchers to mine for key enzymes. This paper reviews recent advances in the genome mining tools, databases and approaches, with a focus on the ways of mining biosynthetic gene clusters (BGCs) of natural products, from classical genome mining to resistance-based and phylogeny-guided mining, and also include a short overview on status and perspective in the discovery of novel natural products. Table and Figures | Reference | Related Articles | Metrics

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Advances of CRISPR gene editing in microbial synthetic biology LI Yang, SHEN Xiaolin, SUN Xinxiao, YUAN Qipeng, YAN Yajun, WANG Jia Synthetic Biology Journal    2021, 2 (1): 106-120.   DOI: 10.12211/2096-8280.2020-039 Abstract （5573）   HTML （475）    PDF（pc） （2241KB）（5167）       Knowledge map    Save With the increase of global consumption on fossil resources for energy products and chemicals and their consequent impact on environment, construction of microbial cell factories for efficient production of biofuels and bio-based chemicals from renewable sources has gained much attention. Pathway engineering of the hosts, such as over-expression of key genes, disruption of competing pathways and integration of heterologous pathways, plays significant role in fulfilling such a purpose. Successful implementation of these pathway engineering strategies requires efficient and accurate gene editing tools. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a powerful gene editing strategy that was found in prokaryotic organisms such as archaea and bacteria, which provide adaptive immunity against foreign elements. When host cells are infected by viruses, a small sequence of the viral genome is integrated into the CRISPR locus to immunize the host cells, and this small sequence is transcribed into small RNA guide that directs the cleavage of the viral DNA by the Cas nuclease. Inspired by the natural talent, many modified CRISPR systems have been developed to modify genes and genomes, including knock-in, knock-down, large deletions, indels, replacements and chromosomal rearrangements. In this review, we briefly comment on the technical basis and advances in CRISPR-related genome editing tools applied for constructing microbial cell factories, with a focus on the CRISPR-based tools for metabolic engineering of the model organisms E. coli and S. cerevisiae. Furthermore, we highlight major challenges in developing CRISPR tools for multiplex genome editing and sophisticated expression regulation. Finally, we propose future perspectives on the application of CRISPR-based technologies for constructing microbial ecosystems toward high production of desired chemicals. We intend to provide insights and ideas for developing CRISPR-related genome editing tools to better serve the construction of efficient microbial cell factories. Table and Figures | Reference | Related Articles | Metrics

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Recent advances in photoenzymatic synthesis MING Yang, CHEN Bin, HUANG Xiaoqiang Synthetic Biology Journal    2023, 4 (4): 651-675.   DOI: 10.12211/2096-8280.2022-056 Abstract （4761）   HTML （410）    PDF（pc） （5786KB）（5021）       Knowledge map    Save 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. Table and Figures | Reference | Related Articles | Metrics

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Advances in synthetic biomanufacturing ZHANG Yuanyuan, ZENG Yan, WANG Qinhong Synthetic Biology Journal    2021, 2 (2): 145-160.   DOI: 10.12211/2096-8280.2020-052 Abstract （4292）   HTML （599）    PDF（pc） （2003KB）（4875）       Knowledge map    Save Synthetic biomanufacturing is a paradigm for material processing and synthesis via synthetic biology. It is expected to completely transform the traditional production mode for medicines, chemicals, food, energy, materials and agriculture in the future, trigger new industrial revolution, lead to new economic growth and reshape carbon-based civilization. In particular, the COVID-19 global spread that has accelerated the reshaping process of the world's economic and social development. In the foreseeable future, human life and production patterns will undergo profound changes in medicines and healthcare, food and agriculture, energy and materials, etc., during which demands for new technologies will promote evolution in the field of biotechnology, and the biomanufacturing industry in the post COVID-19 era is facing unprecedented opportunities for revitalization and new challenges. According to the analysis of the research report from the Ministry of Economy, Trade and Industry of Japan, engineered biological cells and their combination with information & artificial intelligence technologies will become the main driving force for the "post-fourth industrial revolution".Synthetic biomanufacturing has the characteristics of cleanliness, efficiency and renewableness that can reduce the impact of industrial economy on the ecological environment. Here, in this review, we summarize the progress of synthetic biomanufacturing with respect to bulk fermentation products, fine and pharmaceutical chemicals, renewable chemicals and bio-based polymeric materials, natural products, foods and the utilization of C1 raw materials. The technological progress, status and potential of industrial applications of many important bio-based products via synthetic biomanufacturing are analyzed and discussed. The development of synthetic biomanufacturing shows great potentials for building up the ecological route of industrial economy and addressing current issues of economic sustainability in terms of limited substrate, high cost, and poor viability, and to form whole new industry chain with sustainable growth. In the future, with the development of synthetic biology, and the integration of new technologies such as artificial intelligence and big data, more and more bio-based products can be produced via synthetic biomanufacturing. The formation of bioeconomy can be promoted, and the sustainable development of human society will be better served. Table and Figures | Reference | Related Articles | Metrics

