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    31 October 2022, Volume 3 Issue 5
    Comment
    Cell-free multi-enzyme machines for CO2 capture, utilization and its associated challenges
    Jianming LIU, Anping ZENG
    2022, 3(5):  825-832.  doi:10.12211/2096-8280.2022-033
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    Global warming, mainly caused by the emission of carbon dioxide, is becoming a serious problem, and it is urgent to develop efficient carbon dioxide capture and utilization technologies. The use of biomanufacturing technology to fix carbon dioxide is an important research direction in synthetic biology. The mining and in vitro assembly of cell-free multi-enzyme machines have the great potential to facilitate the conversion of carbon dioxide into high-value products. The advantages associated with cell-free biosynthesis, such as clear background, relatively simple metabolic regulation, and high-yield production, make it ideal for biomanufacturing. Recently, the team of James C. Liao designed and developed a novel multi-enzyme molecular machine, and established a reductive glyoxylate-pyruvate synthesis cycle, which can theoretically realize the conversion of 2 molecules of carbon dioxide (bicarbonate) to 1 molecule of glyoxylic acid. They also designed and developed a strategy to control the concentration of cofactors during the reaction to improve the enzyme stabilities. In this comment, we discuss this work from the perspectives of in vitro multi-enzyme assembly and cofactor engineering, and point to associated challenges of carbon dioxide utilization by multi-enzyme machines.

    Invited Review
    Biological carbon fixation: from natural to synthetic
    Lu XIAO, Yin LI
    2022, 3(5):  833-846.  doi:10.12211/2096-8280.2022-042
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    In recent years, the increase in the atmospheric concentration of CO2 has caused serious environmental problems such as climate change, and carbon neutrality is presently a topic of global interest. Achieving carbon neutrality means to convert the atmospheric CO2 into carbon-based compounds. Converting CO2 into organics that can be used by humans is one of the effective ways to utilize CO2. Among them, biological carbon fixation has received great interest. In nature, plants and microbes can fix CO2 through carbon fixation pathways. Researchers have also designed several novel artificial carbon fixation pathways for CO2 fixation. Research on biological carbon fixation has mainly focused on the modification of natural carbon fixation pathways and the design and synthesis of artificial carbon fixation pathways. Since the carbon atom in the CO2 is in the highest oxidation state and the reduction of CO2 into organics requires energy input, the input of reducing power and energy is one of the key factors determining the efficiency of carbon biofixation. This review summarizes the advances achieved in recent years in the engineering of natural carbon fixation pathways and the design and construction of artificial carbon fixation pathways. The efficiencies of the artificial carbon fixation pathways, including the utilization of CO2-derived one carbon compounds, and the natural carbon fixation pathways are compared. Subsequently, we highlight the importance of reducing power and energy supply in the process of artificial biological carbon fixation, including chemical energy such as ATP and reducing power, light energy and electric energy. Finally, we analyze the challenges and trends of biological carbon fixation in terms of pathways and energy, and propose strategies for future research on biological carbon fixation.

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

    Advances in synthetic biology for photosynthetic carbon assimilation
    Yangyang SHENG, Xiumei XU, Qiaohong ZHANG, Lixin ZHANG
    2022, 3(5):  870-883.  doi:10.12211/2096-8280.2022-019
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    With the increase of population and the decrease of cultivated land, human demand for food is increasing. Therefore, it is particularly important to ensure adequate food supply. Photosynthesis is the most important chemical reaction on the earth, which converts inorganic matter into organic matter through light reaction and carbon assimilation. More than 90% of plant dry matter comes from the carbon fixation reaction of photosynthesis. The assimilated organic matter of photosynthesis is the material basis for the formation of crop yield. Therefore, improving the efficiency of crop light energy utilization is an important way to improve crop yield. In recent years, the rapid development of synthetic biology in the fields of energy, materials, health and environment has provided new opportunities for improving plant photosynthetic efficiency. This paper highlights the research progress of synthetic biology in improving the carbon assimilation efficiency of photosynthesis, mainly focusing on: (1) Improving the carboxylation activity of Rubisco enzymes, including identifying Rubisco enzymes with high carboxylation activity, optimizing gene expression regulatory sequences on Rubisco, and co-expressing Rubisco chaperone proteins; (2) Introducing CO2 concentrating mechanisms, including C4 photosynthetic enzymes, cyanobacterial transporter proteins, and cyanobacterial carboxysomes; (3) Reducing photorespiration through the introduction of four photorespiratory branches: chloroplast glycerate bypass, peroxisomal glycerate bypass, chloroplast glycolate oxidation bypass, and 3-hydroxypropionate bypass, and the exploration of new branches of photorespiration; Finally, the new photosynthetic carbon fixation circuit is discussed. The design, transformation, optimization and reorganization of photosynthetic carbon assimilation module through synthetic biology will effectively improve the efficiency of carbon assimilation and ultimately improve crop yield.

