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Table of Content

    31 October 2020, Volume 1 Issue 5
    Contents
    2020, 1(5):  0-0. 
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    Invited Review
    Reading, editing, and writing techniques for genome research
    Hui WANG, Junbiao DAI, Zhouqing LUO
    2020, 1(5):  503-515.  doi:10.12211/2096-8280.2020-013
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    Genome carries the entire genetic information of life. Genome-related researches are ultimate fundamentals for life sciences. Technological development in genomic researches has deepened our understanding of genomes and their function. Obtaining genome sequences through sequencing, studying their function and regulation through editing and creating customer designed genomes through synthesis are three important aspects of genome research. From the first-generation sequencing to the third-generation sequencing, the "reading" technology has greatly reduced the cost and difficulty, while improved the speed, enabling the production of complete genomic information for complex and large genomes. From random mutagenesis to site-specific genome editing and, from ZFN to CRISPR, the genome "editing" technology has improved significantly in efficiency, applicability, and simplicity, providing a wealth of materials to dissect "genotype-phenotype" relationship. Accurate editing and high-throughput editing are moving towards applications in various areas. From viral genome, bacterial genome to yeast genome, and ultimately to human genome, synthetic genomics has moved from simple organisms to many complex organisms. Precise, fast and low-cost synthesis technologies are important for the development of synthetic genomics. This article reviews the histories, features, present status, and applications of technologies for genome sequencing (reading), genome editing (editing) and genome synthesizing (writing). The potential breakthrough of these technologies in the near future is also summarized and prospected. The ability to read, edit and write a genome has been and will continue to advance not only our understanding but also better utilization of living systems.

    Engineering Halomonas spp. for next generation industrial biotechnology (NGIB)
    Jiangnan CHEN, Xiaoning CHEN, Xinyi LIU, Wei WAN, Yixin ZHANG, Zihao ZHANG, Yifei ZHENG, Taoran ZHENG, Xuan WANG, Ziyu WANG, Xu YAN, Xu ZHANG, Fuqing WU, Guoqiang CHEN
    2020, 1(5):  516-527.  doi:10.12211/2096-8280.2020-042
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    China is a country with the largest industrial biotechnology capacity especially fermentation. However, it requires complicated sterilization procedures,high consumption of fresh water and energy, and expensive wastewater treatment processes. Therefore, it is urgent to develop the "Next Generation Industrial Biotechnology (NGIB)". The NGIB based on Halomonas spp. has many advantages, including: (1) energy-saving: no need for sterilization at high temperature and high pressure; (2) water-saving: use seawater instead of fresh water, and water can be recycled; (3) time-saving: production process can be continuous; (4) less investment in equipment: no need to use stainless steel fermentation system, instead, plastic, ceramics or even cement can be used as bioreactors; (5) high concentration of final product, bacteria can produce products at a high concentration; (6) simplification of separation process: increase bacterial volume or surface charge, which is conducive to gravity or self-flocculation precipitation. Based on the newly developed ‘Next Generation Industrial Biotechnology', this paper systematically introduces the latest progress in recent years in the fields such as strengthening technical advantages, development of biobricks and control parts including the porin promoter library, CRISPRi, CRISPR/Cas9 gene editing system, production of new products including poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV), Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)(P3HB4HB) and Bio-surfactant Protein PhaP, optimization of separation process, scale up of fermentation processes, and recycling of wastewater. With the application of synthetic biology, the efficiency of NGIB has been continuously improved, and its advantages are obvious. Nevertheless, NGIB also faces some technical challenges, particularly treatment of salt containing wastewater. NGIB will further improve the recycling strategy of high salt wastewater, develop control modules under high cell density fermentation conditions, strengthen the design of low-cost substrate utilization ways, expand the scale and application fields, and achieve the mass production of various chemicals. The continuous improvement of NGIB will provide strong support for the competitiveness of green bio-manufacturing.

