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| 1 | Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems显示文摘Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However,maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP)yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitrosynthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymaticreaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for thestoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this syntheticmodule, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP),respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6mW/cm^(2),whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0mM FDP from 50 g/L (139mM) maltose by adopting afed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novelstrategies for the high-efficient production of bioelectricity and value-added biochemicals. | Guowei Li Xinlei Wei Ranran Wu Wei Zhou Yunjie Li Zhiguang Zhu Chun You | 2022 | BioDesign Research2022，，1： | 2 |
| 2 | The Role of Synthetic Biology in Atmospheric Greenhouse Gas Reduction: Prospects and Challenges显示文摘The long atmospheric residence time of CO2 creates an urgent need to add atmospheric carbon drawdown to CO2 regulatory strategies.Synthetic and systems biology(SSB),which enables manipulation of cellular phenotypes,offers a powerful approach to amplifying and adding new possibilities to current land management practices aimed at reducing atmospheric carbon.The participants(in attendance:Christina Agapakis,George Annas,Adam Arkin,George Church,Robert Cook-Deegan,Charles DeLisi,Dan Drell,Sheldon Glashow,Steve Hamburg,Henry Jacoby,Henry Kelly,Mark Kon,Todd Kuiken,Mary Lidstrom,Mike MacCracken,June Medford,Jerry Melillo,Ron Milo,Pilar Ossorio,Ari Patrinos,Keith Paustian,Kristala Jones Prather,Kent Redford,David Resnik,John Reilly,Richard J.Roberts,Daniel Segre,Susan Solomon,Elizabeth Strychalski,Chris Voigt,Dominic Woolf,Stan Wullschleger,and Xiaohan Yang)identified a range of possibilities by which SSB might help reduce greenhouse gas concentrations and which might also contribute to environmental sustainability and adaptation.These include,among other possibilities,engineering plants to convert CO2 produced by respiration into a stable carbonate,designing plants with an increased root-to-shoot ratio,and creating plants with the ability to self-fertilize.A number of serious ecological and societal challenges must,however,be confronted and resolved before any such application can be fully assessed,realized,and deployed. | Charles DeLisi Aristides Patrinos Michael MacCracken Dan Drell George Annas Adam Arkin George Church Robert Cook-Deegan Henry Jacoby Mary Lidstrom Jerry Melillo Ron Milo Keith Paustian John Reilly Richard J.Roberts Daniel Segrè Susan Solomon Dominic Woolf Stan D.Wullschleger Xiaohan Yang | 2020 | BioDesign Research2020，，1： | 2 |
| 3 | Revealing CO_(2)-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics显示文摘The majority of marine microbes remain uncultured,which hinders the identification and mining of CO_(2)-fixing genes,pathways,and chassis from the oceans.Here,we investigated CO_(2)-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their ^(13)C-bicarbonate(^(13)C-HCO_(3)^(-))intake via single-cell Raman spectra(SCRS)analysis.The target cells were then isolated by Raman-activated Gravity-driven Encapsulation(RAGE),and their genomes were amplified and sequenced at one-cell resolution.The single-cell metabolism,phenotype and genome are consistent.We identified a notyet-cultured Pelagibacter spp.,which actively assimilates ^(13)C-HCO_(3)^(-),and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO_(2) fixation,a complete gene set for a rhodopsin-based light-harvesting system,and the full genes necessary for carotenoid synthesis.The four proteorhodopsin(PR)genes identified in the Pelagibacter spp.were confirmed by heterologous expression in E.coli.These results suggest that hitherto uncultured Pelagibacter spp.uses light-powered metabolism to contribute to global carbon cycling. | Xiaoyan Jing Yanhai Gong Teng Xu Paul ADavison Craig MacGregor-Chatwin CNeil Hunter La Xu Yu Meng Yuetong Ji Bo Ma Jian Xu Wei E.Huang | 2022 | BioDesign Research2022，，1： | 1 |
| 4 | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research显示文摘The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolitesor therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitativecharacterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N.benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expressionof firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporterprotein and mRNA levels. In addition, we investigated the effects of terminator–promoter combinations on gene expressionand found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest andweakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterizedpromoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expressionstrengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0–362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequencesin N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering. | Chenfei Tian Yixin Zhang Jianhua Li Yong Wang | 2022 | BioDesign Research2022，，1： | 1 |