    Engineering microalgae for photosynthetic biosynthesis: progress and prospect
    Jinyu CUI, Aidi ZHANG, Guodong LUAN, Xuefeng LYU
    2022, 3(5):  884-900.  doi:10.12211/2096-8280.2022-005
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    The development of highly efficient CO2 utilization technologies can alleviate the urgent pressure on the environment and energy, playing a vital role in achieving the goal of “carbon peak and neutrality”. Microalgae are an important group of photoautotrophic microorganisms, providing the main source of primary productivity in the biosphere, and also serving as important model organisms for photosynthesis research. In recent years, microalgae have also been considered as promising chassis for photosynthetic biosynthesis, directly converting solar energy and carbon dioxide into various bio-based products. This technological route is called photosynthetic biomanufacturing, which possesses the advantages of simultaneous carbon fixation and clean production. This review focuses on the perspectives of development models and application scenarios, and suggests trends related to the further development of photosynthetic biomanufacturing. Regarding the efforts to harness and utilize photosynthetic carbon flow in microalgae cells, we summarized and compared three widely adopted strategies, including novel species screening, environmental perturbations, and genetic engineering. The research progress, significant breakthrough, and representative application demonstration of development models were systematically summarized. In the future, the combination of promising chassis cells with desired industrial properties, systematic metabolic engineering to remodel the native metabolism, and specific environmental treatments to maximize synthesis capacities could be expected to generate next-generation advanced microalgae cell factories. The optimized microalgae cell factories with desired photosynthesis and biosynthesis properties could be expected to play important roles in the areas of biomedical therapy, biophotovoltaics, and bioastronautics through interdisciplinary technology cooperation and integrations. Microalgal synthetic biology is also expect to focus on solving emerging problems rising from new application scenarios and larger application scales, including the development and optimization of the synthetic biology toolboxes, engineering chassis cells toward more efficient photosynthesis, the development of anti-biocontamination and biosafety strategies for large-scale cultivation. Taken together, this review provides useful and updated information to facilitate the development of photosynthetic biosynthesis route with carbon fixation and clean production, providing certain feasible solutions for the "carbon peak and neutrality".

    Research progress in carbon neutrality oriented adaptive laboratory evolution of microalgae
    Quanyu ZHAO
    2022, 3(5):  901-914.  doi:10.12211/2096-8280.2021-096
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    Microalgae biotechnology is one of the potential ways to realize carbon peaking and carbon neutrality. At present, microalgae have key problems such as low carbon sequestration efficiency, low photosynthetic transformation efficiency and low content of active components. There are also some technological problems which greatly limit the pace of its industrialization. Most microalgae can not tolerate more than 2% CO2. Apart from 10%~25% CO2, there are other pollutants such as NO x and SO x in industrial flue gas. These flue gas components inhibit the growth of microalgae. If the tolerance of algal strains are not enhanced, microalgae can not achieve the goal of stable carbon sequestration. In order to solve the problems of microalgae industrialization, wastewater resources can be used to meet the water demand in microalgae cultivation, and the economy can be improved by growing high value-added products. It is necessary to construct new algae strains by means of biotechnology such as synthetic biology, and build a new technical route of carbon reduction or negative carbon according to the characteristics of microalgae carbon sequestration and metabolism. Adaptive laboratory evolution (ALE) has made some progress in improving CO2 fixation by microalgae, enhancing wastewater treatment and improving metabolic phenotype. Evolved algal strains resistant to high concentration of carbon dioxide and other environmental stresses have been achieved. However, the efficiency of ALE in microalgae needs to be improved, and there are few studies on the mining of synthetic biological elements based on carbon sequestration, photosynthesis and biosynthesis of active components. In order to overcome the above problems, it is urgent to change the strategy of ALE in microalgae, combined with the application of high-throughput ALE device to speed up the evolution process; Based on the existing evolved strains, the elements of tolerance genes, photosynthesis and biosynthesis of active components will be deeply excavated to lay a foundation for microalgae genetic transformation. It is vital for us to learn carefully from the existing experience of ALE in microorganisms, understand throughly the dynamic process of ALE in microalgae, and explore profoundly the basic law of ALE. Finally, the possible ways of laboratory adaptive evolution to meet the challenge of microalgae carbon neutrality are prospected.