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Enzyme engineering in the age of artificial intelligence KANG Liqi, TAN Pan, HONG Liang Synthetic Biology Journal    2023, 4 (3): 524-534.   DOI: 10.12211/2096-8280.2023-009 Abstract （5144）   HTML （479）    PDF（pc） （1310KB）（4807）       Knowledge map    Save Enzymes have garnered significant attention in both research and industry due to their unparalleled specificity and functionality, and thus opportunities remain for enhancing their physichemical properties and fitness to improve catalytic performance. The primary objective of enzyme engineering is to optimize the fitness of targeted enzymes through various strategies for their modifications, even redesigning. This review provides a comprehensive overview for progress made in enzyme engineering, with a focus on artificial intelligence (AI)-guided design methodology. Several key strategies have been employed in enzyme engineering, including rational design, directed evolution, semi-rational design, and AI-guided design. Rational design relies on an extensive knowledge based on encompassing protein structures and catalytic mechanisms, allowing for purposeful manipulations of enzyme properties. Directed evolution, on the other hand, involves the generation of a library of random variants for subsequent high-throughput screening to identify beneficial mutations. Semi-rational design combines rational design and directed evolution, resulting in a smaller, yet more targeted, library of variants, which mitigates high cost associated with extensive screening of large libraries developed through directed evolution. In recent years, AI technologies, particularly deep neural networks, have emerged as a promising approach for enzyme engineering, and AI-guided methods leverage a vast amount of information regarding protein sequences, multiple sequence alignments, and protein structures to learn key features for correlations. These learned features can then be applied to various downstream tasks in enzyme engineering, such as predicting mutations with beneficial effect, optimizing protein stability, and enhancing catalytic activity. Herewith, we delves into advancements and successes in each of these strategies for enzyme engineering, highlighting the growing impact of AI-guided design on the process. By offering a detailed examination of the current state of enzyme engineering, we aim at providing valuable insight for researchers and engineers to further advance the development and optimization of enzymes for more applications. Table and Figures | Reference | Related Articles | Metrics

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Advances in optogenetics for biomedical research YU Yuanhuan, ZHOU Yang, WANG Xinyi, KONG Deqiang, YE Haifeng Synthetic Biology Journal    2023, 4 (1): 102-140.   DOI: 10.12211/2096-8280.2022-030 Abstract （2323）   HTML （204）    PDF（pc） （5942KB）（4787）       Knowledge map    Save Synthetic biology enables rational design of regulatory molecules and circuits to reprogram cellular behaviors, and its applications to human cells could lead to powerful gene- and cell-based therapies, which are well recognized as central pillars of next-generation medicines. However, the safety of these therapies remains to be assessed, and controllability is a critical issue affecting their safety and limiting their clinical applications. In recent years, optogenetic technologies have been widely used in biomedical applications, which provides new insights for treating intractable diseases due to their distinguishing features of non-invasiveness, reversibility, and spatiotemporal resolution. Light is an ideal inducer to control gene expression, enabling precise and spatiotemporal manipulation of gene expression and cell behaviors by illuminating with light of appropriate intensity and wavelength as a triggering signal to achieve pinpoint spatiotemporal control of cellular activities. With the development of optogenetic toolkits, optogenetics has recently been developed for therapeutic applications. In this review, we summarize various optogenetic tools responsive to different wavelengths and their applications for precise treatment of neurological diseases, tumors, cardiovascular diseases, diabetes, enteric diseases as well as for the optogenetic control of gene transcription, gene editing, gene recombination and organelle movement. At the same time, we introduce recent research progress in portable bioelectronic medicine and artificial intelligence-assisted diagnosis and treatment systems, which are based on optogenetic techniques and the intelligent electronic devices. The rapid development of optogenetics has enormously extended the scope of traditional bioelectronic medicine, and the remote-controllability, reversibility, and negligible toxicity of optical control systems provide a solid foundation for the application of optogenetics in biomedicine. The success of these approaches would have an impact on precision medicine in the future practice. Finally, we also discuss the shortcomings of existing optogenetic tools and the challenges that would be faced in the future clinical applications as well as the prospects of their development. Table and Figures | Reference | Related Articles | Metrics

