Table of Content

31 December 2022, Volume 3 Issue 6

Previous Issue    Next Issue

Invited Review



Design, optimization and application of whole-cell microbial biosensors with engineered genetic circuits

Lu YANG, Nan WU, Rongrong BAI, Weiliang DONG, Jie ZHOU, Min JIANG

2022, 3(6):  1061-1080.  doi:10.12211/2096-8280.2021-021

Asbtract ( 3192 )   HTML ( 333)   PDF (2990KB) ( 2117 )  

Figures and Tables | References | Related Articles | Metrics

Whole-cell microbial biosensors with engineered genetic circuits constructed based on the concept of synthetic biology is an important branch of the biosensor. Whole-cell microbial biosensor is mainly composed of the sensing module, the computing module and the output actuating module. It can sense the concentration of specific substances in the environment and then transfer it to specific signal outputs in time according to certain rules, which shows great potential in bioengineering process control, environmental monitoring, food safety, environmental quality monitoring and disease diagnosis and control, etc. With the improvement of various technologies in synthetic biology and the enrichment of genetic elements, more and more whole-cell microbial sensors based on different response mechanisms, different logic gates and logic circuits have been developed. However, the design and construction of genetically engineered whole-cell microbial biosensor still mainly rely on the empirical method of trial-and error-learning. Therefore, how to design and construct high performance genetically engineered microbial whole-cell biosensors and how to tune its response curves by optimizing genetic elements or circuits to meet the detection requirements of different practical application scenarios is the new and important challenge. Here we reviewed the principle, classification and development process of genetically engineered whole-cell biosensor. We also focused on the design and construction of genetic circuit based on transcription factors and riboswitches, discussed optimization strategies for improving biosensor detection performance including dynamic range, specificity and working range, and then summarized its application progress in different detection fields. The optimization strategies are mainly involved in changing the expression level of genetic elements, adjusting the binding affinity between metabolites and genetic elements, restructuring the position of functional domains, etc. Finally, some challenges, such as biological safety, cumbersome design and construction, and inconvenience to enter the sensor market were discussed. It is expected that emerging technologies such as artificial intelligence, synthetic biology, and droplet microfluidics, will accelerate the development of genetic regulatory elements and the design and construction of novel biosensors.





Recent development of directed evolution in protein engineering

Yanping QI, Jin ZHU, Kai ZHANG, Tong LIU, Yajie WANG

2022, 3(6):  1081-1108.  doi:10.12211/2096-8280.2022-025

Asbtract ( 3614 )   HTML ( 342)   PDF (3627KB) ( 4226 )  

Figures and Tables | References | Related Articles | Metrics

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.





Artificial control of mammalian cell chemotaxis and motility

Wei GUO, Yuhao FU, Yingying FAN, Jialing ZHOU, Xin LI, Ping WEI

2022, 3(6):  1109-1125.  doi:10.12211/2096-8280.2022-029

Asbtract ( 789 )   HTML ( 54)   PDF (2033KB) ( 750 )  

Figures and Tables | References | Related Articles | Metrics

Chemotaxis and migration of mammalian cells are important for life processes. Many key physiological processes rely on cell migration, from embryonic development to bone and angiogenesis, playing key roles in tissue repair, inflammation, immune response and cancer metastasis. In vivo, cells must be able to sense various cues in their environment and move toward or away from them in order to execute morphogenetic programs, generate immune responses, and repair damaged tissue during development. When this process goes awry, it can have devastating consequences. Failure of cells to migrate in an appropriate manner can lead to developmental and immune deficiencies, chronic wounds that never heal, and diseases such as aggressive metastatic cancer, autoimmune disease, and fibrosis. The mechanism of cell migration is to transmit chemical and physical cues in the environment to the intracellular signaling network through surface receptors and mechanosensing molecules, establishing asymmetric biochemical gradients in cells, and activating downstream cytoskeletal regulators to polarize cells. The whole process involves various extracellular environmental cues, receptors that transmit cues to the cell, second messengers, and cytoskeleton regulators. The discovery of new chemotactic regulatory molecules and more detailed mechanisms will lead to a more accurate understanding of biomolecular composition and regulatory network organization during cell chemotaxis, which will provide more help for optimal design. Therefore, a systematic understanding of the process of cell chemotaxis is of great significance for developing the rational design and engineering capabilities of synthetic biology in mammalian cells. It will be an important direction of mammalian cell engineering to engineer the chemotaxis and migration ability of cells to achieve artificial control of cell migration, which can help us explore the mechanism of development, improve the effect of immunotherapy, cure diseases caused by cell chemotaxis disorders, and speed up the repair of tissue damage. This review will review and introduce the chemotactic migration of mammalian cells from the aspects of cell chemotactic migration, environmental cues, molecular mechanisms, engineering, and clinical applications.





