Synthetic biology is moving in the direction of larger and more sophisticated design,which depends heavily on the efficient assembly of genetic modules.Conventional evaluation of the DNA assembly efficiency(AE)require...Synthetic biology is moving in the direction of larger and more sophisticated design,which depends heavily on the efficient assembly of genetic modules.Conventional evaluation of the DNA assembly efficiency(AE)requires transformation,and the whole process requires up to 10 h and is susceptible to various interferences.To achieve rapid and reliable determination of the AE,an alternative transformation-independent method was established using a modified quantitative polymerase chain reaction(qPCR)assay.The AE is represented by the proportion of the ligated fragment,which can be determined within 3 h.This qPCR-based measurement was tested by the commonly used restriction ligation,Golden Gate assembly,and Gibson assembly for the assembly of two or more DNA pieces;the results correlated significantly with the AEs represented by the counting of the colony-forming units(CFUs).This method outperformed the CFU-based measurement by reducing the measuring bias and the random deviations that stem from the transformation process.The method was then employed to investigate the effects of terminal secondary structures on DNA assembly.The results revealed the major effects of the overall properties of the overlap sequence and the negative effects of hairpin structures on the AE,which are relevant for all assembly techniques that rely on homologous annealing of the terminal sequences.The qPCR-based approach presented here should facilitate the development of DNA assembly techniques and the diagnosis of inefficient assemblies.展开更多
The process of metabolic engineering consists of multiple cycles of design,build,test and learn,which is typically laborious and time-consuming.To increase the efficiency and the rate of success of strain engineering,...The process of metabolic engineering consists of multiple cycles of design,build,test and learn,which is typically laborious and time-consuming.To increase the efficiency and the rate of success of strain engineering,novel instrumentation must be applied.Microfluidics,the control of liquid flow in microstructures,has enabled flexible,accurate,automatic,and high-throughput manipulation of cells in liquid at picoliter to nanoliter scale.These attributes hold great promise in advancing metabolic engineering in terms of the phases of design,build,test and learn.To promote the application of microfluidic-based technologies in strain improvement,this review addressed the potentials of microfluidics and the related approaches in DNA assembly,transformation,strain screening,genotyping and phenotyping,and highlighted their adaptations for single-cell analysis.As a result,this facilitates in-depth understanding of the metabolic network,which in turn promote efficient optimization in the following cycles of strain engineering.Taken together,microfluidic-based technologies enable on-chip workflow,and could greatly accelerate the turnaround of metabolic engineering.展开更多
Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid inst...Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid instability.Herein,we conducted genetic modifications at a serial of loci of MG1655 genome,generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways.Using JW128 as the host,the titer of isobutanol reached 5.76 g/L in shake-flask fermentation,and the titer of lycopene reached 1.91 g/L in test-tube fermentation,40-fold and 5-fold higher than that of original MG1655,respectively.These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.展开更多
Microbial fermentation has contributed to 80%of global amino acid production.The key to microbial fermentation is to obtain fermentation strains with high performance to produce target amino acids with a high yield.Th...Microbial fermentation has contributed to 80%of global amino acid production.The key to microbial fermentation is to obtain fermentation strains with high performance to produce target amino acids with a high yield.These strains are primarily derived from screening enormous mutant libraries.Therefore,a high-throughput,rapid,accurate,and universal screening strategy for amino acid overproducers has become a guarantee for ob-taining optional amino acid overproducers.In recent years,the rapid development of various novel screening strategies has been witnessed.However,proper analysis and discussion of these innovative technologies are lacking.Here we systematically reviewed recent advances in screening strategies:the auxotrophic-based strategy,the biosensor-based strategy,and the latest translation-based screening strategy.The design principle,application scope,working efficiency,screening accuracy,and universality of these strategies were discussed in detail.The potential for screening nonstandard amino acid overproducers was also analyzed.Guidance for the improvement of future screening strategies is provided in this review,which could expedite the reconstruction of amino acid overproducers and help promote the fermentation industry to reduce cost,increase yield,and improve quality.展开更多
基金This work was supported by the National Key R&D Program of China(2017YFD0201400)the National Natural Science Foundation of China(21676026)the Fundamental Research Funds for the Central Universities.