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Recent progress of synthetic biology applications in microbial pharmaceuticals research RAO Cong, YUN Xuan, YU Yi, DENG Zixin Synthetic Biology Journal    2020, 1 (1): 92-102.   DOI: 10.12211/2096-8280.2020-036 Abstract （5513）   HTML （559）    PDF（pc） （1980KB）（4680）       Knowledge map    Save Microbial natural products are a major source in the innovation of novel pharmaceuticals, including anti-bacterial, anti-tumor, and immunosuppressive agents for clinical use. Currently, the development of microbial drugs is facing significant challenges due to the growing prevalence of multidrug-resistant bacteria, the continuous emergence of new pathogens and viruses, and the increasing difficulties in the discovery of natural products with new scaffolds. Synthetic biology is an emerging interdisciplinary research area leading to great breakthrough in the field of biomedical sciences in the 21st century, which provides new methods and ideas for drug discovery and development. The application of synthetic biology could unlock the potential of natural product mining, design new biosynthetic routes, and generate much more “unnatural” natural products and structural analogs. This review summarizes the technical innovations of synthetic biology in the field of microbial pharmaceuticals, and its applications in the mining, biosynthesis, and new scaffold generation of aminoglycoside antibiotics, nucleotide antibiotics, ribosomally synthesized and posttranslationally modified peptides, terpenoids, and polyketides in the last five years. Table and Figures | Reference | Related Articles | Metrics

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Application of bacterial quorum sensing system in intercellular communication and its progress in synthetic biology LI Xiaomeng, JIANG Wei, LIANG Quanfeng, QI Qingsheng Synthetic Biology Journal    2020, 1 (5): 540-555.   DOI: 10.12211/2096-8280.2020-043 Abstract （3980）   HTML （253）    PDF（pc） （1959KB）（4366）       Knowledge map    Save Quorum sensing (QS) is a bacterial cell-to-cell communication system. Bacteria sense the density of bacterial population by secreting diffuse small molecular signals, thus causing the coordinated expression of a group of specific genes at the transcriptional level. With continuous research, the quorum-sensing related genetic elements and the regulatory principles have gradually become clear. In recent years, genetic circuits containing components of the bacterial QS system have been constructed through synthetic biology to achieve intra-species and inter-species artificial communication, and these genetic circuits based on QS have great application potential in biotechnology and biomedicine. This paper reviews several relatively clear and representative microbial quorum sensing systems and their functional roles, and introduces the application of genetic circuits based on quorum sensing systems to intra-species and inter-species cell communication, and discusses the microbial quorum sensing system in constructing biological computing tools, regulating the population density and the flow of metabolism of the future development prospect. For intra-cell communication, we mainly introduced the applications of quorum sensing system in the construction of biological computing tools, which are mainly reflected in the research of toggle switches, biosensors and logic gates in synthetic biology. Toggle switches, biosensors, and logic gates designed based on quorum sensing mechanism can better coordinate cell behavior at the population level by combining with some biological control circuits to achieve precise regulation at the spatial, temporal, and population level. For inter-species cell communication, we mainly discuss the influence of introducing quorum sensing system on controlling population density and regulating metabolic flow. Quorum sensing system is used to redistribute the metabolic fluxes of the desired pathways through the recombination of metabolic network, to realize the regulation of population density and co-culture of mixed strains. Meanwhile, it is also found that the combination of QS system and oscillator model has great potential in regulating the synchronicity of microbial complexes. In summary, the in-depth research on the quorum sensing mechanism and application not only lays a certain foundation for elucidating the mechanism of microbial ecological competition and dynamic balance, but also provides an idea for clarifying the regulation mechanism of pathogenic bacteria and developing new disease control strategies. Table and Figures | Reference | Related Articles | Metrics