Recent advances in heterologous production of natural products using Aspergillus oryzae

Jiayu DONG, Min LI, Zonghua XIAO, Ming HU, Yudai MATSUDA, Weiguang WANG

2022, 3(6):  1126-1149.  doi:10.12211/2096-8280.2022-007

Asbtract ( 2512 )   HTML ( 168)   PDF (3318KB) ( 2465 )  

Figures and Tables | References | Related Articles | Metrics

Organic molecules produced by living organisms, generally termed as natural products, are rich sources of pharmaceutical drugs and biopesticides, and fungi are one of the most prolific producers of medicinally important natural products, as represented by penicillins, lovastatin, and cyclosporins. Heterologous expression is a commonly used approach to study the function of biosynthetic genes of natural products, and a number of heterologous hosts have been developed and utilized over the last decades. The filamentous fungus Aspergillus oryzae has long been utilized for the production of fermented food and drinks in East Asia, and intensive genetic and molecular biological studies on the fungus have allowed for its genetic engineering in an efficient manner. Importantly, A. oryzae is known to possess a relatively clean metabolic background with a low level of secondary metabolite production, providing an attractive feature as a heterologous host. Furthermore, unlike prokaryotic and yeast hosts, most coding sequences of fungal biosynthetic proteins can be directly introduced into A. oryzae in their intact form without removing introns, which simplifies the transformation procedures. Collectively, A. oryzae is a robust platform for heterologous production of natural products, which not only facilitates the elucidation of the biosynthetic pathway of a given natural product but also allows the activation of silent or cryptic biosynthetic gene clusters. Thus, the A. oryzae host has been widely utilized for biosynthetic studies, genome mining, and synthetic biology of fungal natural products. It should be noted that more than ten biosynthetic genes can readily be introduced into the fungus, indicating that the majority of fungal biosynthetic gene clusters can be easily transferred to the A. oryzae host. This review first provides the general transformation procedure of A. oryzae and the molecular biological tools available for the fungus. Next, recent successful applications of this fungal host for the heterologous production of natural products are summarized. With the recent rapid advance in molecular biology, such as the development of genome editing tools, we believe that the heterologous expression of biosynthetic genes in A. oryzae will be performed in a much faster and more versatile manner in the near future, which would ultimately lead to the discovery of useful natural products for drug leads and other applications.





Rewiring and application of Pichia pastoris chassis cell

Qi LIU, Zhilan QIAN, Lili SONG, Chaoying YAO, Mingqiang XU, Yanna REN, Menghao CAI

2022, 3(6):  1150-1173.  doi:10.12211/2096-8280.2022-039

Asbtract ( 2674 )   HTML ( 312)   PDF (2981KB) ( 2376 )  