| 5 | Biodesign Research to Advance the Principles and Applications of Biosystems Design显示文摘Over the course of civilization,humans have increasingly expanded their freedom to live a better life.In comparison with the primitive society,our modern society has many more choices of life-supporting resources,such as yearround food supply,permanent shelters,diverse energy sources,and effective preventive and curing medicine.However,our society is currently still heavily relying on the resources provided by Mother Nature,which cannot meet the future global needs in terms of both quantity and quality under the pressure of population growth,natural resource reduction,and environmental deterioration.For example,the food sources originating from plants,animals,or microbes do not have the nutrition balance for optimal human health[1–3].Climate change and environmental deterioration threaten the food security[4–6].Increasingly,infectious diseases(e.g.,HIV/AIDS),genetic diseases(e.g.,cancer),and improper lifestyle-related disorders(e.g.,obesity)become more prevalent and remain challenging to be prevented,controlled,and cured.Conventional medical technologies and modern medicine development are also meeting the ceiling. | Xiaohan Yang Lei S.Qi Alfonso Jaramillo Zong-Ming(Max)Cheng | 2019 | BioDesign Research2019，，1： | 1 |
| 6 | Rebooting Synthetic Phage-Inducible Chromosomal Islands:One Method to Forge Them All显示文摘Phage-inducible chromosomal islands(PICIs)are a widespread family of mobile genetic elements,which have an important role in bacterial pathogenesis.These elements mobilize among bacterial species at extremely high frequencies,representing an attractive tool for the delivery of synthetic genes.However,tools for their genetic manipulation are limited and timing consuming.Here,we have adapted a synthetic biology approach for rapidly editing of PICIs in Saccharomyces cerevisiae based on their ability to excise and integrate into the bacterial chromosome of their cognate host species.As proof of concept,we engineered several PICIs from Staphylococcus aureus and Escherichia coli and validated this methodology for the study of the biology of these elements by generating multiple and simultaneous mutations in different PICI genes.For biotechnological purposes,we also synthetically constructed PICIs as Trojan horses to deliver different CRISPR-Cas9 systems designed to either cure plasmids or eliminate cells carrying the targeted genes.Our results demonstrate that the strategy developed here can be employed universally to study PICIs and enable new approaches for diagnosis and treatment of bacterial diseases. | Rodrigo Ibarra-Chávez Andreas F.Haag Pedro Dorado-Morales Iñigo Lasa JoséR.Penadés | 2020 | BioDesign Research2020，，1： | 1 |
| 7 | In-Depth Computational Analysis of Natural and Artificial Carbon Fixation Pathways显示文摘In the recent years,engineering new-to-nature CO_(2)-and C1-fixing metabolic pathways made a leap forward.New,artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham(CBB)cycle.The question remains how to best predict their in vivo performance and what actually makes one pathway“better”than another.In this context,we explore aerobic carbon fixation pathways by a computational approach and compare them based on their specific activity and yield on methanol,formate,and CO_(2)/H_(2)considering the kinetics and thermodynamics of the reactions.Besides pathways found in nature or implemented in the laboratory,this included two completely new cycles with favorable features:the reductive citramalyl-CoA cycle and the 2-hydroxyglutarate-reverse tricarboxylic acid cycle.A comprehensive kinetic data set was collected for all enzymes of all pathways,and missing kinetic data were sampled with the Parameter Balancing algorithm.Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway-specific activities.The specific activities of the reductive glycine pathway,the CETCH cycle,and the new reductive citramalyl-CoA cycle were predicted to match the best natural cycles with superior productsubstrate yield.However,the CBB cycle performed better in terms of activity compared to the alternative pathways than previously thought.We make an argument that stoichiometric yield is likely not the most important design criterion of the CBB cycle.Still,alternative carbon fixation pathways were paretooptimal for specific activity and product-substrate yield in simulations with C1 substrates and CO_(2)/H_(2)and therefore hold great potential for future applications in Industrial Biotechnology and Synthetic Biology. | Hannes Löwe Andreas Kremling | 2021 | BioDesign Research2021，，1： | 1 |