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Cell-free protein synthesis: from basic research to engineering applications HOU Jiaqi, JIANG Nan, MA Lianju, LU Yuan Synthetic Biology Journal    2022, 3 (3): 465-486.   DOI: 10.12211/2096-8280.2021-064 Abstract （3962）   HTML （292）    PDF（pc） （2624KB）（4276）       Knowledge map    Save Cell-free protein synthesis (CFPS), also known as in vitro gene expression, is a multifunctional technique used to complement cell-based protein expression, which is at the core of cell-free synthetic biology. Since the CFPS system does not require a living cell, it can simulate the entire cellular transcription and translation process in vitro in a controlled environment, and allows for an in-depth study of individual components and biological networks. Therefore, as a platform technology, it is expected to overcome the loopholes caused by the limitations of cell membranes in the current in vivo manufacturing systems, which has a broad research prospect in fundamental and applied scientific research. The cell-free operation is simple and easy to control, and its advantages over in vivo protein expression include its nature with open systems, eliminating the dependence on living cells and using all system energy for the production of the target proteins. This article reviews the composition of CFPS systems and their development based on different component types, including different biological extracts or purified transcription and translation components. Furthermore, different CFPS reaction patterns are introduced, including batch and continuous exchange modes, and the research progress of CFPS systems in genetic circuits, protein engineering, and the construction of artificial life is described. Among them, the genetic circuit research progress mainly summarizes the latest applications and contributions of cell-free technology in the prototype design, biosensors, and in vitro metabolic engineering. The protein engineering research progress lists the advantages and advances of the CFPS systems for producing membrane proteins, virus-like particles, post-translational modifications, unnatural amino acid incorporation and protein evolution. In the construction of artificial "living systems", the synthesis of bacteriophages and the construction of artificial cells have opened up a novel frontier field. Finally, the opportunities and challenges of the CFPS platforms for future scientific research and industrial applications are highlighted. Table and Figures | Reference | Related Articles | Metrics

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Research advances in synthetic microbial communities QU Zepeng, CHEN Moxian, CAO Zhaohui, ZUO Wenlong, CHEN Ye, DAI Lei Synthetic Biology Journal    2020, 1 (6): 621-634.   DOI: 10.12211/2096-8280.2020-012 Abstract （4181）   HTML （447）    PDF（pc） （2495KB）（4271）       Knowledge map    Save Synthetic microbial communities are an emerging research field at the intersection of synthetic biology and microbiome. A synthetic microbial community is created artificially by co-culturing of multiple species under a well-defined condition. Synthetic communities that retain the key features of their natural counterparts can act as a model system to study the ecology and function of microbial communities in a controlled way. This review covers important topics and research progress in synthetic microbial communities. We start with a summary of ecological factors that shape the structure of microbial communities, including interactions among microbes, host metabolism and immunity and environmental conditions. We then discuss the methods and experimental techniques in design-build-test-learn (DBTL) cycle, used to study synthetic microbial communities. In addition, we review the potential applications of synthetic microbiome in human health, agriculture, industrial production and environmental remediation. Finally, we summarize key scientific questions for future studies of synthetic microbial communities,including how to construct a controllable and stable microbial interaction network, how to characterize and manage the spatial structure of microbial communities, and how to accurately shape the function of microbial communities. Table and Figures | Reference | Related Articles | Metrics

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Enzymatic synthesis of 2'-fucosyllactose： advances and perspectives SHI Ran, JIANG Zhengqiang Synthetic Biology Journal    2020, 1 (4): 481-494.   DOI: 10.12211/2096-8280.2020-033 Abstract （2762）   HTML （126）    PDF（pc） （2801KB）（4134）       Knowledge map    Save Human milk oligosaccharides (HMOs) constitute a unique group of endogenous indigestible carbohydrates in human breast milk. HMOs play a crucial role in infant health and growth. As the most abundant HMO, 2'-fucosyllactose (Fucα1, 2Galβ1, 4Glc, 2'-FL) has been approved for infant formulas, dietary supplements and medical foods in the United States and European Union. 2'-FL has been synthesized by chemical, enzymatic synthesis and cell factory approaches, and currently mainly produced by cell factory approach. The crucial factors for 2'-FL production are the reduction of the cost of L-fucose, the discovery of novel α1,2-fucosyltransferases, and the maintenance of the balance between intracellular GDP-L-fucose level and the growth of engineering strain. Also, the construction of antibiotic-free system (such as antibiotic-free Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) is still a challenge for the synthesis of 2'-FL. In this review, the research progress of enzymatic synthesis of 2'-FL was particularly presented. 2'-FL could be enzymatically synthesized using α-1,2-fucosyltransferases or α-L-fucosidases. α-1,2-Fucosyltransferases catalyze the transformation of a fucose from a GDP-L-fucose to a lactose. The main disadvantage for 2'-FL synthesis by fucosyltransferase is the requirement for an expensive nucleotide donor. Also, α-L-fucosidases have been studied extensively since they catalyze the synthesis of 2'-FL through a transglycosylation reaction and often possess a higher availability and activity, in comparison with fucosyltransferases. The discovery of efficient transfucosidases and the availability of appropriate, fucosylated donor substrates will promote the application of α-L-fucosidases in the synthesis of 2'-FL. In the near future, enzymatic synthesis is expected to become a method for industrial production of 2'-FL. Table and Figures | Reference | Related Articles | Metrics