Figures and Tables | References | Related Articles | Metrics

Microbial chassis hosts are important platforms for green and sustainable biomanufacturing. Pichia pastoris has served as a preferred chassis for heterologous protein expression and fermentation production, which is attributed to its numerous advantages in expression capacity, post-translational modification, high cell density culture, and extracellular product purification. Moreover, as an industrial methylotrophic yeast, P. pastoris effectively utilizes cheap and widely sourced methanol as the sole carbon source, making it a potential biotransformation platform for C1 compounds. Recently, scientists have endowed this nonconventional yeast as an efficient microbial cell factory for biosynthesis of small molecule products beyond its traditional role of a protein expression workhorse. The growing of synthetic biology and biopharmaceutical technology has promoted the rapid development on the genetic rewiring of P. pastoris chassis host. A series of engineering strategies have been developed to break the restrictions and bottlenecks of P. pastoris in both academic and industrial applications. This allowed the updated chassis versions adapting to diversified application scenarios. In this review, we briefly introduce the advances and current status of P. pastoris. We describe the development and application of this chassis from the genetic manipulation technology, regulation of gene expression, and metabolic engineering. We summarize the establishment and characterization of synthetic biological techniques, regulatory parts and devices, novel expression platform, and bioconversion system in P. pastoris. We emphasize the CRISPR-mediated gene editing, transcription regulation, rewiring of natural transcription system, and the design of artificial biosystems. Then the production of glycoprotein and the synthesis of natural products based on alcohols are concisely summarized. Also, the advantages and limitations of this host in practical application are analyzed and discussed. Finally, we propose the research directions for further updating versions of P. pastoris and provide a perspective on their future application scenarios.





Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology

Jiayu LIU, Zhihan YANG, Lei YANG, Liying ZHU, Zhengming ZHU, Ling JIANG

2022, 3(6):  1174-1200.  doi:10.12211/2096-8280.2022-022

Asbtract ( 1639 )   HTML ( 132)   PDF (4313KB) ( 1279 )  

Figures and Tables | References | Related Articles | Metrics

As an important industrial microorganism and a novel probiotic, Clostridium tyrobutyricum is a superior strain for metabolizing various substrates to produce butyric acid under anaerobic condition, presenting a great potential for the valorization of agricultural wastes. Consequently, this bacterium with high yield of butyric acid has also been widely used in other fields, such as fine chemical production and human health. However, there are still many challenges in the construction of highly productive and robust C. tyrobutyricum cell factories. For example, the genetic transformation efficiency is rather low, due to the presence of restriction-modification systems. Gene editing tools are less developed and strain construction suffers from tedious processes and low efficiency. Moreover, genetic modification of C. tyrobutyricum is limited to a single mode of metabolic regulation, either knockout or overexpression, which is far behind the conventional model hosts. In recent years, with the continuous rapid development of synthetic biology and the collection of increasing amounts of C. tyrobutyricum bioinformatics data, a variety of research strategies and techniques, particularly the gene editing systems, have been employed to design and construct C. tyrobutyricum cell factories for efficient production of various fine chemicals. In this paper, we firstly provide an overview of the unique physiological properties of C. tyrobutyricum, including substrate range, environmental adaptability, butyric acid synthesis pathway, as well as the one-carbon gas fixation and energy metabolism pathways. Subsequently, we summarize the systems biology methods as well as the genetic manipulation tools for the modification of C. tyrobutyricum chassis cell, such as biological elements, the conjugation system, and the CRISPR/Cas system. Meanwhile, we discuss the static and dynamic metabolic regulation strategies and two types of quorum sensing systems (agr-type andRRNPP-type) as well as their applications in the synthesis of fine chemicals in C. tyrobutyricum. Finally, we prospect the trends for the creation of C. tyrobutyricum chassis cell, in terms of enhancing genetic transformation efficiency, expanding gene editing tools, designing and reconstructing gene circuits, establishing high-throughput screening platforms, and utilizing one-carbon gas.





Recent progress in the molecular genetic modification tools of Clostridium

Jiaxin LIU, Chi CHENG, Xinqi LI, Chaojun WANG, Ying ZHANG, Chuang XUE

2022, 3(6):  1201-1217.  doi:10.12211/2096-8280.2022-041

Asbtract ( 1161 )   HTML ( 97)   PDF (1942KB) ( 1158 )  