    Optimization and upgradation of microalgal photosynthesis for carbon peak and carbon neutrality goals
    Song WANG, Sha WU, Yanan JIANG, Zhangli HU
    2022, 3(5):  915-931.  doi:10.12211/2096-8280.2022-031
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    Cyanobacteria evolved oxygenic photosynthesis approximately 2.5 billion years ago, which has gradually changed the composition of the atmosphere since then. In order to cope with rising greenhouse gas emission and severe environmental pollution, microalgae, important in carbon sequestration via photosynthesis in the ecosystem, has attracted growing attention. Although microalgae can outcompete terrestrial higher plants in terms of photosynthetic rate and solar energy conversion efficiency, the potentials of microalgal photosynthesis have not been materialized yet. In this paper, we reviewed the progress in modifications of microalgal photosynthesis and its related pathways. The employed approaches include modifications of light-harvesting antenna, manipulations of the expression levels of the key enzymes in Calvin-Benson-Bassham cycle, construction of photorespiratory bypass, as well as the engineering of carbon-concentrating mechanism to increase the photosynthetic efficiency in microalgae. The bottlenecks in the employed approaches have also been discussed. To further improve the photosynthetic capacity of microalgae, it is necessary to screen new components with higher photosynthetic performance from other species, especially the ones adapted to extreme conditions. Transcription factors and microRNAs may also play important roles in regulating photosynthetic efficiency and biomass accumulation of microalgae. While the construction of alternative carbon-fixation pathways and photorespiratory bypass can accelerate carbon fixation, the introduction of synthetic pathways, by which photosynthetic end-products can be consumed at a higher rate, can mitigate the sink limitation on photosynthesis. The development of synthetic biology provides unprecedented opportunities to generate microalgae species with higher energy conversion and carbon fixation rate, more resistance to photodamage but less production of reactive oxygen species. This paper proposes to construct high-efficiency engineering strains for carbon-sequestration by selection of optimal chassis, elucidation of regulatory mechanisms of carbon fixation, introduction of exogenous metabolic pathways and modifications of endogenous metabolic network. It can be expected that the further improvement of the carbon-sequestration ability of microalgae will effectively reduce carbon emissions and make substantial contributions to the achievement of China's goals for carbon sequestration.