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Artificial intelligence-assisted protein engineering BIAN Jiahao, YANG Guangyu Synthetic Biology Journal    2022, 3 (3): 429-444.   DOI: 10.12211/2096-8280.2021-032 Abstract （5086）   HTML （491）    PDF（pc） （2486KB）（4125）       Knowledge map    Save Protein engineering is one of the important research fields of synthetic biology. However, de novo design of protein functions based on rational design is still challenging, because of the limited understanding on biological fundamentals such as protein folding and the natural evolution mechanism of enzymes. Directed evolution is capable of optimizing protein functions effectively by mimicking the principle of natural evolution in the laboratory without relying on structure and mechanism information. However, directed evolution is highly dependent on high-throughput screening methods, which also limits its applications on proteins which lack high-throughput screening methods. In recent years, artificial intelligence has been developed very rapidly for integrating into multidisciplinary fields. In synthetic biology, artificial intelligence-assisted protein engineering has become an efficient strategy for protein engineering besides rational design and directed evolution, which has shown unique advantages in predicting the structure, function, solubility of proteins and enzymes. Artificial intelligence models can learn the internal properties and relationships from given sequence-function data sets to make predictions on properties for virtual sequences. In this article, we review the application of artificial intelligence-assisted protein engineering. With the basic and process of the strategy introduced, three key points that affect the performance of the predictive model are analyzed: data, molecular descriptors and artificial intelligence algorithms. In order to provide useful tools for researchers who want to take advantage of this strategy, we summarize the main public database, diverse toolkits and web servers of the common molecular descriptors and artificial intelligence algorithms. We also comment on the functions, applications and websites of several artificial intelligence-assisted protein engineering platforms, through which a complete prediction task including protein sequences representation, feature analysis, model construction and output can be completed easily. Finally, we analyze some challenges that need to be solved in the artificial intelligence-assisted protein engineering, such as the lack of high-quality data, deviation in data sets and lacking of the universal models. However, with the development of automated gene annotations, ultra-high-throughput screening technologies and artificial intelligence algorithms, sufficient high-quality data and appropriate algorithms will be developed, which can enhance the performance of artificial intelligence-assisted protein engineering and thus facilitate the development of synthetic biology techniques. Table and Figures | Reference | Related Articles | Metrics

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Plant synthetic biology for carbon peak and carbon neutrality YANG Jianzhao, ZHU Xinguang Synthetic Biology Journal    2022, 3 (5): 847-869.   DOI: 10.12211/2096-8280.2022-034 Abstract （2747）   HTML （214）    PDF（pc） （2330KB）（4119）       Knowledge map    Save Synthetic biology is an interdisciplinary research field, for which complete quantitative research systems have been established in bacteria, yeast, and mammalian cells. However, synthetic biology in plants is still at its infancy. Plant synthetic biology can play important roles in synthesizing plant natural products, developing molecular farming, improving photosynthesis to increase light energy utilization efficiency, designing carbon farming plants, and building plant factories. In the current efforts in creating a carbon neutral society, plant synthetic biology can help to address challenges of food shortage, energy crisis, and environmental pollution. Specifically, innovative methods can be developed to reduce the emission of CO2 and pollutants through plant production of high value products, whose industrial production is mostly associated with high CO2 emission. Moreover, plant synthetic biology can be used to optimize plant production through minimizing carbon emissions and reducing the use of chemical fertilizers and pesticides. Furthermore, plants specialized in carbon capturing, such as high photosynthetic efficiency, large root systems, and high resistance to degradation, should be developed as well. Various options for increased photosynthetic efficiency, such as optimizing the antenna size of photosystem, converting C3 to C4 photosynthesis, introducing CO2 concentrating mechanisms, and establishing the photorespiration bypasses into C3 crops, holds the potential to dramatically increase the carbon capturing capacity for improved productivity. In the future, in addition to crops, trees and algae can also be engineered to become efficient carbon sinks. Photosynthetic algae are expected to become a source of clean energy and industrial production system with zero or negative carbon emissions. In the long term, a complete plant factory system, which has optimal control of light, temperature, CO2, water, and nutrient, will be developed to achieve optimal plant growth and production while maintaining maximal carbon capturing capacity. Finally, artificial photosynthesis also promises to be an ideal solution as an energy production system. These aspects will be facilitated by the rapid development of plant synthetic biology tools, including biological part standardization, genetic circuits design, and directed evolution. This paper summarizes the major progresses of plant synthetic biology and prospects the major roles of plant synthetic biology in the future efforts in carbon emission peak and carbon neutrality. Table and Figures | Reference | Related Articles | Metrics