| 8 | Plant Biosystems Design for a Carbon-Neutral Bioeconomy显示文摘Our society faces multiple daunting challenges including finding sustainable solutions towards climate change mitigation;efficient production of food,biofuels,and biomaterials;maximizing land-use efficiency;and enabling a sustainable bioeconomy.Plants can provide environmentally and economically sustainable solutions to these challenges due to their inherent capabilities for photosynthetic capture of atmospheric CO2,allocation of carbon to various organs and partitioning into various chemical forms,including contributions to total soil carbon.In order to enhance crop productivity and optimize chemistry simultaneously in the above-and belowground plant tissues,transformative biosystems design strategies are needed.Concerted research efforts will be required for accelerating the development of plant cultivars,genotypes,or varieties that are cooptimized in the contexts of biomass-derived fuels and/or materials aboveground and enhanced carbon sequestration belowground.Here,we briefly discuss significant knowledge gaps in our process understanding and the potential of synthetic biology in enabling advancements along the fundamental to applied research arc.Ultimately,a convergence of perspectives from academic,industrial,government,and consumer sectors will be needed to realize the potential merits of plant biosystems design for a carbon neutral bioeconomy. | Udaya C.Kalluri Xiaohan Yang Stan D.Wullschleger | 2020 | BioDesign Research2020，，1： | 1 |
| 9 | Diverse Systems for Efficient Sequence Insertion and Replacement in Precise Plant Genome Editing显示文摘CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes.However,the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes.Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success.A recent study has significantly improved NHEJ-and HDR-mediated sequence insertion and replacement using chemically modified donor templates.Together with other newly developed precise editing systems,such as prime editing and CRISPR-associated transposases,these technologies will provide new avenues to further the plant genome editing field. | Yingxiao Zhang Yiping Qi | 2020 | BioDesign Research2020，，1： | 1 |
| 10 | Reconfiguring Plant Metabolism for Biodegradable Plastic Production显示文摘For decades,plants have been the subject of genetic engineering to synthesize novel,value-added compounds.Polyhydroxyalkanoates(PHAs),a large class of biodegradable biopolymers naturally synthesized in eubacteria,are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic pathways.It was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic wastes.However,after three decades of effort synthesizing PHAs,and in particular the simplest form polyhydroxybutyrate(PHB),and seeking to improve their production in plants,it has proven very difficult to reach a commercially profitable rate in a normally growing plant.This seems to be due to the growth defects associated with PHA production and accumulation in plant cells.Here,we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been encountered.Then,from the point of view of plant synthetic biology,we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production,with the goal of maximizing PHA yield while minimizing growth inhibition.Specifically,we suggest genetic elements that can be considered in genetic circuit design,approaches for nuclear genome and plastome modification,and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways. | Haiwei Lu Guoliang Yuan Steven H.Strauss Timothy J.Tschaplinski Gerald A.Tuskan Jin-Gui Chen Xiaohan Yang | 2020 | BioDesign Research2020，，1： | 1 |
| 11 | Advancing How We Learn from Biodesign to Mitigate Risks with Next-Generation Genome Engineering显示文摘In the last decade,the unprecedented simplicity and flexibility of the CRISPR-Cas system has made it the dominant transformative tool in gene and genome editing.However,this democratized technology is both a boon and a bane,for which we have yet to understand the full potential to investigate and rewrite genomes(also named“genome biodesign”).Rapid CRISPR advances in a range of applications in basic research,agriculture,and clinical applications pose new risks and raise several biosecurity concerns.In such a fast-moving field of research,we emphasize the importance of properly communicating the quality and accuracy of results and recommend new reporting requirements for results derived from next-generation genome engineering. | Paul E.Abraham Jessy L.Labbé Amber A.McBride | 2020 | BioDesign Research2020，，1： | 1 |
