The third-generation(3G)biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon(C1)sources,such as CO_(2),formate,and methanol,fueled by renewable ener...The third-generation(3G)biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon(C1)sources,such as CO_(2),formate,and methanol,fueled by renewable energies like light and electricity.This promising technology represents an important step toward sustainable development,which can help address some of the most pressing environmental challenges faced by modern society.However,to establish processes competitive with the petroleum industry,it is crucial to determine the most viable pathways for C1 utilization and productivity and yield of the target products.In this review,we discuss the progresses that have been made in constructing artificial biological systems for 3G biorefineries in the last 10 years.Specifically,we highlight the representative works on the engineering of artificial autotrophic microorganisms,tandem enzymatic systems,and chemobio hybrid systems for C1 utilization.We also prospect the revolutionary impact of these developments on biotechnology.By harnessing the power of 3G biorefinery,scientists are establishing a new frontier that could potentially revolutionize our approach to industrial production and pave the way for a more sustainable future.展开更多
Globally,agriculture depends on industrial nitrogen fertilizer to improve crop growth.Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution.A potential solution would be to ha...Globally,agriculture depends on industrial nitrogen fertilizer to improve crop growth.Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution.A potential solution would be to harness nitrogenases—enzymes capable of converting atmospheric nitrogen N2 to NH3 in ambient conditions.It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants,or bacteria that form symbiotic relationships with crops,to support growth and reduce dependence on industrially produced fertilizer.This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.展开更多
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...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.展开更多
High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels.Gene transcription is tightly regulated by promoters and terminators.Promo...High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels.Gene transcription is tightly regulated by promoters and terminators.Promoters determine the timing,tissues and cells,and levels of the expression of genes.Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally,e.g.,the 3′-end processing,stability,translation efficiency,and nuclear to cytoplasmic export of mRNAs.The promoter and terminator combination affects gene expression.In the present article,we review the function and features of plant core promoters,proximal and distal promoters,and terminators,and their effects on and benchmarking strategies for regulating gene expression.展开更多
Probiotics are the treasure of the microbiology fields.They have been widely used in the food industry,clinical treatment,and other fields.The equivocal health-promoting effects and the unknown action mechanism were t...Probiotics are the treasure of the microbiology fields.They have been widely used in the food industry,clinical treatment,and other fields.The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic’s developed applications.In recent years,various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics.As important genome editing tools,CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics.The high efficiency,flexibility,and specificity are the advantages of using CRISPR-Cas systems.Here,we summarize the classification and distribution of CRISPR-Cas systems in probiotics,as well as the editing tools developed on the basis of them.Then,we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems.Finally,we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.展开更多
Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis.In this study,we investigated multidimensional strategies to improve the carotenoid synthesis in the indu...Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis.In this study,we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae.First,we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply.Second,we restricted the consumption of farnesyl pyrophosphate(FPP)by the down-regulation of squalene synthase using the PEST degron.Third,we further explored the human lipid binding/transfer protein saposin B(hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids.Last,the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation.By combining the abovementioned strategies,the final engineered yeast produced 166.79±10.43 mg/Ⅰβ-carotene in shake flasks,which was nearly 5-fold improvement of the parental carotenoid-producing strain.Together,we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.展开更多
