Table of Content

31 December 2023, Volume 4 Issue 6

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Invited Review



Progress in synthetic biology research of Clostridium thermocellum for biomass energy applications

Yan XIAO, Yajun LIU, Yin′gang FENG, Qiu CUI

2023, 4(6):  1055-1081.  doi:10.12211/2096-8280.2023-046

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Biomass, including agricultural and forestry waste, energy plants, and microalgae, possesses both "energy" and "substance" properties, making it a promising renewable resource that can potentially replace fossil fuels. The efficient lignocellulose bioconversion relies on the development of effective biocatalysts. Clostridium thermocellum (also known as Ruminiclostridium thermocellum, Hungateiclostridium thermocellum, and Acetivibrio thermocellus) is a thermophilic anaerobic bacterium that can efficiently degrade lignocellulosic biomass. Over the past two decades, extensive research and development have led to the potential of using C. thermocellum as a cell factory to produce various energy and chemicals from lignocellulose. C. thermocellum has been used to produce ethanol, butanol, isobutanol, hydrogen, lactic acid, medium/short-chain fatty acid esters, and fermentable sugars from lignocellulosic biomass. The degradation and utilization process of lignocellulosic biomass by C. thermocellum mainly involves substrate recognition and hydrolysis through the cellulosome, hydrolysate uptake through ABC transporters, and intracellular metabolism via atypical glycolytic pathways. C. thermocellum possesses dynamic regulation of cellulosome production adapting extracellular substrates, which enables thehigh capability of degrading various lignocellulosic substrates. The cellulosome consists of non-catalytic scaffoldins and multiple enzymatic subunits with distinct catalytic activities and has broad applications in synthetic biology as well as lignocellulose degradation. In addition to lignocellulose refinery, the thermophilic C. thermocellum also has great potential in synthetic biology research under high-temperature conditions. Several genetic manipulation tools have been developed for C. thermocellum, although greater challenges have been encountered compared to model organisms such as Escherichia coli. The genetic tools include homologous recombination technology, Thermotargetron technology, and CRISPR/Cas systems, which enable gene knockout, insertion, replacement, mutation, and expression regulation of target genes in the strain. C. thermocellum has been used as the whole-cell biocatalyst for lignocellulose bioconversion through consolidated bioprocessing (CBP) and consolidated bio-saccharification (CBS). CBS follows the concept of sugar platform construction and shows great potential in real-world applications. The synthetic biology research targeting the CBS strategy still requires future development. For example, we need to explore new genetic tools and thermophilic functional elements for C. thermocellum and improve the efficiency of gene editing. We need to strengthen research on the genetic, physiological, and metabolic aspects of C. thermocellum, and the molecular mechanisms underlying lignocellulose degradation. It is noteworthy that, as a strict anaerobe, C. thermocellum cannot be used as the chassis for catalyzing oxygen-involved reactions. Selecting suitable metabolic pathways and target products will be the focus in future developments of synthetic biology based on C. thermocellum. Therefore, we need to investigate additional target pathways and products for synthetic biology development. In recent years, automation methods and artificial intelligence (AI) technologies are being developed rapidly and have been applied in various synthetic biology research fields. Such technologies may also be employed to promote research on thermophilic and anaerobic microorganisms.





Progress in the construction of microbial cell factories for efficient biofuel production

Xiongying YAN, Zhen WANG, Jiyun LOU, Haoyu ZHANG, Xingyu HUANG, Xia WANG, Shihui YANG

2023, 4(6):  1082-1121.  doi:10.12211/2096-8280.2023-047

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Biofuels are important supplements and alternatives to fossil fuels, which can alleviate the current global energy crisis and environmental pollution. Using microbes mined from nature or engineered in the lab to produce biofuels from renewable biomass of both economic and social benefits has become a major direction of sustainable biomanufacturing. It is necessary to develop robust microbial cell factories through synthetic biology for efficient and economic biofuel production, combining the strategy of systems biology to understand and design the synthetic pathways for biofuels and regulatory networks in microbes. This review discussed the major types of biofuels, the corresponding metabolic pathways, and current progress for producing these biofuels, including bioethanol, higher alcohols, biodiesel, fatty acid derivatives and isoprenoid derivatives. The strategies to understand, construct, and engineer synthetic microbial chassis as cell factories for diverse biofuel production were summarized, especially from substance metabolism, energy balance, physiological modification, and information regulation. In addition, current status and challenges for microbial biofuel production were analyzed. The insufficient understanding of natural biosynthetic pathways and the functions of biological components, lack of genetic manipulation tools for non-model biofuel chassis cells, low efficiency of gene editing, incompatibility between different heterologous pathways and chassis cells, toxicity of heterologous products and metabolic intermediates to cell factories, inhibition of many stress factors when using cheap renewable resources as raw materials, and engineering obstacles in industrial scale-up are the barriers and challenges to the industrial biofuel production. However, the rapid development of artificial intelligence and bioinformatics provides new solutions to these challenges. Finally, this review proposed future directions and key tasks based on the need for biofuel commercialization, emphasizing the combination of information technology and biotechnology as the trend in developing biofuel cell factories, which can provide tools and resources for strain engineering and accelerate the industrialization process of biofuels.