Figures and Tables | References | Related Articles | Metrics

Clostridium are Gram-positive, strictly anaerobic, endospore-forming bacteria that produce a variety of chemicals, including butanol, which is now a promising new biofuel. Improving the fermentation titer and yield of Clostridium by genetic modification has always been an important challenge that needs to be broken through, but it has long been hindered by the limitation of genetic manipulation tools of Clostridium. In recent years, with the continuous development of molecular biology, gene editing tools for Clostridium have been continuously developed. Many genetic manipulation tools such as plasmid-based gene overexpression, antisense RNA technology, transposon-based mutagenesis, group Ⅱ intron-mediated gene inactivation, and homologous recombination-based or CRISPR/Cas-mediated gene editing technology have been developed. Various operations such as target gene insertion, deletion, substitution, point mutation, and gene expression level regulation have been accomplished in Clostridium. In this review, we summarize the research progress in the molecular genetic modification tools of Clostridium, and especially discuss the potential application of new technologies, such as recombinase-based gene editing technology. Although the application of the recombinase system in Clostridium is rarely reported and discussed, the future application value and significance of this technology should be paid attention to. In the future, optimization of the existing molecular genetic modification technologies in Clostridium is still imperative, such as overcoming the low efficiency of homogeneous recombination in Clostridium, improving the stability and transformation efficiency of plasmids, solving the off-target problem of antisense RNA technology and type Ⅱ intron technology, reducing the toxicity of Cas9 protein, and so on. At the same time, new gene editing technologies should be developed, focusing on emerging technologies including CRISPR/Cas-mediated multi-locus editing systems, phage recombinase-mediated multiplex genome editing, targeted or random multi-copy gene integration, and so on. It is believed that with the development and improvement of genetic modification tools, Clostridium will be able to fully each its potential biorefinery capacity and make an important contribution to the green biosynthesis of bioenergy and bio-based chemicals.





Progress in microalgae chloroplast organelle factory development

Zhen ZHU, Jing TIAN, Jing JIANG, Wangyin WANG, Xupeng CAO

2022, 3(6):  1218-1234.  doi:10.12211/2096-8280.2022-044

Asbtract ( 1227 )   HTML ( 105)   PDF (1589KB) ( 1117 )  

Figures and Tables | References | Related Articles | Metrics

Microalgae are important solar-driven CO₂ biotransformation organisms. The chloroplasts of microalgae are important organelles for carbon assimilation and subsequent synthesis of carbohydrates, fatty acids, natural pigments, and amino acids. Unlike higher plant cells, which have multiple relatively small chloroplasts, most microalgae only have one large chloroplast that accounts for more than 50% of the cell volume. This makes it more conducive for us to obtain pure strains and it is expected to be used in food, aquatic industry, medicine, chemical products, biofuels and other fields by the establishment of microalgal chloroplast organelle factories. The construction of microalgae chloroplast organelle factories is one of the potential ways to achieve “carbon neutrality” by means of synthetic biology. The researches on microalgae transformation and microalgae chloroplast organelle factory are still in their infancy. There are still a lot of scientific and technical questions to be answered before microalgae chloroplast organelle factory can be applied at a large scale. In this mini review, the current progress of chloroplast transformation and expression technology in microalgae has been systematically summarized and briefly analyzed, and different approaches are compared, especially regarding the transformation strategies, i.e., direct chloroplast transformation and the transformation of nuclear-encoded chloroplast-targeted genes based on chimeric chloroplast transit peptides. The direct transformation strategy targeting the chloroplast genome is widely used. In Chlamydomonasreinhardtii, the most studied species, more than 100 different proteins have been successfully expressed. However, the chloroplast genome has limited insertion sites and the available regulation machineries on the exogenous genes' expression are rare. By using chloroplast signal peptides, more than 90% of native chloroplast proteins are nuclear-encoded and controllably delivered to the chloroplast. In recent years, the strategy of nuclear transformation and chloroplast-targeting expression based on chimeric chloroplast signal peptide has gained attention, having shown advantages in carbon fixation and oil production regulation. Some perspectives were also discussed. In the global effort for carbon neutralization, the microalgae chloroplast organelle factories will be good carriers to convert CO₂ to complex biomass by artificial and natural hybrid photosynthesis as a solution to food, energy, and environmental problems.