Plants are complex systems hierarchically organized and composed of various cell types.To understand the molecular underpinnings of complex plant systems,single-cell RNA sequencing(scRNA-seq)has emerged as a powerful ...Plants are complex systems hierarchically organized and composed of various cell types.To understand the molecular underpinnings of complex plant systems,single-cell RNA sequencing(scRNA-seq)has emerged as a powerful tool for revealing high resolution of gene expression patterns at the cellular level and investigating the cell-type heterogeneity.Furthermore,scRNA-seq analysis of plant biosystems has great potential for generating new knowledge to inform plant biosystems design and synthetic biology,which aims to modify plants genetically/epigenetically through genome editing,engineering,or re-writing based on rational design for increasing crop yield and quality,promoting the bioeconomy and enhancing environmental sustainability.In particular,data from scRNA-seq studies can be utilized to facilitate the development of high-precision Build-Design-Test-Learn capabilities for maximizing the targeted performance of engineered plant biosystems while minimizing unintended side effects.To date,scRNA-seq has been demonstrated in a limited number of plant species,including model plants(e.g.,Arabidopsis thaliana),agricultural crops(e.g.,Oryza sativa),and bioenergy crops(e.g.,Populus spp.).It is expected that future technical advancements will reduce the cost of scRNA-seq and consequently accelerate the application of this emerging technology in plants.In this review,we summarize current technical advancements in plant scRNA-seq,including sample preparation,sequencing,and data analysis,to provide guidance on how to choose the appropriate scRNA-seq methods for different types of plant samples.We then highlight various applications of scRNA-seq in both plant systems biology and plant synthetic biology research.Finally,we discuss the challenges and opportunities for the application of scRNA-seq in plants.展开更多
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 sel...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.展开更多
Human life intimately depends on plants for food,biomaterials,health,energy,and a sustainable environment.Various plants have been genetically improved mostly through breeding,along with limited modification via genet...Human life intimately depends on plants for food,biomaterials,health,energy,and a sustainable environment.Various plants have been genetically improved mostly through breeding,along with limited modification via genetic engineering,yet they are still not able to meet the ever-increasing needs,in terms of both quantity and quality,resulting from the rapid increase in world population and expected standards of living.A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches.This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems.Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes.From this perspective,we present a comprehensive roadmap of plant biosystems design covering theories,principles,and technical methods,along with potential applications in basic and applied plant biology research.We highlight current challenges,future opportunities,and research priorities,along with a framework for international collaboration,towards rapid advancement of this emerging interdisciplinary area of research.Finally,we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception,trust,and acceptance.展开更多
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 ...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.展开更多
Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity.Disease-resistant...Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity.Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology.Nevertheless,enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects.Upstream open reading frames(uORFs)are translational control elements located in the 5′untranslated region(UTR)of eukaryotic mRNAs and may repress the translation of downstream genes.To investigate the function of three uORFs from the 5′-UTR of ACCELERATED CELL 11(uORFsACD11),we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation.Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels.Importantly,uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves.When coupled with the PATHOGENESIS-RELATED GENE 1(PR1)promoter,the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2(AtLecRKVI.2)during pathogen attack and enhance plant resistance to Phytophthora capsici.These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack,while for ensuring lower levels of protein expression at normal conditions.展开更多
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 th...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.展开更多
Lignin is one of the most widespread organic compounds found on earth,boasting a wealth of aromatic molecules.The use of lignin feedstock for biochemical productions is of great importance for achieving"carbon ne...Lignin is one of the most widespread organic compounds found on earth,boasting a wealth of aromatic molecules.The use of lignin feedstock for biochemical productions is of great importance for achieving"carbon neutrality."In recent years,a strategy for lignin valorization known as the"bio-funnel"has been proposed as a means to generate a variety of commercially valuable chemicals from lignin-derived compounds.The implementation of biocatalysis and metabolic engineering techniques has substantially advanced the biotransformation of depolymerized lignin into chemicals and materials within the supply chain.In this review,we present an overview of the latest advancements in microbial upcycling of depolymerized lignin into value-added chemicals.Besides,the review provides insights into the problems facing current biological lignin valorization while proposing further research directions to improve these technologies for the extensive accomplishment of the lignin upcycling.展开更多