State-of-the-art for alcohol dehydrogenase development and the prospect of its applications in bio-based furan compounds valorization

Xiangshi LIU, Yilu WU, Peng ZHAN, Tianhao HUANG, Di CAI, Peiyong QIN

2023, 4(6):  1122-1139.  doi:10.12211/2096-8280.2023-059

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Alcohol dehydrogenase (ADH) is found widely in living cells, where it catalyzes the oxidation or reduction between hydroxyl group and carbonyl group. It also catalyzes redox reactions of a variety of organic compounds with high selectivity. In recent years, with the research progress of the catalytic mechanism, structural information, molecular modification, and reaction systems, ADH has shown great promise in the highly selective catalysis of various bio-based platform compounds. One example is the oxidation or reduction of furan derivatives such as furfural and 5-hydroxymethylfurfural, which are important sustainable building blocks for bio-jet fuels and biomaterials that are derived from tandem hydrolysis, isomerization, and dehydration of hemicelluloses and celluloses fractions in lignocellulose matrixes. This review focuses on the cutting-edge technologies in molecular design and directional engineering of ADH. In addition, the intensification of cofactor regeneration processes, including chemical-driven, enzyme-driven, and photo/electricity-driven pathways were also summarized. These methods could be the solutions to the negative aspects, such as expensive cost, poor stability, and the poor circulating efficiency of the nicotinamide cofactors that are indispensable assisted in typical ADH catalysis process. Moreover, the latest research progresses of ADH in catalysis of bio-based furans platform chemicals were also discussed. Apart from using ADH solely for the activation of the hydroxyl and carbonyl groups in the biobased furan derivates and the production of oxidative and reductive products, there is also of great promise in cascade ADH catalysis and other chemical or biological catalysis processes in one-pot under relatively mild conditions to valorize the furan derivates into valuable fine chemicals. Meanwhile, the whole-cell catalytic process that involves ADH and in vivo cofactors regeneration also possesses potentials in biobased furans valorization, with the advantages of low catalyst loading and processing costs. Overall, the researches of ADH in catalysis biological furans valorization has entered a new stage. With the further exploration of the potential applications of ADH, its role in the transformation of biomass resources will be increasingly important, particularly in the industrial process in the future.





Research progress and prospects in lipid metabolic engineering of eukaryotic microalgae

Han SUN, Jin LIU

2023, 4(6):  1140-1160.  doi:10.12211/2096-8280.2023-044

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Microalgae represent a diverse group of photosynthetic organisms that are widely found in various ecosystems on the Earth. They play a crucial role in carbon dioxide bio-fixation. Apart from their efficient growth through photosynthesis, many microalgae can also grow robustly under heterotrophic and mixotrophic conditions for high biomass production. Due to their high lipid content and the presence of diverse fatty acid and lipid species, microalgae have a wide range of applications in industries of energy, chemicals, and food. However, the high production cost associated with microalgae-based bioenergy poses a significant challenge for large-scale implementation. To overcome this, there is a growing interest in engineering microalgae to enhance lipid biosynthesis and accumulation, which holds promise for improving the economic feasibility of microalgal lipid production. This requires a better understanding of lipid metabolism and regulation in microalgae. This article provides an overview of recent advances in the elucidation of lipid metabolic pathways, the roles of key enzyme genes involved in lipid metabolism, and the transcriptional regulation of lipid metabolic pathways under different cultivation conditions in eukaryotic microalgae. It also summarizes strategies for metabolic engineering aiming for manipulating lipid biosynthesis-related enzymes, transcription factors, and competing pathways to increase lipid content and/or modify fatty acid composition in microalgae. Integrated analysis of genomics, transcriptomics, and proteomics data can help identify crucial nodes and key regulators in lipid metabolism, facilitating the identification of potential targets for metabolic engineering. Furthermore, the rapid development of genetic tools and gene editing technologies has significantly improved transformation efficiency and enabled precise gene modification, providing a foundation for genetic engineering of microalgae. By reshaping energy and carbon metabolic pathways, it becomes possible to design and optimize lipid biosynthesis processes in microalgae for a better production. Further research and exploration in genetic tools, gene editing technologies, metabolic pathway regulation, and large-scale implementation are of utmost importance for driving the research and development of microalgal lipid engineering.





Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

Huili SUN, Jinyu CUI, Guodong LUAN, Xuefeng LYU

2023, 4(6):  1161-1177.  doi:10.12211/2096-8280.2023-051

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The utilization of solar energy and carbon dioxide by cyanobacterial cell factories for photosynthetic ethanol production represents a promising and sustainable route towards green biofuels. Ethanol is one of the most representative products of the cyanobacterial photosynthetic biomanufacturing technology. Cyanobacterial ethanol production systems could serve as models for developing and optimizing advanced synthetic biology and metabolic engineering strategies. While most known cyanobacterial species lack the ability to synthesize and accumulate ethanol, the introduction and overexpression of heterologous pyruvate decarboxylase and alcohol dehydrogenase (heterologous or native) are required to enable ethanol synthesis in cyanobacteria. In the past decades, the performance of ethanol-producing cyanobacterial cell factories has been significantly improved through systematic optimization of proteins, pathways, chassis cells, and cultivation techniques. Cyanobacterial ethanol production technology has yielded the highest titer, productivity, and carbon partitioning ratio among all current cyanobacterial biomanufacturing systems. Recent advances have led to further improved efficiency of cyanobacterial ethanol photosynthetic production. Based on extensive systems biology data and rapidly developing computer modeling technologies, more accurate simulations of cyanobacterial physiological characteristics and metabolic networks have become possible. These simulations facilitate the identification of potential modification targets, thereby enhancing ethanol production capacity and guiding the design of next-generation alcohol-producing cell factories. With a more comprehensive understanding of cyanobacteria physiology and metabolism, systematic genome modifications and pathway optimizations have been performed, resulting in further improved ethanol productivity and final titers. Concurrently, efforts have been made to improve model strains and evaluate newly emerging non-conventional strains to establish more robust and efficient ethanol production processes. In conclusion, this review summarizes and compares three technological routes of light-driven carbon fixation and ethanol production in cyanobacteria, introduces the technological development trajectory and basic status of efficient light-driven carbon fixation for ethanol synthesis by cyanobacteria, provides valuable and up-to-date insights to facilitate the development of more promising cyanobacterial ethanol photosynthetic production technologies and explores future challenges and directions in this dynamic field.





Progress on bio-fixation and utilization of CO₂ in acetogens driven by chemical energy

Zhiqiong WEN, Yuzhen LI, Jin′gang ZHANG, Feifei Wang, Xiaoqing MA, Fuli LI

2023, 4(6):  1178-1190.  doi:10.12211/2096-8280.2023-045

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To promote the development of carbon utilization and biomanufacturing industry, one-carbon substrates are expected to be the next generation feedstocks and attracted much attention because of their abundance and availability for value-added products. Acetogens can naturally use syngas (CO, CO₂ and H₂) through the classical Wood-Ljungdahl pathway to support their growth and produce value-added chemicals such as acetic acid and ethanol. Compared to chemical catalysis, this biorefinery process has the advantages of low requirements for feedstocks, mild reaction conditions and high product selectivity. In some acetogens, both CO and H₂ can act as energy sources to provide the reducing equivalents required for growth and metabolism during gas fermentation. In particular, acetogens utilizing CO and H₂ have distinct patterns of energy metabolism. Compared to H₂, CO is a more thermodynamically favorable energy source for gas fermentation. In general, different acetogens during autotrophic growth with syngas as carbon source and energy source can produce different products. However, the low solubility of gases in liquid medium, as well as the poor mass transfer and diffusion, limits the energy supply during the fermentation, resulting in slow growth rate of microorganisms, low raw material utilization, and low target product yield. Therefore, improving syngas utilization efficiency and enlarging products spectrum of acetogens are both of great importance to meet the demands of industrialization. In recent years, with the development of synthetic biology and molecular genetic tools, modification of acetogen strains has been well designed. In this review, we briefly described the natural metabolic pathways such as ethanol and acetone metabolism pathways, and summarized the progress of acetogens in autotrophic fermentation using syngas as energy sources to produce value-added products such as ethanol, 2,3-butanediol, acetone and isoprepanol. For engineering the acetogens, we summarized the pathways to produce natural and non-natural chemicals. Finally, the energy capture and utilization of acetogens was prospected, with the aim to facilitate the future advancement of biorefineries.