    Recent development in catalytic mechanisms and applications of microbial epoxide hydrolases
    Fei CAO, Yongquan LI, Xuming MAO
    2020, 1(5):  528-539.  doi:10.12211/2096-8280.2020-004
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    Microbial epoxide hydrolases (EHs, EC 3.3.2.3) catalyze the enantioselective ring opening of racemic epoxides to the chiral epoxides and vicinal diols. EHs have high catalytic efficiency and chemical-, region-, and stereo-selectivity without cofactors, and are conducive to the synthesis of high-purity chiral compounds by biotransformation. Since they were discovered, EHs have become very important catalysts and powerful biosynthetic elements for the green synthesis of chiral drugs in industry. In the last 10 years, with the rapid development of genomics, molecular biology, chemical biology and structural biology, researchers have found a variety of EHs from various microorganisms with potential applications. At the same time, researchers have also paid a lot of attention to the enzymatic properties, protein structure and catalytic mechanism of microbial EHs. This review introduces the currently well-understood catalytic mechanism of microbial EHs, such as ArEH from Agrobacterium radiobacter and limonene 1,2-EH from Rhodococcus erythropolis. Moreover, it summarizes 30+ microbial EHs with potential values, which were newly discovered in the last decade based on genome mining of microorganisms, along with the cognate biosynthetic pathways for natural product production. At least 15 crystal structures of new microbial EHs have been elucidated, helping people further understand the catalytic mechanisms at atomic levels and laying the foundation of their evolutions and applications. In addition, the applications of EHs in the field of pharmaceuticals and synthetic biology are also highlighted. Nowadays, researchers are mainly focusing on the improvement of catalytic activity, specificity and efficiency, as well as enantio-purity and yield of the products from microbial EHs with two main strategies: random or rational mutations of enzymes and strains, and artificial design of enzyme cascades within chassis cells. In the future, the synthesis of chiral drugs catalyzed by EHs will commence on the evolution of enzymatic activity, and assembly of multi-enzyme instead of single-enzyme in a cell factory for efficient biocatalysis.

    Application of bacterial quorum sensing system in intercellular communication and its progress in synthetic biology
    Xiaomeng LI, Wei JIANG, Quanfeng LIANG, Qingsheng QI
    2020, 1(5):  540-555.  doi:10.12211/2096-8280.2020-043
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    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.

    Advances in genome evolution of Saccharomyces cerevisiae
    Siyang XIA, Lihong JIANG, Jin CAI, Lei HUANG, Zhinan XU, Jiazhang LIAN
    2020, 1(5):  556-569.  doi:10.12211/2096-8280.2020-044
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    Due to our limited knowledge of the complicated cellular networks, genome evolution has played critical roles in the construction and optimization of microbial cell factories, especially for those complex traits regulated by multi-genes and for organisms with few genetic engineering tools. Directed genome evolution mimics natural evolution in the laboratory via iterative rounds of genetic diversification and functional screening or selection to isolate evolved mutants with the desirable phenotypes. Genome evolution has been found to be one of the most effective synthetic biology tools for systematic modification and optimization of Saccharomyces cerevisiae, one of the most important chassises in metabolic engineering. This review summarized the advances and applications of genome evolution techniques in the construction and optimization of efficient S. cerevisiae cell factories. Firstly, random mutagenesis based genome evolution strategies, including chemical/physical mutagenesis, genome shuffling, transposon mediated mutagenesis, global transcriptional machinery engineering, recombinase mediated mutagenesis, as well as adaptive laboratory evolution, are introduced. Then, the recently developed trackable genome-scale engineering techniques, including YOGE (yeast oligo-mediated genome engineering), eMAGE (eukaryotic multiplex automated genome engineering), RAGE (RNAi-assisted genome evolution), CHAnGE (CRISPR/Cas9- and homology-directed-repair-assisted genome-scale engineering), MAGIC (multi-functional genome-wide CRISPR system), and MAGESTIC (multiplexed accurate genome editing with short, trackable, integrated cellular barcodes), are discussed in details. In addition, the applications of these irrational and semi-rational genome evolution techniques in engineering yeast cell factories to expand substrate utilization, enhance product formation, and improve cellular properties, are also presented. Finally, the challenges and future directions of genome evolution, particularly when in combination with the high-throughput screening methodologies, are prospected.