The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzym...The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise;however,a thorough comparison and evaluation of their bactericidal efficacy are lacking.This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes,with the goal of harnessing them for antibacterial therapy.First,we examined the bactericidal activity of spanins,endolysins,and holins derived from 2 Escherichia coli model phages,T1 and T7.Among these,T1-spanin exhibited the highest bactericidal activity against E.coli.Subsequently,we expressed T1-spanin within bacterial cells and assessed its bactericidal activity.T1-spanin showed potent bactericidal activity against all clinical isolates tested,including bacterial strains of 111 E.coli,2 Acinetobacter spp.,3 Klebsiella spp.,and 3 Pseudomonas aeruginosa.In contrast,T1 phage-derived endolysin showed bactericidal activity against E.coli and P.aeruginosa,yet its efficacy against other bacteria was inferior to that of T1-spanin.Finally,we developed a phage-based technology to introduce the T1-spanin gene into target bacteria.The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria.The potent bactericidal activity exhibited by spanins,combined with the novel phage synthetic technology,holds promise for the development of innovative antimicrobial agents.展开更多
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 esp...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.展开更多
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,of...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.展开更多
Many applications in plant biology requires editing genomes accurately including correcting point mutations,incorporation of single-nucleotide polymorphisms(SNPs),and introduction of multinucleotide insertion/deletion...Many applications in plant biology requires editing genomes accurately including correcting point mutations,incorporation of single-nucleotide polymorphisms(SNPs),and introduction of multinucleotide insertion/deletions(indels)into a predetermined position in the genome.These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems,which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence.However,low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient.Another kind of technology known as base editing can perform precise editing;however,only certain types of modifications can be obtained,e.g.,C/G-to-T/A and A/T-to-G/C.Recently,a new type of genome-editing technology,referred to as“prime editing,”has been developed,which can achieve various types of editing such as any base-to-base conversion,including both transitions(C→T,G→A,A→G,and T→C)and transversion mutations(C→A,C→G,G→C,G→T,A→C,A→T,T→A,and T→G),as well as small indels without the requirement for inducing double-stranded break in the DNA.Because prime editing has wide flexibility to achieve different types of edits in the genome,it holds a great potential for developing superior crops for various purposes,such as increasing yield,providing resistance to various abiotic and biotic stresses,and improving quality of plant product.In this review,we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.展开更多
Plants adapt to their changing environments by sensing and responding to physical,biological,and chemical stimuli.Due to their sessile lifestyles,plants experience a vast array of external stimuli and selectively perc...Plants adapt to their changing environments by sensing and responding to physical,biological,and chemical stimuli.Due to their sessile lifestyles,plants experience a vast array of external stimuli and selectively perceive and respond to specific signals.By repurposing the logic circuitry and biological and molecular components used by plants in nature,genetically encoded plant-based biosensors(GEPBs)have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected.GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development.GEPBs are also useful for environmental monitoring,plant abiotic and biotic stress management,and accelerating design-build-test-learn cycles of plant bioengineering.With the advent of synthetic biology,biological and molecular components derived from alternate natural organisms(e.g.,microbes)and/or de novo parts have been used to build GEPBs.In this review,we summarize the framework for engineering different types of GEPBs.We then highlight representative validated biological components for building plant-based biosensors,along with various applications of plant-based biosensors in basic and applied plant science research.Finally,we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.展开更多
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 phosphoryl...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.展开更多
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 bio...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.展开更多
基金the National Key R&D Program of China(2022YFA0912002)Zhejiang Provincial Postdoctoral Research Grant(103110456582201)Competitive Research Funding Program(WU2022A006)in Center for Synthetic Biology and Integrated Bioengineering at Westlake University,and Westlake Education Foundation.
文摘The third-generation(3G)biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon(C1)sources,such as CO_(2),formate,and methanol,fueled by renewable energies like light and electricity.This promising technology represents an important step toward sustainable development,which can help address some of the most pressing environmental challenges faced by modern society.However,to establish processes competitive with the petroleum industry,it is crucial to determine the most viable pathways for C1 utilization and productivity and yield of the target products.In this review,we discuss the progresses that have been made in constructing artificial biological systems for 3G biorefineries in the last 10 years.Specifically,we highlight the representative works on the engineering of artificial autotrophic microorganisms,tandem enzymatic systems,and chemobio hybrid systems for C1 utilization.We also prospect the revolutionary impact of these developments on biotechnology.By harnessing the power of 3G biorefinery,scientists are establishing a new frontier that could potentially revolutionize our approach to industrial production and pave the way for a more sustainable future.