Electro-assisted carbon dioxide biotransformation

Weisong LIU, Kuncheng ZHANG, Huijuan CUI, Zhiguang ZHU, Yiheng ZHANG, Lingling ZHANG

2023, 4(6):  1191-1222.  doi:10.12211/2096-8280.2023-041

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The increasing emission of CO₂ has resulted in severe climate problems, prompting global actions to reduce CO₂ emission or fix the atmospheric CO₂. In 2020, China set targets for carbon peaking and carbon neutrality, making it an urgent need to develop carbon-fixation technologies. Attributed to the rapid emergence of synthetic biology in recent years, CO₂ biotransformation through biochemical reactions catalyzed by enzymes and microbes has achieved a series of significant progress, in the design and engineering of enzymes, metabolic pathway, as well as the construction of in vitro/vivo systems. Many products, such as fuels, amino acids, starch, single-cell proteins, bio-based plastics, and other biocommodities, have been synthesized. Consequently, CO₂ is considered as the resource for third-generation biomanufacturing. The crucial step in CO₂ biotransformation is the activation of CO₂ molecules through the introduction of external energy. Compared to light, heat, and chemical energy, electrical energy is favored due to cost effectiveness, miniaturized apparatus, and convenience, attracting significant attention from both academia and industry. Electrical energy can be utilized in two ways for CO₂ biotransformation. In one way, CO₂ is electro-activated directly and biotransformed. In the other way, electrical energy facilitates the production of C1 intermediate such as formate, carbinol, CO, and the C₁ intermediates are then transformed by coupled microorganisms or enzymes, or the production of reducing forces such as NADH and H₂, which participate essentially in CO₂ biotransformation. This review comprehensively introduces research advancements in both approaches, analyzes potential carbon-fixation mechanisms, and discusses the advantages and disadvantages of different methods. Furthermore, the review proposes potential synthetic biology strategies to address efficiency concerns in CO₂ biotransformation, such as mining highly active carbon-fixing enzyme, enzyme engineering to improve the electron transfer efficiency between the enzyme and the electrode, metabolic engineering to enrich products of carbon-fixing microorganisms and improve the carbon-fixing efficiency, aiming to enable practical applications and the achievement of carbon neutrality goals.





Microbial conversion and in vitro enzymatic catalysis for carbon dioxide utilization: a review

Wei YE, Rui LI, Weihong JIANG, Yang GU

2023, 4(6):  1223-1245.  doi:10.12211/2096-8280.2023-050

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Carbon dioxide (CO₂) is the main greenhouse gas, but it also represents an abundant, cost-effective, safe, and easily accessible carbon resource. Driven by the national goals to achieve "carbon peaking and carbon neutrality", there has been an increasing interest in recent years in effective reduction of CO₂ emission and utilization of this one-carbon resource, thereby accelerating the development of carbon capture, utilization, and storage (CCUS) technologies. Biological conversion plays a major role in CCUS. This approach enables the transformation of CO₂ into desired products through either direct biological catalysis or in combination with chemical catalysis (using CO₂-derived organic compounds such as methanol, formic acid, and acetic acid). Thus, biological CO₂ fixation and conversion represents a promising solution for both utilization of greenhouse gas or industrial waste gases and sustainable production of bulk chemicals and fuels. However, the current state of biotransformation technology for CCUS is still in its infancy, leaving ample room for improvement in the efficiency, yield, and cost-effectiveness. In this review, we briefly summarize recent advances in biological utilization of CO₂, highlight the characteristics and limitations of the existing technologies, and also propose future research directions. Our aim is to provide a valuable reference to researchers in this field. Overall, industrial application of CO₂ bioconversion remains in its nascent phase. Although industrial-scale ethanol production through syngas (CO₂/CO) fermentation by chemoautotrophic bacteria has made significant strides, there is still a need for further improvement in the conversion efficiency of CO₂. In addition, gas fermentation should consider more value-added products beyond ethanol to enhance the economic viability of this technology. Other CO₂ bioutilization technologies, such as the coupling of chemical and biological conversion and in vitro enzymatic catalysis, have yet to bridge the gap to large-scale applications. Therefore, further optimization of these technical systems and reduction of production cost are essential to meet the needs of industrial applications.