    Protein engineering of nicotinamide coenzyme-dependent oxidoreductases for coenzyme preference and its application in synthetic biology
    Meixia LIU, Qiangzi LI, Dongdong MENG, Xinlei WEI, Chun YOU
    2020, 1(5):  570-582.  doi:10.12211/2096-8280.2020-038
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    Nicotinamide-based coenzyme NAD(P) is the most common electron mediator in the redox processes in living organisms. As NAD and NADP play an important role in catabolism and anabolism, it is essential to ensure their supply and consumption, as well as to maintain their balance in synthetic biological systems. Synthetic pathways that fail to match coenzyme supply with demand will probably result in low product yield and slow volumetric productivity. To solve the problem of coenzyme imbalance, the best strategy is to change the coenzyme preference of oxidoreductases in the pathway by protein engineering and then replace the wild-type enzymes with the mutants. In addition, biomimetic coenzymes can be designed and used to replace natural nicotinamide-based coenzymes for redox reactions due to the low cost and better stability. However, most of the oxidoreductases in nature have no or little activity on biomimetic coenzymes. Therefore, this review first focuses on the methodology and research progress of natural nicotinamide-based coenzyme engineering, with its application in improving product yield and decreasing production cost. Studies on the utilization of protein engineering technology for the switching of coenzyme preference from natural to biomimetic coenzymes are also presented, indicating a broad application prospect of biomimetic coenzymes in the construction of both in vivo bioorthogonal redox pathways and in vitro synthetic enzymatic pathways. Despite some general rules have been proposed for natural coenzyme engineering, coenzyme engineering for changing the preference of oxidoreductases on biomimetic coenzymes remains its early stage due to the significant differences in structures and sizes among various natural and biomimetic coenzymes. Nevertheless, with the increasing numbers of resolved high-resolution protein crystal structures and homogeneous oxidoreductase sequences, the development of novel high-throughput screening methods, as well as the design of more biomimetic coenzymes with improved properties, the modification of coenzyme preference from natural to biomimetic coenzymes will become a prior direction of coenzyme engineering in the future.

    Synthetic biology approaches to improve druggability of natural products
    Qing WANG, Yijun CHEN
    2020, 1(5):  583-592.  doi:10.12211/2096-8280.2020-019
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    As an important source of clinical medicine and drug candidates, natural products originated from microorganisms and plants have a variety of biological activities, such as anti-infection, anticancer, immunosuppression and others. However, the physiochemical properties of natural products are usually not favorable for drug discovery and development, which has seriously limited the development of natural products for clinical applications. These hurdles include low aqueous solubility, lower potency, complexed structural analogs, and limited availability. Because all drugs should possess certain degree of aqueous solubility, the inherited low aqueous solubility of natural products markedly limits their druggability. Meanwhile, the efficacy of natural products is generally low, which requires significant improvements for therapeutic usefulness. Furthermore, the accumulation of various structural analogs of natural products leads to the difficulty of quality control for desired natural products. Moreover, in many cases, natural products are easily obtained or accessed for preclinical and clinical evaluations and subsequent clinical supply. Nevertheless, traditional strategy for natural product isolation has resulted in highly repeated rediscovery and the waste of time and resources, failing to deliver valuable bioactive leads and drug candidates. Recently, synthetic biology has become an emerging and valuable tool to address these limitations. Through the combination of genetic engineering, metabolic engineering, bioinformatics, systems biology, synthetic organic chemistry and computational biology, synthetic biology has been explored to improve various properties of natural products.In this review, we focus on the major factors that hinder the druggability of natural products and briefly summarize the progress made by approaches of synthetic biology in recent years. Based on structure-activity analysis, the structures of natural products can be modified or optimized by enzymes with different functions to produce favorable derivatives. Meanwhile, the manipulation of the synthetic and regulatory elements, the construction of a series of modules and the optimization of metabolic fluxes can significantly promote the production of natural product derived molecules. Moreover, de novo design of biosynthetic pathways under artificial regulation of the transcription and metabolism in coupling with suitable hosts and heterologous expression can further expand the biosynthetic potential towards natural products for their druggability. Given the diversity of structure and activity, natural products will continue to be an important source of bioactive compounds and new drugs in the future. With the rapid and prosperous development of synthetic biology technologies, together with the assistance of pharmaceutical sciences and computational technologies, a new era of natural product discovery and engineering can be foreseen.