| 12 | Transporter Engineering in Microbial Cell Factory Boosts Biomanufacturing Capacity显示文摘Microbial cell factories (MCFs) are typical and widely used platforms in biomanufacturing for designing and constructingsynthesis pathways of target compounds in microorganisms. In MCFs, transporter engineering is especially significant forimproving the biomanufacturing efficiency and capacity through enhancing substrate absorption, promoting intracellular masstransfer of intermediate metabolites, and improving transmembrane export of target products. This review discusses thecurrent methods and strategies of mining and characterizing suitable transporters and presents the cases of transporterengineering in the production of various chemicals in MCFs. | Xiaodong Lv Haijie Xue Lei Qin Chun Li | 2022 | BioDesign Research2022，，1： | 0 |
| 13 | Data-Driven Synthetic Cell Factories Development for Industrial Biomanufacturing显示文摘Revolutionary breakthroughs in artificial intelligence (AI) and machine learning (ML) have had a profound impact on a widerange of scientific disciplines, including the development of artificial cell factories for biomanufacturing. In this paper, wereview the latest studies on the application of data-driven methods for the design of new proteins, pathways, and strains. Wefirst briefly introduce the various types of data and databases relevant to industrial biomanufacturing, which are the basis fordata-driven research. Different types of algorithms, including traditional ML and more recent deep learning methods, are alsopresented. We then demonstrate how these data-based approaches can be applied to address various issues in cell factorydevelopment using examples from recent studies, including the prediction of protein function, improvement of metabolicmodels, and estimation of missing kinetic parameters, design of non-natural biosynthesis pathways, and pathway optimization.In the last section, we discuss the current limitations of these data-driven approaches and propose that data-driven methodsshould be integrated with mechanistic models to complement each other and facilitate the development of synthetic strains forindustrial biomanufacturing. | Zhenkun Shi Pi Liu Xiaoping Liao Zhitao Mao Jianqi Zhang Qinhong Wang Jibin Sun Hongwu Ma Yanhe Ma | 2022 | BioDesign Research2022，，1： | 0 |
| 14 | Improving the Efficiency and Orthogonality of Genetic Code Expansion显示文摘The site-specific incorporation of the noncanonical amino acid(ncAA)into proteins via genetic code expansion(GCE)has enabled the development of new and powerful ways to learn,regulate,and evolve biological functions in vivo.However,cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge.In this review,we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components,constructing genomically recoded organism(GRO),utilization of unnatural base pairs(UBP)and quadruplet codons(four-base codons),and spatial separation of orthogonal translation. | Xian Fu Yijian Huang Yue Shen | 2022 | BioDesign Research2022，，1： | 0 |
| 15 | Perspective:The Rapidly Expanding Need for Biosecurity by Design显示文摘Advancing biotechnologies are revolutionizing not only health and medicine, but also many different sectors such as agriculture,energy, chemistry, and textiles. As synthetic biology is leveraged as a programmable platform for the creation and biodesign ofhigh-value biological medicines, foods, and commodities, the world is facing new territory in terms of ensuring the safety andsecurity of both novel and engineered biological organisms, as well as the biological and digital platforms in which they aredesigned. Biosecurity practices and policies have traditionally revolved around preventing the misuse of biological pathogens,primarily through controlling access to pathogens. The advent of biodesign capabilities, such as gene editors, gene synthesiscapabilities, and genetic engineering, requires a reevaluation of traditional biosecurity policies to mitigate risks associated withsuch engineering of biological entities. Here, features of “Biosecurity by Design” approaches are described, including theapplication of risk/benefit analysis and risk mitigation, post-COVID opportunities, and ethical global norms in the progressionof biodesign and growing bioeconomies. | Diane DiEuliis | 2022 | BioDesign Research2022，，1： | 0 |
| 16 | An Overview of Antiviral Peptides and Rational Biodesign Considerations显示文摘Viral diseases have contributed significantly to worldwide morbidity and mortality throughout history.Despite the existence of therapeutic treatments for many viral infections,antiviral resistance and the threat posed by novel viruses highlight the need for an increased number of effective therapeutics.In addition to small molecule drugs and biologics,antimicrobial peptides(AMPs)represent an emerging class of potential antiviral therapeutics.While AMPs have traditionally been regarded in the context of their antibacterial activities,many AMPs are now known to be antiviral.These antiviral peptides(AVPs)have been shown to target and perturb viral membrane envelopes and inhibit