基金E.B.is supported by a BBSRC Doctoral Training Partnership Studentship(Project Reference:1949015).
文摘Globally,agriculture depends on industrial nitrogen fertilizer to improve crop growth.Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution.A potential solution would be to harness nitrogenases—enzymes capable of converting atmospheric nitrogen N2 to NH3 in ambient conditions.It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants,or bacteria that form symbiotic relationships with crops,to support growth and reduce dependence on industrially produced fertilizer.This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.
基金supported by the Deutsche Forschungsgemeinschaft(DFG),grant KR 2963/3-1.
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.
基金the USDA Floriculture and Nursery Research Initiative(FNRI)grant 8020-21000-071-23Sthe USDA National Institute of Food and Agriculture(NIFA)Hatch project 02913the Center for Bioenergy Innovation(CBI),which is a U.S.Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.Oak Ridge National Laboratory is managed by UT-Battelle,LLC for the U.S.DOE under contract number DE-AC05-00OR22725。
文摘High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels.Gene transcription is tightly regulated by promoters and terminators.Promoters determine the timing,tissues and cells,and levels of the expression of genes.Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally,e.g.,the 3′-end processing,stability,translation efficiency,and nuclear to cytoplasmic export of mRNAs.The promoter and terminator combination affects gene expression.In the present article,we review the function and features of plant core promoters,proximal and distal promoters,and terminators,and their effects on and benchmarking strategies for regulating gene expression.
基金the National Key Research and Development Program of China under grant number 2022YFA0912201the National Natural Science Foundation of China under grant number 32270090+1 种基金the Foundation of Hubei Hongshan Laboratory under grant numbers 2021hszd013 and 2021hszd022the LongYun Program for College of Life Science and Technology,Huazhong Agricultural University.
文摘Probiotics are the treasure of the microbiology fields.They have been widely used in the food industry,clinical treatment,and other fields.The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic’s developed applications.In recent years,various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics.As important genome editing tools,CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics.The high efficiency,flexibility,and specificity are the advantages of using CRISPR-Cas systems.Here,we summarize the classification and distribution of CRISPR-Cas systems in probiotics,as well as the editing tools developed on the basis of them.Then,we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems.Finally,we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.
基金support from the National Natural Science Foundation of China(32270087)the Natural Science Foundation of Fujian Province of China(2020J05011)+2 种基金Guangdong Basic and Applied Basic Research Foundation(2021A1515110340)Xiamen University(0660X2510200)Daan Gene(20223160A0063),and ZhenSheng Biotech.
文摘Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis.In this study,we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae.First,we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply.Second,we restricted the consumption of farnesyl pyrophosphate(FPP)by the down-regulation of squalene synthase using the PEST degron.Third,we further explored the human lipid binding/transfer protein saposin B(hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids.Last,the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation.By combining the abovementioned strategies,the final engineered yeast produced 166.79±10.43 mg/Ⅰβ-carotene in shake flasks,which was nearly 5-fold improvement of the parental carotenoid-producing strain.Together,we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.
基金supported by the Center for Bioenergy Innovation(CBI),which is a U.S.Department of Energy(DOE)Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science,and the DOE Genomic Science Program,as part of the Secure Ecosystem Engineering and Design Scientific(SEED)Focus Area.Oak Ridge National Laboratory is man-aged by UT-Battelle,LLC for the U.S.DOE under Contract Number DE-AC05-00OR22725This material is based on work supported by the U.S.Department of Energy,Ofice of Science,Biological and Environmental Research Program under Award Number DE-SC0023338 to CRB.