    The legal issues about commercialization of food products employing synthetic biology strategies
    Li DU, Meng WANG
    2020, 1(5):  593-608.  doi:10.12211/2096-8280.2020-041
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    With continuous advances in basic researches related to synthetic biology, it's potential for application in various fields has become manifest, and its commercial value keeps increasing. In the field of food manufacturing, using synthetic biology tools can improve the food's nutritional value, provide consumers with more options for their diets, and alleviate future food supply shortage. At present, several kinds of food products using synthetic biology strategies have been put into the market in European countries and in the United States. However, the issues of biosafety, food safety, and ethical concerns of using synthetic biology to produce food remain controversial. How to balance relevant benefits and potential risks challenges domestic authorities as well as international communities. Based on a review of recent investment and financing situation in the field of food products using synthetic biology tools, this paper intends to discuss the relevant policies and legislation in Europe, the United States, and China for the commercialization of such products. The case of "Impossible Hamburger" (an artificial bio-food that has been approved and listed in the United States) is analyzed in depth, to examine the impact of legislation on the commercialization of related products. The paper concludes with suggestions on improvement of the legal framework for supporting and regulating the commercialization of synthetic biology-based food products in China.

    Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster
    Ruxin LIU, Lei DU, Xiaoqing XU, Jinpeng DING, Wei ZHANG, Shengying LI
    2020, 1(5):  609-620.  doi:10.12211/2096-8280.2020-032
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    Modern agriculture urgently demands for green biological pesticides. Polyoxins are a class of nucleoside antibiotics with a broad spectrum of biological activities. Polyoxins show remarkable potency towards diverse crop diseases due to their competitive inhibition of the chitin synthetase's activity during the building of fungal cell wall and insect crust. This study aimed to construct a high polyoxin B-producing strain, which is one of the most bioactive ingredients in polyoxin derivatives. First, a high polyoxin B-producing mutant strain Pol-12, showing a 1.2-fold higher yield of polyoxin B than the wild-type strain, was obtained from the random mutants generated by ultraviolet mutagenesis of the starting strain Streptomyces ansochromogenes that was isolated from soil and stored by this laboratory. Second, the polyoxin biosynthetic gene cluster pol was directly cloned into p15A vector by ExoCET direct cloning method; and the original promoter and the kasOp* strong promoter were respectively added upstream of the first gene of pol. Third, the resulting shuttle vectors were used to transform the Pol-12 strain by interspecies conjugation and the gene cluster pol was integrated into the chromosome by integrase phiC31, leading to the pol-duplicated strains S. ansochromogenes Pol-12::Pori-pol (M1) and S. ansochromogenes Pol-12::PkasOp*-pol (M2). Compared with Pol-12, the yield of polyoxin B was increased by 22 and 33 times in M1 and M2, respectively. These results indicate that UV mutagenesis together with genetic engineering breeding can be applied in construction of high polyoxin-producing strains. Increase of the copy number of biosynthetic gene cluster and strong promoter insertion are effective for titer-improvement of polyoxin B.