Table of Content

28 February 2021, Volume 2 Issue 1

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2021, 2(1):  0. 

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Invited Review



Design of biomolecular sequences by artificial intelligence

Ye WANG, Haochen WANG, Minghao YAN, Guanhua HU, Xiaowo WANG

2021, 2(1):  1-14.  doi:10.12211/2096-8280.2020-074

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Based on the concept of learning from nature， transforming and transcending nature， the core of synthetic biology is to optimize， reconstruct and recombine genetic elements in order to build synthetic biological systems that meet our needs. Obtaining desirable biological components is the basis for building and controlling synthetic biological systems. Recently， synthetic biomolecules have been widely used in areas such as metabolic engineering and gene therapy. How to search for biomolecular sequences with specific biological functions from the vast sequence library is a challenge for synthetic biology. With the rapid development of artificial intelligence， intelligent algorithms have shown great potentials in mining complex biological characteristics and designing biomolecules. In this review， the applications of deep generative models for the design of different artificial biological sequences are analyzed from the perspective of exploring new drug molecules， nucleic acid fragment sequences and protein sequence spaces under the guidance of complex feature rules discovered by deep learning technology. Furthermore， combined with the application cases in the design of small molecular compounds， nucleic acids and proteins， the directed optimization strategies for designing artificial biomolecules are summarized and analyzed. In order to evaluate the model-designed molecular sequences， this review systematically analyzes the schemes for sequence design evaluation from different perspectives in applications. As an important information writing carrier of synthetic life systems， how the artificial biological sequence interacts with the complex multi-level regulation in the cell is still an important issue to be studied. In the future， the intelligent design of artificial biological sequence needs to consider the characteristics of biological systems with multi-level regulation that is often coupling. Through the design of biological sequences at different levels， different regulation in natural biological systems should be elucidated at different levels properly for an overall intelligent adaptation and optimization of biological sequences and cell chassis environments.



Computational protein design: perspectives in methods and applications

Fan CAO, Yaoxi CHEN, Yangyang MIAO, Lu ZHANG, Haiyan LIU

2021, 2(1):  15-32.  doi:10.12211/2096-8280.2020-067

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In computational protein design， the amino acid sequence of a protein is rationally chosen through computations so that the resulting molecule is of desired structure and function. Systematic methods for computational protein design have been developed and validated in increasing number of experiments. Exhibiting strong potential for broad applications， computational protein design has been considered as an important enabling technology for Synthetic Biology. Here we briefly review the history of methods for computational design， which are divided into three sections about heuristic design that based on rules， automatic optimization of amino acid sequences， and de novo main chain design respectively. In the next chapter， we introduce the basic approaches and strategies in details. In proteins energy calculation methods， we introduce physical energy terms and statistical energy terms. Based on these energy calculation methods， we introduce sequence and structure design methods including automated optimization of amino acid sequences， de novo design of polypeptide backbones （with fragment assembling method or sequence independent backbone potentials）， designing new interfaces for inter-molecule recognition such as protein-ligand interfaces and protein-protein interfaces， and the concept of negative design. Besides the history and detail of computational protein design methods that mentioned above， we also briefly discuss examples of using computational protein design to support application studies， including enhancing protein structural stability and redesign or de novo design of enzymes， vaccines and protein materials that related to interfaces design. These examples not only present current studies using the computational protein design methods， but also enlighten us on more broader applications in the future. Finally， we analyze some problems that need to be solved in the protein computational design method， such as inefficient in design accuracy， difficulty in characterizing polar interactions， and the need to consider the environment of non-aqueous solvents. We also discuss some aspects of possible application in synthetic biology like biological logic gates design and biosensor design， and application prospects in the medical field such as antibodies， vaccine design， etc.



Enzymatic ligation technologies for the synthesis of pharmaceutical peptides and proteins

Xinyu YANG, Tong ZHU, Ruifeng LI, Bian WU

2021, 2(1):  33-45.  doi:10.12211/2096-8280.2020-064

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As a hot-spot of synthetic biology, chemical protein synthesis and modification have been widely applied to generate and functionalize naturally inaccessible proteins to meet scientific or pharmaceutical demands. Breaking away from the restrictions on the amino acids utilized in ribosomal protein synthesis, protein synthesis, and modification through the ligation of synthetic peptides have provided a platform for preparing the unnatural proteins that contain hundreds of residues, which realizes the artificial design of protein on the atomic scale. Although several chemical ligation methods have been reported, they are confronted with the defects like limited junction choices and complicated substrate preparations, impelling the scientists to find out alternative solutions and develop enzymatic strategies fit for different applications. As a group of popular peptide ligation methods, enzymatic ligation strategies not only expand our understanding of protein, but also exhibit great advantages in the industrial production of pharmaceutical peptides.Sortase A (SrtA) has been extensively utilized for protein terminal modification. The researchers from various fields have attempted to develop novel applications based on SrtA, for instance, characterizing the surface proteins of eukaryotic cells and expanding the choices of phage capsid proteins. Butelase 1 from a cyclopeptide-producing plant (Clitoria ternatea) is a highly efficient peptide ligase for peptide cyclization. Large proteins like GFP could be cyclized with excellent yield (>95%) as well, and Butelase 1 can be used for synthesizing peptide dendrimers, preparing protein thioesters, and modifying bacterial surfaces as well. Subtilisin-derived ligase is engineered from subtilisin and thought as a powerful tool for peptide cyclization, protein synthesis and terminal modification. In recent years, based on a calcium-independent subtilisin variant, a thermostable and organic solvent-tolerant peptide ligase was constructed and termed Peptiligase, which was further engineered afterward. Omniligase-1 is a universal ligase bearing broad sequence compatibility, while Thymoligase is specially designed for the production of the pharmaceutic peptide Thymosin-α1. These Peptiligase variants exhibit great advantages for industrial applications and have been considered as the most impressive enzymatic approach to date. The development of the existing enzymatic methods is anticipated to be ameliorated through enzyme engineering, to which rational design and directed evolution have contributed a lot. At the moment of computational protein design communicating with chemical protein synthesis, we anticipate that more peptide ligases would be discovered or designed. They may serve for generations of previously hard-to-access modified proteins.