文摘Plants are complex systems hierarchically organized and composed of various cell types.To understand the molecular underpinnings of complex plant systems,single-cell RNA sequencing(scRNA-seq)has emerged as a powerful tool for revealing high resolution of gene expression patterns at the cellular level and investigating the cell-type heterogeneity.Furthermore,scRNA-seq analysis of plant biosystems has great potential for generating new knowledge to inform plant biosystems design and synthetic biology,which aims to modify plants genetically/epigenetically through genome editing,engineering,or re-writing based on rational design for increasing crop yield and quality,promoting the bioeconomy and enhancing environmental sustainability.In particular,data from scRNA-seq studies can be utilized to facilitate the development of high-precision Build-Design-Test-Learn capabilities for maximizing the targeted performance of engineered plant biosystems while minimizing unintended side effects.To date,scRNA-seq has been demonstrated in a limited number of plant species,including model plants(e.g.,Arabidopsis thaliana),agricultural crops(e.g.,Oryza sativa),and bioenergy crops(e.g.,Populus spp.).It is expected that future technical advancements will reduce the cost of scRNA-seq and consequently accelerate the application of this emerging technology in plants.In this review,we summarize current technical advancements in plant scRNA-seq,including sample preparation,sequencing,and data analysis,to provide guidance on how to choose the appropriate scRNA-seq methods for different types of plant samples.We then highlight various applications of scRNA-seq in both plant systems biology and plant synthetic biology research.Finally,we discuss the challenges and opportunities for the application of scRNA-seq in plants.
基金This work was supported by the National Key Research and Development Program of China(2018YFA0900600)the Strategic Priority Research Program“Molecular Mech-anisms of Plant Growth and Development”of the Chinese Academy of Sciences(CAS)(XDB27020202)+5 种基金the National Natural Science Foundation of China(22077129,32070328,and 41876084)the Natural Science Foundation of Shanghai Municipal Science and Technology Committee(21ZR1470900)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-002-15),and the Program of Shanghai Academic Research Leader(20XD1404400)This work was also financially supported by the Construction of the Registry and Data-base of Bioparts for Synthetic Biology of the CAS(ZSYS-016)the International Partnership Program of the CAS(153D31KYSB20170121)the National Key Laboratory of Plant Molecular Genetics,Shanghai Institute of Plant Physiology and Ecology,CAS.
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.
基金The writing of this manuscript was supported by the Center for Bioenergy Innovation,a U.S.Department of Energy(DOE)Bioenergy Research Center supported by the Biological and Environmental Research(BER)program,the Laboratory Directed Research and Development program of Oak Ridge National Laboratory,and the U.S.DOE BER Genomic Science Program,as part of the Secure Ecosystem Engineering and Design Scientific Focus Area and the Plant-Microbe Interfaces Scientific Focus AreaYY is supported by NSF Plant Genome Research Project Grant(1740874)and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04659 and Accession#1016432.HY is supported by Nonprofit Research Projects(CAFYBB2018ZY001-1)of Chinese Academy of Forestry+3 种基金CTT acknowledges the financial support from the NSF CAREER award(NSF#1553250)and the DOE BER Genomic Science Program(DE-SC0019412)PMS acknowledges support from the Joint BioEnergy Institute which is supported by the U.S.DOE Office of Science,BER program under Contract No.DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of EnergyDL acknowledges financial support through the National Science Foundation(NSF)under Award Number 1833402.AJM acknowledges financial support from the UK Biotechnology and Biological Sciences Research Council(grants BB/M006468/1 and BB/S015531/1)the Leverhulme Trust(grant RPG-2017-402).
文摘Human life intimately depends on plants for food,biomaterials,health,energy,and a sustainable environment.Various plants have been genetically improved mostly through breeding,along with limited modification via genetic engineering,yet they are still not able to meet the ever-increasing needs,in terms of both quantity and quality,resulting from the rapid increase in world population and expected standards of living.A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches.This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems.Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes.From this perspective,we present a comprehensive roadmap of plant biosystems design covering theories,principles,and technical methods,along with potential applications in basic and applied plant biology research.We highlight current challenges,future opportunities,and research priorities,along with a framework for international collaboration,towards rapid advancement of this emerging interdisciplinary area of research.Finally,we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception,trust,and acceptance.