Progress in the bioconversion of biogas into sustainable aviation fuel

Chenyue ZHANG, Yingqun MA, Xing WANG, Rongzhan FU, Jiwei HUANG, Xiufu HUA, Daidi FAN, Qiang FEI

2023, 4(6):  1246-1258.  doi:10.12211/2096-8280.2023-048

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Biogas primarily composed of methane (CH₄) and carbon dioxide (CO₂) is recognized as a clean and renewable energy source with potential to replace fossil fuels. Currently, the most common way to utilize biogas is by using combined heat and power (CHP) units to generate electricity and heat. However, burning CH₄ in biogas releases an equivalent amount of CO₂, resulting in a lower carbon-atom economy. To enhance the carbon-utilization efficiency of biogas and reduce greenhouse gas emission, this review suggests a novel route of biological converting both CH₄ and CO₂ in biogas produced from anaerobic digestion of food wastes for sustainable aviation fuel (SAF) production with applications of synthetic biology techniques and biomanufacturing strategies. Photoautotroph microorganisms and aerobic methanotrophs are used to convert CO₂ and CH₄ in biogas, respectively. Primary pathways and key enzymes for lipid biosynthesis from CO₂ and CH₄ by photoautotrophic microbes and methanotrophic bacterial and strategies for carbon flux improvement are introduced and discussed. As the precursor of SAF, the lipids produced by aforementioned microbes need to undergo recovery, pre-treatment, and upgrading procedures. The effects of different technologies developed for lipid recovery (flocculation, dissolved air flotation (DAF), centrifugation, coagulation, filtration) and upgrading (Hydrogenated Esters and Fatty Acids (HEFA), Fischer-Tropsch (F-T), Alcohol-to-jet (ATJ), Hydroprocessed Fermented Sugars (HFS) on efficiency and operation cost are evaluated. Besides, physical properties of SAF derived from different raw materials are compared. The global warming potential of SAF production using different feedstock by HEFA are summarized and the reduction of greenhouse emission can be up to 80% comparing with petroleum-based ones. This review discusses the metabolic pathways, biosynthesis strategies, fermentation technology, recovery and upgrading processes for the production of biogas-derived SAF. It also provides an outlook on strategies to improve the economic efficiency of microbes-based SAF manufacturing and guideline for commercial applications of biotechnology in fuel production.





Biophotovoltaics: an environmentally friendly technology for solar energy utilization

Huawei ZHU, Yin LI

2023, 4(6):  1259-1280.  doi:10.12211/2096-8280.2023-039

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Biophotovoltaics (BPV) is an environmentally friendly power generation technology that uses self-renewing photosynthetic microorganisms to absorb solar energy and convert it into electricity. BPV is an energy transduction process that involves photochemical reactions occurring in photosynthetic cells, extracellular electron transfer occurring at cell-electrode interfaces, and electrical current generation occurring in bioelectrochemical systems. However, the intrinsic light-dependent exoelectrogenic activity of photosynthetic microorganisms is extremely weak, which hampers the electrical outputs of BPV systems. In recent years, different electron transfer strategies have been developed to more efficiently extract photosynthetic electrons. These include the exogenous electron mediators-based strategy, conductive nanomaterials-based strategy, and synthetic microbial consortia-based strategy. Among them, the exogenous electron mediators-based strategy could improve the instantaneous efficiency; however, the improvement tends to be unstable. The conductive nanomaterials-based strategy can hardly achieve a targeted distribution of nanomaterials, and the cost of nanomaterials is also an issue. The synthetic microbial consortia-based strategy shows great potential in enhancing the power output and prolonging the lifetime of BPV systems. This review gives an overview of the development history of BPV technology, and summarizes the fundamental principles, advantages, and disadvantages of different strategies for electron extraction. Moreover, we also discuss different biotic/abiotic approaches that have been taken to improve the electrical outputs of BPV systems. These approaches mainly include broadening available photosynthetic materials, remolding intracellular metabolism and electron transfer, developing advanced electrodes with new materials and structures, and designing high-performance devices with novel configurations. Furthermore, we envision the real-world applications of BPV technology in the future, e.g., running small electronic sensors on environmental and agricultural internet of things, driving hydrogen production, and even being applied in space scientific research. To this end, the scale-up of BPV systems is required to amplify the output voltage and output power. Lastly, we propose that it is crucial to understand the molecular mechanisms behind the exoelectrogenesis of photosynthetic microorganisms in order to facilitate the advancement of BPV technology. One possible approach is to utilize synthetic biology to reconstruct transmembrane molecular wires using multi-heme cytochromes or nanowires.