    Challenges and opportunities in the research of Synechococcus chassis under the context of carbon peak and neutrality
    Fei TAO, Tao SUN, Yu WANG, Ting WEI, Jun NI, Ping XU
    2022, 3(5):  932-952.  doi:10.12211/2096-8280.2021-104
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    CO2 is both the primary greenhouse gas and an abundant carbon resource. Highly efficient CO2 utilization technologies, which can alleviate the urgent pressure of energy and environment, are considered as the crucial reliances for getting the goal of “carbon peak and neutrality”. Photoautotrophic cyanobacteria can directly convert CO2 into organic compounds only using solar energy. It is the main microbial chassis for developing light-driven cell factories that can produce useful compounds by capturing CO2. As a typical representative of cyanobacteria, Synechococcus possesses many advantages: fast growth rate, clear genetic background, and low nutritional requirements. It is currently a hotspot of cyanobacterial synthetic biology. In the context of “carbon peak and neutrality,” research of Synechococcus chassis is ushering unprecedented opportunities. This review discusses the rationality and opportunities in developing cyanobacterial chassis from the perspectives of natural evolution, historical geologic limitations, climate dependence, and energy conversion efficiency. The application potentials in energy production, chemical manufacturing, and carbon sequestration are proposed and discussed. The metabolic potential of cyanobacteria is also discussed for their carbon fixation, light utilization, and biodiversity. Then, we systematically review the significant research advances in cyanobacterial chassis development and application. First, we describe the recently developed gene-editing methods of cyanobacteria, which are very important for constructing and remodeling cyanobacteria chassis. The feasibility of developing base editing technology that can facilitate multiplex editing in cyanobacteria is discussed. The CRISPRi technology for Synechococcus is also summarized. Second, we review the adaptive evolution in cyanobacteria. Researches on direct chassis evolution based on continuous cultivation and genetic element evolution based on phage and error-prone PCR are summarized. We also discuss the potential of adaptive evolution in cyanobacteria. Third, we review the stress tolerance of Synechococcus, especially the resistance to multiple stresses. The reported genetic elements responsible for stress factors, such as intense light, alkali, low pH, high temperature, and high salinity, are described. Some newly identified chassis are discussed on their unique characteristics. We propose some strategies for the directed engineering, which are practible for enhancing the stress tolerance of Synechococcus. Fourth, we review the progress of cyanobacterial cell factories and describe the recent production of various compounds by cyanobacteria, including bulk chemicals and fine chemicals. Moreover, we review new methods for developing cyanobacterial cell factories. The coculture method is discussed on its advantages and applications. The nanoparticle-mediated NADP regeneration is also reviewed for its application in enhancing the efficiency of the cyanobacterial cell factories. The existing problems and challenges are also listed with corresponding proposed solutions and coping strategies. We believe that the in-depth exploration of these problems and challenges will promote the advancement of cyanobacterial synthetic biology. It is expected that breakthroughs will soon be made in light energy capture, carbon fixation, stress resistance, and metabolic reprogramming. It is also expected that we can eventually design and build an efficient photosynthetic chassis surpassing natural evolution, based on which next-generation light-driven microbial factories can be constructed. This will significantly propel the realization of “carbon neutrality”.

    From CO2 to value-added products—carbon neutral microalgal green biomanufacturing
    Zhongliang SUN, Hui CHEN, Qiang WANG
    2022, 3(5):  953-965.  doi:10.12211/2096-8280.2022-023
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    Currently, our world is facing the dual pressure of carbon emission reduction and resource shortage. China has also put forward a goal of reaching CO2 emission peak by 2030 and achieving carbon neutrality by 2060. At present, the production and manufacture of fuels and bulk chemical products mainly rely on petrochemical refining, which is facing the challenges of high risk of production safety, great pressure of environmental protection, and contradiction between supply and demand of oil and gas resources. In this context, the use of microalgae for direct CO2 fixation is expected to establish large-scale biomanufacturing with CO2 as raw material and sunlight as energy source, this is a new manufacturing mode that breaks away from the route of petrochemical industry, and has the typical characteristics of low carbon, recyclable, green, and clean. This emerging green industry is of strategic significance for solving the current issues of food security and energy shortages, through sustainable production of food, energy, chemicals, and pharmaceuticals. In addition, microalgae possess great potential in environment protection, thanks to their strong stress resistance, effective remediation of eutrophic elements such as nitrogen and phosphorus from wastewater, and simultaneous removal of SO x and NO x during CO2 utilization in flue gas. Therefore, compared with heterotrophic chassis cells, microalgae-based synthetic biology and bio-manufacturing also play a role in carbon sequestration and emission reduction, and microalgae have then attracted much attention in recent years as “green cell factories”. From the perspective of light-driven autotrophy, we summarize the latest progress of microalgae as a cell factory, introduce chassis transformation strategies, and then look into the future development of this technology. In particular, improved genetic manipulation and larger cultivation scales are critical for microalgae to serve as high efficiency chassis for synthetic biology, whereas promising directions include establishment of standardized systems for algal genome editing, deep understanding of metabolic flux and control for robust biosynthesis, as well as improvement of biomass productivity and photosynthesis efficiency. All in all, this review provides a useful reference to establish controllable and replicable processes for microalgae green biomanufacturing.