Review of research on unspecific peroxygenases (UPOs)

Mingyuan LAI, Jian WEI, Jianhe XU, Huilei YU

2022, 3(6):  1235-1249.  doi:10.12211/2096-8280.2022-028

Asbtract ( 2732 )   HTML ( 135)   PDF (2023KB) ( 1625 )  

Figures and Tables | References | Related Articles | Metrics

The selective insertion of oxygen species through unactivated C—H bonds is one of the most challenging tasks in organic synthesis. Fungal unspecific peroxygenases (UPOs) are a class of highly glycosylated thioheme enzymes that catalyze reactions including hydroxylation of unactivated C—H bonds in n-alkanes, epoxidation of alkenes and aromatics, oxidation of heteroatom (N, S) compounds, ether cleavage, N-dealkylation, deacylation and one-electron oxidation of phenols. As one of the most promising oxidases in synthetic chemistry,UPOs use H₂O₂ as the oxygen donor and the electron acceptor, and do not require cofactors other than heme. This paper reviews the classification and development process of UPOs, and focuses on the heterologous expression, selectivity engineering and H₂O₂in situ regeneration of UPOs. Since the first discovery of UPOs from Agrocybe aegerita in 2004, UPOs have attracted much attention due to the advantages described above. However, the difficulties of heterologous expression and poor selectivity of UPOs still limit their development. The difficulty of heterologous expression makes it hard to mine new variants of UPOs, and native UPOs are difficult to be characterized and applied in biocatalysis due to the slow growth rate of their hosts. In the past two years, important breakthroughs have been made in the heterologous expression of UPOs through the modification or replacement of signal peptides, revealing the important role of signal peptides in this process. However, the specific role of signal peptides in the secretory expression and three-dimensional structure formation of UPOs remain elusive. With the in-depth research on the mechanism of signal peptides affecting the heterologous expression of UPOs and the development of artificial intelligence (AI) algorithms, the combination of genome mining and signal peptide prediction will be the key for discovering new UPOs. The poor selectivity of UPOs also hinders the development and application of UPOs. This paper reviews different types of reactions that UPOs catalyze, and reveals the problem that UPOs have broad substrate range but poor selectivity. In-depth research on the structure-function relationship of UPOs and the development of protein structure prediction algorithms will help the engineering of UPOs and lay a foundation for solving the problem of poor substrate selectivity. This paper also compares several methods for the in situ regeneration of H₂O₂, and concluded that the multi-enzyme cascade method is the most economical and practical method for the in situ regeneration of H₂O₂.





Designing, building and rapid prototyping of biosynthesis module based on cell-free system

Shiming TANG, Jiyuan HU, Suiping ZHENG, Shuangyan HAN, Ying LIN

2022, 3(6):  1250-1261.  doi:10.12211/2096-8280.2022-024

Asbtract ( 1382 )   HTML ( 158)   PDF (1933KB) ( 1037 )  

Figures and Tables | References | Related Articles | Metrics

Bio-based chemical production has drawn attention regarding the realization of a sustainable society. With the development of metabolic engineering and synthetic biology, it is possible to make more efficient biosynthesis and scale-up commercial production of useful metabolites by metabolic pathway modification. Usually,some microorganisms are utilized as platforms by increasing the expression of desired genes and/or decreasing, that of undesired genes. Precise control of natural metabolism is, however, still challenging due to the complicated regulatory architecture at the levels of transcription, translation, and post-translation. Hence, various strategies of design and construction of biosynthesis modules have been proposed to optimize and expedite the design-build-test cycles of developing biosynthetic system for renewable chemical synthesis in vitro to avoid laborious and expensive ways for the optimization of metabolism pathway,strain and biocatalysts for each new product. In this review, we discuss the strategies of modules design and their rapid prototyping based on cell-free protein synthesis for assembling biosynthetic pathway in vitro. Biosynthetic modules could be sets of enzymes that catalyze a cascade reaction for a specific purpose or chemical, containing the conversion of starting materials to intermediary metabolites, biosynthesis of target products from the intermediates, cofactor balance and phosphorylation. Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel, enabling easier pathway design with high modularity. Multiple modules could then been designed by different groups for different purpose with the help of metabolic pathway database,computational design tool and some general module design rules. Cell-free protein synthesis was here utilized to build and rapidly prototype the functionality of biosynthesis pathway and module. The further application of machine learning methods might also contribute to better “precision-robustness” design and construction of these modules. This process is also called cell-free pathway engineering which has been proved to be a powerful and flexible enabling technology, providing simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.