Advances and applications of gene editing and transcriptional regulation in electroactive microorganisms

Yaru CHEN, Yingxiu CAO, Hao SONG

2023, 4(6):  1281-1299.  doi:10.12211/2096-8280.2023-056

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Electroactive microorganisms (EAMs) engage in bidirectional electron exchange with extracellular electron acceptors/donors through the extracellular electron transfer (EET) pathways, resulting in the generation or consumption of electric current. EAMs have been widely applied in many microbial electrochemical technologies, such as biogeochemical cycling of Earth elements, bioremediation of environmental pollutants, electricity production, biosensing, biomining, and microbial electrosynthesis of chemicals, rendering EAMs a focal point in the global pursuit of environmental conservation and low-carbon economy. However, there are still substantial limitations in the practical applications of EAMs. For example, microbial fuel cells encounter a capped upper limit in power density, and the CO₂ reduction rate in microbial electrosynthesis remains below the desired threshold for practical applications. Overcoming these challenges necessitates enhancement in the bidirectional EET rate of EAMs. Nonetheless, complex phenotypes such as elevated EET efficiency often correlate with the expression of multiple genes. To obtain high-performance EAM strains, a deep comprehension of the genotype-phenotype relationship in EAMs and more nuanced manipulation at the genomic level are imperative. This review provides a comprehensive summary of the latest advances in genome editing and transcriptional regulation in EAMs. The main focus is on the CRISPR (clustered regularly interspaced short palindromic repeat)-based biotechnologies developed in model EAMs, such as Shewanellaoneidensis and Geobactersulfurreducens, and a few other representative EAMs. The genome editing techniques to be discussed include (CRISPR-assisted) homologous recombination, CRISPR-associated transposase systems, and base editing. Similarly, transcriptional regulation tools involve CRISPR-based interference (CRISPRi) and activation (CRISPRa) systems. Strategies and advancements related to multiplexed editing and regulation are thoroughly summarized. Subsequently, the review delves into the applications of these technologies in both fundamental and applied scientific domains. On the fundamental science front, efforts are directed toward unveiling factors related to EET and uncovering hidden genotypes. In the realm of application, green electricity-producing microbial fuel cells, bioremediation of nuclear waste, heavy metals, and azo dyes are discussed. Finally, current challenges and future directions in the genetic engineering of EAMs are discussed.



Research Article



Metabolic flux analysis of Shewanella sp. MR-4 with different terminal electron receptors

Qingxiang ZHENG

2023, 4(6):  1300-1320.  doi:10.12211/2096-8280.2022-052

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Shewanella is ubiquitous in natural environments, known to show various unique activities, such as metal reduction and trimethylamine production. The metal reduction capability expands the range of electron acceptors and enables its application in producing current in microbial fuel cells. Previous work primarily focused on the model organism Shewanella oneidensis MR-1, based on which the metabolic models were constructed for three other strains including Shewanella sp. MR-4. Interestingly, the genetic similarity does not always coincide with metabolic phenotype similarity between these two strains, suggesting that potential regulatory effects affecting cellular phenotypes are present in Shewanella sp.MR-4. This study analyzed the metabolic flux distribution of Shewanella sp. MR-4 when Fe³⁺, fumaric acid, nitric acid and dimethyl sulfoxide were used as electron acceptors under anaerobic conditions. The intracellular metabolic flux distribution was significantly changed with different electron acceptors. The formation rate of acetic acid was decreased, while the metabolic flux of the tricarboxylic acid (TCA) cycle was increased with Fe³⁺ and nitric acid as electron acceptors, compared to that with fumaric acid. The anaplerotic reaction in the TCA cycle was also dramatically altered: the phosphoenolpyruvate carboxylase-catalyzed reaction or malic enzyme-catalyzed reaction is the only anaplerotic reaction with fumaric acid or Fe³⁺ and nitric acid as electron acceptor, respectively. Based on the metabolic flux map, quantitative analysis was performed to derive the synthesis and consumption rates of reducing agent NADH, and the change of electron transfer. It was found that NADH metabolism varies significantly with the electron acceptors tested. The results provide a theoretical basis for the future metabolic engineering of Shewanella and experimental evidences that can be used for future study of different phenotypes unaccounted for in previous models of Shewanella sp. MR-4 and MR-1.