    Applications of regulatory engineering in photosynthetic cyanobacteria
    Zhengxin DONG, Tao SUN, Lei CHEN, Weiwen ZHANG
    2022, 3(5):  966-984.  doi:10.12211/2096-8280.2022-012
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    Energy shortage and environmental pollution are the limiting factors for the development of human society. Photosynthetic cyanobacteria have attracted increasing attention due to their ability to use solar energy for the fixation of CO2 for green production of biofuels and chemicals under low nutrient conditions. So far, nearly 100 kinds of fuels and chemicals have been synthesized in photosynthetic cyanobacteria directly from CO2, which is expected to promote CO2 utilization and contribute to “carbon neutrality”. However, the industrial applications of photosynthetic cyanobacteria are restricted by factors such as their slow growth rate, low biomass, low product yield, and poor environmental robustness. Regulatory engineering, enabling global regulation of metabolic networks and multi-level regulation of genes expression, is a powerful tool to address these challenges. In this paper, we firstly introduce the classification, mechanism, and function of three major regulatory systems, including two-component signal transduction systems (TCS), regulatory small RNAs (sRNAs), and σ factor in photosynthetic cyanobacteria. Subsequently, we briefly discuss the functions of regulatory elements in the regulatory systems of photosynthetic cyanobacteria related to the tolerance against high salt, short-chain alcohols, light stress, metal ions, oxidative stress, as well as heat and drought, and the regulation of carbon metabolism in the production of the target components. We systematically review the applications of regulatory engineering, based on engineered regulatory system elements, in improving the robustness of photosynthetic cyanobacteria under the above stress conditions and in optimizing the carbon fluxes towards product biosynthesis. Finally, we conclude with the future research focuses of regulatory engineering in photosynthetic cyanobacteria, highlighting research areas such as functional elucidation of the regulatory systems, toolbox development, multi-gene regulations, protein engineering of the regulatory systems, and systematic regulatory engineering. In a word, the regulatory systems are expected to be artificially designed through regulatory engineering to achieve accurate control of the global metabolic network to improve the robustness and cell growth of photosynthetic cyanobacteria, and more importantly the titer, yield, and productivity of the target components for industry-scale applications.

    Advances in the study on the modification of carbon dioxide metabolic pathways in plants
    Menglin SHI, Lin ZHOU, Qing WANG, Lei ZHAO
    2022, 3(5):  985-1005.  doi:10.12211/2096-8280.2022-002
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    Reducing carbon emissions is a major strategic decision in the process of sustainable development in China. In order to achieve the ambitious goal of “carbon peak, carbon neutrality” on schedule, China needs to make a breakthrough in improving the ecological carbon sink capacity. Plant photosynthesis is beneficial to increase the Earth's carbon sink by fixing atmospheric carbon dioxide or inorganic carbon to produce organic compounds, while CO2 is released in the process of photorespiration and respiration which reduce carbon sinks by degrading the organic compounds into other substrates. Energy utilization rate of the above-mentioned CO2 natural metabolic process is low, and those processes are difficult to be artificially modified and improved in plants due to the characters and limitations of the relevant enzymes. Therefore, reconstructing new artificial metabolic pathways with synthetic biology in plants is expected to greatly improve the plant CO2 fixation capacity, which is one of the effective ways to solve the bottleneck of humanity development in the future. In the present review, we introduce the metabolic pathways associated with CO2 fixation and release which are involved in plant photosynthesis, photorespiration and respiration, respectively, and then point out the potential targets that could be used for modification by synthetic biology. In each section, we mainly discuss the artificial carbon fixation pathways that have been implemented in plants and their underlying principles. Especially, the modification of photorespiration is particularly discussed and several pathways are mentioned in details which shed lights on the design of artificial pathway in the future. Then we compare the capacity of each pathway in carbon fixation and limitation. Finally, we propose the key questions of designing and synthesizing novel carbon fixation pathway in plants, and the zero-carbon releasing design is mainly discussed. The development trend of transformation of plant CO2 metabolic pathway by synthetic biology is also forecasted.