Research progress of synthetic biology in the field of protein functional materials

Pan WANG, Chenhui ZHU, Jing ZHAO, Daidi FAN

2021, 2(1):  46-58.  doi:10.12211/2096-8280.2020-061

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Protein functional materials have good biocompatibility, degradability and versatility, so they have great application values in the fields of medicine, military and textile. However, due to their unique characteristics of high molecular weight, high-frequency amino acids and special post-translation modifications, there are bottlenecks in its low expression rate in artificial cell synthesis, poor compatibility between functional elements and chassis cells, and unstable structure and efficacy, which seriously limit the efficient production and application of these proteins. Synthetic biology, as the third biotechnology revolution, has the advantages of renewable resources, low pollution, easy control and directional design of biological macromolecules. With the gradual excavation and analysis of the functional principles and novel design concepts of protein functional materials, and the development of alternative materials with excellent performance under specific conditions, it has brought revolutionary changes to human social life. It has been reported that such protein functional materials have great application value in cancer diagnosis and treatment, regenerative medicine, gene delivery system, data storage and so on. Although synthetic biology has broadened the range of potential applications of protein functional materials, there are still some limitations. This requires us to carry out research from two different perspectives, biology and materials science. On the one hand, we should establish a common technology platform to form a complete upper, middle and downstream research system. On the other hand, we should establish targeted research based on the characteristics of different protein materials to produce materials with better performance. This article introduces the application and development of protein functional materials. Taking the efficient protein synthesis and functional requirements as the guide, the synthesis strategy of protein functional materials are explained from the aspects of protein molecule orientation design, cell factory construction and adaptation control, and protein material processing applications, in order to realize their functional directional enhancement and industrial production. The limitations of synthetic biology oriented protein functional materials in biology and materials science are prospected, which lay a foundation for the wide application of protein functional materials with excellent properties.



Progress and perspectives on developing Zymomonas mobilis as a chassis cell

Yongfu YANG, Binan GENG, Haoyue SONG, Qiaoning HE, Mingxiong HE, Jie BAO, Fengwu BAI, Shihui YANG

2021, 2(1):  59-90.  doi:10.12211/2096-8280.2020-071

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Zymomonas mobilis, the only microorganism known to use the Entner-Doudoroff (ED) pathway anaerobically, can produce ethanol naturally from glucose, fructose and sucrose with many desirable traits such as ethanol production at high rate and yield and merit with biosafety (generally regarded as safe, GRAS), which has attracted more attention to be engineered as cell factories to produce biofuels and other bio-based products from lignocellulosic biomass. With the rapid development of novel technologies such as next-generation sequencing (NGS) and CRISPR-Cas genome editing as well as the accumulation of knowledge from studies on its physiology and modifications through metabolic engineering and systems biology, it is necessary to summarize accomplishments achieved recently to further explore the advantages of Z. mobilis, expediting the development and deployment of the robust synthetic microbial chass for the goals of "build to understand" and "build to apply" in the systems and synthetic biology era. In this review, we critically comment on the unique physiological characteristics of Z. mobilis and its potentials as a synthetic chassis to be engineered as microbial cell factories for producing diverse biochemicals economically, with a focus on the advances and challenges of developing efficient and effective tools and techniques for engineering this bacterium, taking advantages of methodologies developed with system biology and synthetic biology as well as metabolic engineering. We also prospect on future research for developing Z. mobilis as an attractive microbial chassis to be able to fix CO₂ and N₂ for biochemical production through genome optimization and metabolic engineering to advance the principles of synthetic biology and explore its potentials on biotechnological applications, which need unceasing effort to improve, develop and deploy efficient and effective genome editing tools, strategies for fine-tuning metabolic and regulatory pathways timely and spatially, as well as automatic high-throughput screening and quantification approaches.