文摘Synthetic biology is moving in the direction of larger and more sophisticated design,which depends heavily on the efficient assembly of genetic modules.Conventional evaluation of the DNA assembly efficiency(AE)requires transformation,and the whole process requires up to 10 h and is susceptible to various interferences.To achieve rapid and reliable determination of the AE,an alternative transformation-independent method was established using a modified quantitative polymerase chain reaction(qPCR)assay.The AE is represented by the proportion of the ligated fragment,which can be determined within 3 h.This qPCR-based measurement was tested by the commonly used restriction ligation,Golden Gate assembly,and Gibson assembly for the assembly of two or more DNA pieces;the results correlated significantly with the AEs represented by the counting of the colony-forming units(CFUs).This method outperformed the CFU-based measurement by reducing the measuring bias and the random deviations that stem from the transformation process.The method was then employed to investigate the effects of terminal secondary structures on DNA assembly.The results revealed the major effects of the overall properties of the overlap sequence and the negative effects of hairpin structures on the AE,which are relevant for all assembly techniques that rely on homologous annealing of the terminal sequences.The qPCR-based approach presented here should facilitate the development of DNA assembly techniques and the diagnosis of inefficient assemblies.
基金This work was supported by the Recruitment Programfor Young Professionals(1000 Plan)the Financial Aid Project for Outstanding Young Teachers of Beijing Institute of Technology(2015YG1607).
文摘The process of metabolic engineering consists of multiple cycles of design,build,test and learn,which is typically laborious and time-consuming.To increase the efficiency and the rate of success of strain engineering,novel instrumentation must be applied.Microfluidics,the control of liquid flow in microstructures,has enabled flexible,accurate,automatic,and high-throughput manipulation of cells in liquid at picoliter to nanoliter scale.These attributes hold great promise in advancing metabolic engineering in terms of the phases of design,build,test and learn.To promote the application of microfluidic-based technologies in strain improvement,this review addressed the potentials of microfluidics and the related approaches in DNA assembly,transformation,strain screening,genotyping and phenotyping,and highlighted their adaptations for single-cell analysis.As a result,this facilitates in-depth understanding of the metabolic network,which in turn promote efficient optimization in the following cycles of strain engineering.Taken together,microfluidic-based technologies enable on-chip workflow,and could greatly accelerate the turnaround of metabolic engineering.
基金The work finished in Beijing Institute of Technology was supported by the National Key R&D Program of China(grant No.2019YFA0904104)the National Natural Science Foundation of China(Grant No.21676026)the Fundamental Research Funds for the Central Universities.
文摘Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid instability.Herein,we conducted genetic modifications at a serial of loci of MG1655 genome,generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways.Using JW128 as the host,the titer of isobutanol reached 5.76 g/L in shake-flask fermentation,and the titer of lycopene reached 1.91 g/L in test-tube fermentation,40-fold and 5-fold higher than that of original MG1655,respectively.These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.
基金supported by the National Key R&D Program of China(2019YFA0906500)the National Natural Science Foundation of China(32000059)+1 种基金the Innovation Team Project of Colleges and Universities in Jinan(2019GXRC033)the Fundamental Research Funds for the Central Universities.
文摘Microbial fermentation has contributed to 80%of global amino acid production.The key to microbial fermentation is to obtain fermentation strains with high performance to produce target amino acids with a high yield.These strains are primarily derived from screening enormous mutant libraries.Therefore,a high-throughput,rapid,accurate,and universal screening strategy for amino acid overproducers has become a guarantee for ob-taining optional amino acid overproducers.In recent years,the rapid development of various novel screening strategies has been witnessed.However,proper analysis and discussion of these innovative technologies are lacking.Here we systematically reviewed recent advances in screening strategies:the auxotrophic-based strategy,the biosensor-based strategy,and the latest translation-based screening strategy.The design principle,application scope,working efficiency,screening accuracy,and universality of these strategies were discussed in detail.The potential for screening nonstandard amino acid overproducers was also analyzed.Guidance for the improvement of future screening strategies is provided in this review,which could expedite the reconstruction of amino acid overproducers and help promote the fermentation industry to reduce cost,increase yield,and improve quality.