various stages of the viral life cycle,from preattachment inhibition through viral release from infected host cells.Rational design of AMPs has also proven effective in identifying highly active and specific peptides and can aid in the discovery of lead peptides with high therapeutic selectivity.In this review,we highlight AVPs with strong antiviral activity largely curated from a publicly available AMP database.We then compile the sequences present in our AVP database to generate structural predictions of generic AVP motifs.Finally,we cover the rational design approaches available for AVPs taking into account approaches currently used for the rational design of AMPs. | Ying-Chiang J.Lee Jaden D.Shirkey Jongbeom Park Karishma Bisht Alexis J.Cowan | 2022 | BioDesign Research2022，，1： | 0 |
| 17 | Activating Silent Glycolysis Bypasses in Escherichia coli显示文摘All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocksand reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarilyemployed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model andidentified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas(EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal andthe other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defectiveEMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerasedeletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression ofmethylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the “serine shunt” whichconverts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, toexplore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution studyusing an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals theflexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism. | Camillo Iacometti Katharina Marx Maria Hönick Viktoria Biletskaia Helena Schulz-Mirbach Beau Dronsella Ari Satanowski Valérie A.Delmas Anne Berger Ivan Dubois Madeleine Bouzon Volker Döring Elad Noor Arren Bar-Even Steffen N.Lindner | 2022 | BioDesign Research2022，，1： | 0 |
| 18 | Design of Protein Segments and Peptides for Binding to Protein Targets显示文摘Recent years have witnessed a rise in methods for accurate prediction of structure and design of novel functional proteins. Designof functional protein fragments and peptides occupy a small, albeit unique, space within the general field of protein design. Whilethe smaller size of these peptides allows for more exhaustive computational methods, flexibility in their structure and sparsity ofdata compared to proteins, as well as presence of noncanonical building blocks, add additional challenges to their design. Thisreview summarizes the current advances in the design of protein fragments and peptides for binding to targets and discussesthe challenges in the field, with an eye toward future directions. | Suchetana Gupta Noora Azadvari Parisa Hosseinzadeh | 2022 | BioDesign Research2022，，1： | 0 |
| 19 | Dawn of a New Era for Membrane Protein Design显示文摘A major advancement has recently occurred in the ability to predict protein secondary structure from sequence using artificialneural networks. This new accessibility to high-quality predicted structures provides a big opportunity for the protein designcommunity. It is particularly welcome for membrane protein design, where the scarcity of solved structures has been a majorlimitation of the field for decades. Here, we review the work done to date on the membrane protein design and set outestablished and emerging tools that can be used to most effectively exploit this new access to structures. | Shahin Sowlati-Hashjin Aanshi Gandhi Michael Garton | 2022 | BioDesign Research2022，，1： | 0 |
| 20 | iGEM 2021:A Year in Review显示文摘The international Genetically Engineered Machine (iGEM) Foundation has continued to promote synthetic biology educationthroughout its 2021 competition. The 2021 Virtual iGEM Jamboree was the culmination of the competition’s growth, with 350projects from 7314 innovators globally. Collegiate, high school, and community lab teams applied their ideas to the Registry ofStandard Biological Parts, designing biological systems that provide solutions to an international scope of issues. Theenvironmental, diagnostics, and therapeutics tracks continue to be the most prevalent focal points for projects, as students deviseapproaches to detrimental impacts of climate change and the COVID-19 pandemic. The competition exemplifies high standards ofhuman practices, biosafety, and biosecurity through responsible biological engineering. As the iGEM Foundation continuespioneering STEM education into the future, equal developments of the competition’s economic accessibility, global diversity, andlong-term impact are necessary to allow a larger range of thinkers to access the power of synthetic biology. | Hannah Moon | 2022 | BioDesign Research2022，，1： | 0 |
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