Host-circuit coupling: toward a new framework for genetic circuit design

Pan CHU, Jingwen ZHU, Wenqi HUANG, Chenli LIU, Xiongfei FU

2021, 2(1):  91-105.  doi:10.12211/2096-8280.2020-046

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With the rapid development of synthetic biology and increasing demand for complicated artificial life systems， increased complexity and scale have been observed in the design of synthetic gene circuits， resulting in more unpredictable behaviors. Traditionally， applying engineering principles into genetic circuit design employs mainly the bottom-up strategy from individual parts to their potential assembly into biological parts/circuits/systems. This strategy involves best fitting intrinsic parameters of individual parts through considering interactions among genetic parts， which requires extensive trial-and-error to tweak the circuits’ properties. Recent research reveals coupling between synthetic circuits and host cells， which originates， on the one hand， from the global regulation of host cell physiology on the circuit gene expression， and on the other hand， from the competition for and depleting of the cellular resources including gene expression machinery and metabolic pools. This coupling not only alters the physiology of host cells， but also influences the functionality of the circuits. Therefore， incorporating the physiological states of host cells into the framework of genetic circuits design may improve the predictability of the circuits’ behaviors for rational design. Facing challenges from the host-circuits coupling， several strategies have been proposed， including parts orthogonalization and device modularity， which show their potentials in unraveling the tangle of hosts and circuits. In this review， we comment on mechanism underlying the coupling between prokaryotic host cells and genetic circuits that have been widely reported in recent years. Two categories of biophysical models， coarse-grained and whole-cell models， are presented， which help us to understand， predict and evaluate the effect of the host-circuit coupling and the counter-intuitive phenomena as well. Meanwhile， attempts to reduce the coupling effect by orthogonalization and modular design strategies are summarized. With the development of genome read-editing-writing techniques and deployment of automatic high-throughput screening and analysis， we prospect on the genetic circuits design： （1） Excavating of high-quality genetic parts， （2） Quantitative method for characterizing parts， and （3） Integrating multi-level omics data to mine for hidden regulator networks between circuits and hosts， and （4） Developing accurate and robust predicting framework.



Advances of CRISPR gene editing in microbial synthetic biology

Yang LI, Xiaolin SHEN, Xinxiao SUN, Qipeng YUAN, Yajun YAN, Jia WANG

2021, 2(1):  106-120.  doi:10.12211/2096-8280.2020-039

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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.



Regulatory requirements for food and feed produced with genetically modified microorganisms and case studies for EU authorization

Xiaolian WEI, Zhiling QIAN, Qiaoqiao CHEN, Hongwei YU

2021, 2(1):  121-133.  doi:10.12211/2096-8280.2020-079

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With the rapid development of genetic modification technology in recent years, emerging biotechnology represented by gene recombination, gene editing, synthetic biology, etc., has laid the key foundation for subversive changes in the food industry. Genetically modified microorganisms (GMMs) are involved in the production of a variety of food and feed. At present, the application of GMMs in fermentation has become more and more extensive. The marketing of food and feed products produced by fermentation of GMMs falls under different legislative instruments for different countries. In the industrialization process using GMMs, it is necessary to consider the requirements of the laws and regulations in the countries for production and export. Different countries have different historical and cultural backgrounds, technical levels and public awareness of genetically modified foods. Therefore, there are different attitudes for the supervision of foods produced with GMMs. This article mainly discusses the regulatory requirements of food and feed produced with GMMs. For these products, if the components from genetically modified microorganisms (usually GMMs and recombinant DNA) can be detected in the final products, they will be classified as genetically modified foods (GMF) in the European Union and the United States. Therefore, they need to meet the regulatory requirements of relevant laws and regulations on genetically modified foods. In this article the different definitions of genetically modified food in European Union, the United States of America and China is firstly introduced, followed by a brief summary of the supervision requirements of genetically modified food in these three countries. Secondly, the EU, which has the strictest regulatory requirements, is examined for its regulatory system for GMF and the authorization requirements for food and feed fermented with GMMs. Finally, genetically modified Escherichia coli is taken as an example to study several EU authorization cases. From these cases, recommendatory notes on applying for EU authorization of GMM fermented food and feed are briefly summarized.



Design and experimental research of new robot for clone selection

Wei ZHU, Wenling ZHAO, Kai HE

2021, 2(1):  134-144.  doi:10.12211/2096-8280.2020-059

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Clone-selecting is a prerequisite for gene sequencing, protein expression, high-throughput screening and so on. The operation is to select clones that meet specific requirements from medium, and then inoculate them for culture, which is performed so far mainly by manual operation. With rapid growth in demand for cloning selection at research institutes and pharmaceutical companies, the labor-intensive and less efficient selection can no longer meet the market demand, and robot is expected to be a solution. At present, challenges with such a robot include its clone-selecting efficiency and accuracy as well as the survival rate of selected clones during the culture. In this article, the design for a new type of clone-selecting robot is reported, which employs a cylinder to drive the gripping system with disposable needles connected to the pneumatic claw for clone-gripping. Moreover, an integrated system based on industrial computer control with the HexSight imagining technology was developed for position and identification. The robot was tested experimentally by selecting E. coli clones, which could position and identify the colonies, with an identification time of 130 ms and positioning accuracy of 0.03 mm for inoculating them into 96-well plates for culture.