    Construction and enhancement of enzymatic bioelectrocatalytic systems
    Xinyu CUI, Ranran WU, Yuanming WANG, Zhiguang ZHU
    2022, 3(5):  1006-1030.  doi:10.12211/2096-8280.2022-018
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    Enzymatic bioelectrocatalysis, as a green and efficient catalytic technology, combines the advantages of enzymatic catalysis and electrocatalysis to enable the interconversion between chemical energy and electrical energy. It has received extensive attention in the fields of bioelectricity generation, electric energy storage, CO2 fixation, biosensing and monitoring, and so on. This review analyzes the recent developments and challenges of enzymatic bioelectrocatalysis. From the perspective of synthetic biology, the structure and function of oxidoreductases which catalyze many biological electron transfer reactions with high speed, selectivity and specificity, and the basic elements of enzymatic bioelectrocatalytic systems are introduced in detail. Strategies of enzyme engineering are discussed, including directed evolution, rational design, and the introduction of non-natural structural components. In addition, the construction of multienzyme complex modules and the enhancement of electron transfer at the biology-abiotic interface, both of which can improve the system performance, are presented. The issues of electron transfer and energy conversion efficiency are further highlighted, and the oriented immobilization of enzymes, electron transfer mechanism, and electrode material modification are discussed. Furthermore, some recent applications of enzymatic bioelectrocatalysis in the frontier fields of synthetic biology are summarized, including enzymatic fuel cells, biosensors, and enzymatic electrosynthesis. Taken together, this review proposes the future directions of engineering bioelectroactive parts, broadening reaction redox potentials, and scaling up reaction systems, in order to further boost the performance of enzymatic bioelectrocatalysis as well as increase its applicability.

    Design and construction of electroactive cells by synthetic biology strategies
    Zixuan YOU, Feng LI, Hao SONG
    2022, 3(5):  1031-1059.  doi:10.12211/2096-8280.2022-014
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    Bioelectrocatalytic systems (BESs) employ redox reactions of electroactive cells on electrodes to integrate biocatalysis and electrocatalysis, providing a green, economical and sustainable approach for energy and chemical production. Electroactive cells, including exoelectrogens that release electrons to the environment and electrotrophs that obtain electrons from the environment, have the ability to exchange electrons in inward and outward directions with the external environment and play a central role as microbial electrocatalysts in BESs. In the last decade, with in-depth researches on the electron transfer mechanisms including direct and indirect electron transfer pathways of electroactive cells, BESs with electroactive cells as the core component have been widely used in ecological environment management, green energy development, high-value chemical synthesis, and so on. However, the inefficient energy conversion rate of wild-type electroactive cells remains a major bottleneck for large-scale applications of BESs. This bottleneck is mainly caused by narrow available substrate spectrum, slow intracellular electron generation rate, weak bidirectional electron transfer capability, unstable biofilm structure, and low microbial electrosynthesis efficiency. Thus, this review focuses on the latest research progresses in the synthetic biology engineering of electroactive cells in the past five years. By disassembling and analyzing the bidirectional electron transfer mechanisms, the synthetic biology strategies are classified and summarized for electrogenic cells and electrophic cells, respectively. Electrogenic cells can be engineered via strengthening the intracellular electron production and extracellular transfer efficiency, with a special focus on broadening the substrate spectrum, improving intracellular electron release, accelerating extracellular electron transfer and strengthening electroactive biofilms formation. In terms of electrophic cells, they can be engineered via enhancing electrophic electron uptake and conversion as well as product synthesis efficiency, specifically promoting extracellular electron uptake and conversion and regulating cellular metabolic pathways to electrosynthesize chemicals and biofuels. Finally, perspectives on further engineering of electroactive cells and potential applications of BESs are proposed.