基金This manuscript has been authored by UT-Battelle,LLC,under Contract No.DE-AC05-00OR22725 with the U.S.DOE Oak Ridge National Laboratory is managed by UT-Battelle,LLC,for the U.S.DOE under Contract Number DE-AC05-00OR22725.
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.
基金the National Natural Science Foundation of China(31625023,31721004,and 32072507)the Fundamental Research Funds for the Central Universities(KYT202001).
文摘Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity.Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology.Nevertheless,enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects.Upstream open reading frames(uORFs)are translational control elements located in the 5′untranslated region(UTR)of eukaryotic mRNAs and may repress the translation of downstream genes.To investigate the function of three uORFs from the 5′-UTR of ACCELERATED CELL 11(uORFsACD11),we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation.Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels.Importantly,uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves.When coupled with the PATHOGENESIS-RELATED GENE 1(PR1)promoter,the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2(AtLecRKVI.2)during pathogen attack and enhance plant resistance to Phytophthora capsici.These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack,while for ensuring lower levels of protein expression at normal conditions.
基金the National Key R&D Program Young Scientists Project of China(No.2021YFD1900400 to XYJ)the National Science Foundation of China(No.32270109 to XYJ)+3 种基金BM acknowledges the support of the Key Instrument Development Program from Chinese Academy of Sciences(YJKYYQ20170017)WEH acknowledges finance and instrumentation support from the Engineering and Physical Sciences Research Council(EP/M002403/1 and EP/M02833X/1)and Natural Environment Research Council(NE/M002934/1)WEH,PAD,CMC,and CNH acknowledge financial support from the Biotechnology and Biological Sciences Research Council(BBSRC UK),award number BB/M000265/1CNH and PAD are supported by the European Research Council Synergy Award 854126.We also thank NSFC(31770112)for the financial support.
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.
基金supported by the National Key Research and Development Program of China(2022YFC2104600)the National Natural Science Foundation of China(nos.32270087,32241040,and 31970314)+3 种基金the Natural Science Foundation of Fujian Province of China(no.2020J05011)Guangdong Basic and Applied Basic Research Foundation(no.2021A1515110340)Xiamen University(no.0660X2510200)the Fundamental Research Funds for the Central Universities(no.20720220086),and ZhenSheng Biotech.
文摘Lignin is one of the most widespread organic compounds found on earth,boasting a wealth of aromatic molecules.The use of lignin feedstock for biochemical productions is of great importance for achieving"carbon neutrality."In recent years,a strategy for lignin valorization known as the"bio-funnel"has been proposed as a means to generate a variety of commercially valuable chemicals from lignin-derived compounds.The implementation of biocatalysis and metabolic engineering techniques has substantially advanced the biotransformation of depolymerized lignin into chemicals and materials within the supply chain.In this review,we present an overview of the latest advancements in microbial upcycling of depolymerized lignin into value-added chemicals.Besides,the review provides insights into the problems facing current biological lignin valorization while proposing further research directions to improve these technologies for the extensive accomplishment of the lignin upcycling.
基金supported by the Japan Agency for Medical Research and Development under grant numbers JP23wm0325065,JP22fk0108532,JP21fk0108496,and JP21 wm0325022 to K.K.grant number JP21gm1610002 to L.C.and K.K.JSPS KAKENHI grants numbers 21H02110 and 21K19666 to K.K.