Identification of RiPPs precursor peptides and cleavage sites based on deep learning

Jingwei LYU, Zixin DENG, Qi ZHANG, Wei DING

2022, 3(6):  1262-1276.  doi:10.12211/2096-8280.2022-016

Asbtract ( 1187 )   HTML ( 102)   PDF (3013KB) ( 1014 )  

Figures and Tables | References | Related Articles | Metrics

Genome sequencing data showed explosive growth attributed to the rapid development of DNA sequencing technology. Ribosomally synthesized and post-translationally modified peptides are a kind of natural peptide product that gradually came into people's view in the last decade. These compounds are widely distributed in nature, diverse in structure and bioactivity, and are important sources of natural drugs. The discovery of RiPPs mainly relies on low-throughput biological experiments, which are accurate but costly. With the development of new information technologies, bioinformatics tools such as antiSMASH and RIPP-Prism can greatly accelerate the process of RiPPs mining. However, methods based on gene homology, such as searching for conserved biosynthetic enzymes, are still unable to effectively identify novel RiPPs with different biosynthetic mechanisms. Here, for the first time, based on the natural language processing pre-training model BERT, four deep learning models that can fully rely on sequence data to identify RiPPs instead of homology and genomic context information are proposed and trained on the same RiPPs dataset. Through verification and comparison of these models, the best model BERiPPs performs well on the RiPPs identification track and is as accurate as the homology-based method. BERiPPs can identify RiPPs precursor peptides and RiPPs classes in an unbiased manner regardless of the genomic background, extending the range of novel RiPPs captured by approximately 60% compared to homology-based approaches. By combining BERiPPs with a conditional random field, the prediction of the cleavage site of the leader peptide can be indirectly generated with high accuracy by the recognition of each amino acid label in the sequence. The deep learning based on the pre-training model provides the possibility for high-throughput mining of novel RiPPs in a manner different from that of the gene context-dependent methods and reveals the underlying biological relationship between precursor peptides and modified enzymes.





Efficient synthesis of gentamicin and its related products in industrial chassis cells

Liangliang WU, Yingying CHANG, Zixin DENG, Tiangang LIU

2022, 3(6):  1277-1291.  doi:10.12211/2096-8280.2022-004

Asbtract ( 1471 )   HTML ( 151)   PDF (3905KB) ( 1296 )  

Figures and Tables | References | Related Articles | Metrics

Gentamicin is a kind of aminoglycoside antibiotic, widely used to treat severe Gram-negative bacterial infections. As an important secondary metabolite produced by Micromonospora echinospora, its biosynthetic pathway has been studied for years and scientists have a clear understanding for the biosynthetic gene cluster. In order to increase the titer of gentamicin, this study used the industrial strain M. echinospora J1-020 to determine the gentamicin synthetic gene cluster and established a stable genetic manipulation method. On this basis, three promoters with strong (kasOp*), medium (rpsLp-cf ), and weak (ermE*) strengths were used to evaluate the optimal overexpression level of phosphotransferase GenP, and the corresponding attB/attP site integration mutant strains YC002, YC003, YC001 was then constructed. After shaking flask fermentation, the results showed that the titer of gentamicin C component of YC001, YC002 and YC003 strains were increased by 16.9 % [(1178±39)mg/L], 30.8 % [(1319±29)mg/L] and 18.8%[(1198±46)mg/L] respectively, compared with the original strain [(1008±57)mg/L]. At the same time, combined with the impurity content, it was determined that the medium-strength promoter has the best effect on controlling the overexpression of gene genP in the above three strains, so that the corresponding overexpression strain YC004 of the genP stably integrated in the genome was constructed through homologous recombination. After shaking flask fermentation, the results showed that the titer of gentamicin C component increased by 34.5 %[(1427±37)mg/L]. Then, using M. echinospora J1-020 as the chassis, the genQ knockout strain YC005 was constructed to produce the G418 as the single component. The results showed that the titer of G418 was 460 mg/L. Finally, the gene genP overexpression strain YC004 as the starting strain, in which genB4 and genK were knocked out, was used to construct a double knockout mutant YC007 in order to produce sisomicin as a single component. After shaking flask fermentation, the titer of sisomicin was 1046 mg/L. It is expected that overproduction strains of various aminoglycoside antibiotics can be readily constructed by rational metabolic engineering strategies in the industrial chassis.