文摘The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise;however,a thorough comparison and evaluation of their bactericidal efficacy are lacking.This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes,with the goal of harnessing them for antibacterial therapy.First,we examined the bactericidal activity of spanins,endolysins,and holins derived from 2 Escherichia coli model phages,T1 and T7.Among these,T1-spanin exhibited the highest bactericidal activity against E.coli.Subsequently,we expressed T1-spanin within bacterial cells and assessed its bactericidal activity.T1-spanin showed potent bactericidal activity against all clinical isolates tested,including bacterial strains of 111 E.coli,2 Acinetobacter spp.,3 Klebsiella spp.,and 3 Pseudomonas aeruginosa.In contrast,T1 phage-derived endolysin showed bactericidal activity against E.coli and P.aeruginosa,yet its efficacy against other bacteria was inferior to that of T1-spanin.Finally,we developed a phage-based technology to introduce the T1-spanin gene into target bacteria.The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria.The potent bactericidal activity exhibited by spanins,combined with the novel phage synthetic technology,holds promise for the development of innovative antimicrobial agents.
基金the National Basic Research Program of China(973 Program)(2018YFA0901800)the National Natural Science Foundation of China(22138006,21736002,and 22078020).
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.
基金The workshop was supported by a grant from the Alfred P.Sloan Foundationby the Boston University(BU)Program in Bioinformatics and Systems Biology,and by the BU College of Engineering。
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.
基金This manuscript has been authored by UT-Battelle,LLC,under Contract No.DE-AC05-00OR22725 with the U.S.Department of Energy,Oak Ridge National Laboratory is managed by UT-Battelle,LLC,for the U.S.Department of Energy under Contract Number DEAC05-00OR22725。
文摘Many applications in plant biology requires editing genomes accurately including correcting point mutations,incorporation of single-nucleotide polymorphisms(SNPs),and introduction of multinucleotide insertion/deletions(indels)into a predetermined position in the genome.These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems,which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence.However,low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient.Another kind of technology known as base editing can perform precise editing;however,only certain types of modifications can be obtained,e.g.,C/G-to-T/A and A/T-to-G/C.Recently,a new type of genome-editing technology,referred to as“prime editing,”has been developed,which can achieve various types of editing such as any base-to-base conversion,including both transitions(C→T,G→A,A→G,and T→C)and transversion mutations(C→A,C→G,G→C,G→T,A→C,A→T,T→A,and T→G),as well as small indels without the requirement for inducing double-stranded break in the DNA.Because prime editing has wide flexibility to achieve different types of edits in the genome,it holds a great potential for developing superior crops for various purposes,such as increasing yield,providing resistance to various abiotic and biotic stresses,and improving quality of plant product.In this review,we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.
基金the Biological and Environmental Research(BER)program.Oak Ridge National Laboratory is managed by UT-Battelle,LLC for the U.S.Department of Energy under Contract Number DE-AC05-00OR22725The support to Chang-jun Liu was partially from the DOE Office of Basic Energy Sciences,specifically through the Physical Biosciences program of the Chemical Sciences,Geosciences and Biosciences Division,under contract number DE-SC0012704.
文摘Plants adapt to their changing environments by sensing and responding to physical,biological,and chemical stimuli.Due to their sessile lifestyles,plants experience a vast array of external stimuli and selectively perceive and respond to specific signals.By repurposing the logic circuitry and biological and molecular components used by plants in nature,genetically encoded plant-based biosensors(GEPBs)have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected.GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development.GEPBs are also useful for environmental monitoring,plant abiotic and biotic stress management,and accelerating design-build-test-learn cycles of plant bioengineering.With the advent of synthetic biology,biological and molecular components derived from alternate natural organisms(e.g.,microbes)and/or de novo parts have been used to build GEPBs.In this review,we summarize the framework for engineering different types of GEPBs.We then highlight representative validated biological components for building plant-based biosensors,along with various applications of plant-based biosensors in basic and applied plant science research.Finally,we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.
基金the National Key Research and Development Program of China(Grant number 2021YFA0910601)the National Natural Science Foundation of China(Grant numbers 32022044 and 32001027).
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.
基金the National Key Research and Development Program of China(grant number 2018YFA0900300)the International Partnership Program of Chinese Academy of Sciences(grant number 153D31KYSB20170121)Youth Innovation Promotion Association CAS,and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(grant numbers TSBICIP-PTJS-001 and TSBICIP-CXRC-018).
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.
