Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the is...Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.展开更多
Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in...Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology,including synthesis and assembly of a genome,DNA storage,gene editing,molecular evolution and de novo design of function proteins,cell and gene circuit engineering,cell-free synthetic biology,artificial intelligence(AI)-aided synthetic biology,as well as biofoundries.We also introduce the concept of quantitative synthetic biology,which is guiding synthetic biology towards increased accuracy and predictability or the real rational design.We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.展开更多
Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two ...Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.展开更多
The conversion of r-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps ofketolation are catalyzed by β-carotene hydroxylase （CrtZ） and β-carotene ketolase （Crt...The conversion of r-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps ofketolation are catalyzed by β-carotene hydroxylase （CrtZ） and β-carotene ketolase （CrtW） respectively. Here, astaxanthin biosynthesis path- way was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high r-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE Scl 18060 with CrtW from Brevundimonas vesicu- laris DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Scl 18060 was replaced from FBAlp to TEFlp, the astaxanthin yield was increased by 30.4% （from 3.4 to 4.5 mg/g DCW）. In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtWwere observed at early exponential phase in medium with 4% （w/v） glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% （w/v） glucose, the transcription level of both crtZ and crtW were actually decreased during the whole timecourse, consequently leading to no significant improve- ment on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance lation by employing the key desired compounds accumu- enzyme（s） in microbes.展开更多
Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as...Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE,different levels of loss of heterozygosity(LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome Ⅹ, whole chromosome LOH of synthetic chromosome Ⅹ, and duplication of chromosome Ⅷ(trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.展开更多
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different ch...Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.展开更多
The rapid development of synthetic biology has conferred almost perfect modification on single cells,and provided methodological support for synthesizing microbial consortia,which have a much wider application potenti...The rapid development of synthetic biology has conferred almost perfect modification on single cells,and provided methodological support for synthesizing microbial consortia,which have a much wider application potential than synthetic single cells.Co-cultivating multiple cell populations with rational strategies based on interacting relationships within natural microbial consortia provides theoretical as well as experimental support for the successful obtaining of synthetic microbial consortia,promoting it into extensive research on both industrial applications in plenty of areas and also better understanding of natural microbial consortia.According to their composition complexity,synthetic microbial consortia are summarized in three aspects in this reviewand are discussed in principles of design and construction,insights and methods for analysis,and applications in energy,healthcare,etc.展开更多
Environmental sustainability is an increasingly important issue in industry.As an environmentally friendly and sustainable way,constructing microbial cell factories to produce all kinds of valuable products has attrac...Environmental sustainability is an increasingly important issue in industry.As an environmentally friendly and sustainable way,constructing microbial cell factories to produce all kinds of valuable products has attracted more and more attention.In the process of constructing microbial cell factories,systems biology plays a crucial role.This review summarizes the recent applications of systems biology in the design and construction of microbial cell factories from four perspectives,including functional genes/enzymes discovery,bottleneck pathways identification,strains tolerance improvement and design and construction of synthetic microbial consortia.Systems biology tools can be employed to identify functional genes/enzymes involved in the biosynthetic pathways of products.These discovered genes are introduced into appropriate chassis strains to build engineering microorganisms capable of producing products.Subsequently,systems biology tools are used to identify bottleneck pathways,improve strains tolerance and guide design and construction of synthetic microbial consortia,resulting in increasing the yield of engineered strains and constructing microbial cell factories successfully.展开更多
Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination ...Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.展开更多
Varied environmental stress can affect cell growth and activity of the cellular catalyst.Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype,meanwhile,it is a challenge ...Varied environmental stress can affect cell growth and activity of the cellular catalyst.Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype,meanwhile,it is a challenge to dissect the underlying genetic mechanism.Here,using SCRaMbLE,a genome scale tool to generate random structural variations,a total of 222 evolved yeast strains with enhanced environmental tolerances were obtained in haploid or diploid yeasts containing six synthetic chromosomes.Whole genome sequencing of the evolved strains revealed that these strains generated different structural variants.Notably,by phenotypic-genotypic analysis of the SCRaMbLEd strains,we find that a deletion of gene YFR009W(GCN20)can improve salt tolerance of Saccharomyces cerevisiae,and a deletion of gene YER056C can improve 5-flucytosine tolerance of Saccharomyces cerevisiae.This study shows applications of SCRaMbLE to accelerate phenotypic evolution for varied environmental stress and to explore relationships between structural variations and evolved phenotypes.展开更多
Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the...Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the main gene targets in the upstream pathways to investigate their influences on the downstream resver- atrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.144-3.74 mg.L-1, 2.27 times higher than the initial production in YPD medium （4% glucose）. The newly discovered gene targets TRP1 expressing phosphoribosy- lanthranilate isomerase, AR03 expressing 3-deoxy-D- arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.展开更多
Currently,the establishment of synthetic microbial consortia with rational strategies has gained extensive attention,becoming one of the important frontiers of synthetic biology.Systems biology can offer insights into...Currently,the establishment of synthetic microbial consortia with rational strategies has gained extensive attention,becoming one of the important frontiers of synthetic biology.Systems biology can offer insights into the design and construction of synthetic microbial consortia.Taking the high-efficiency production of 2-keto-L-gulonic acid(2-KLG)as an example,we constructed a synthetic microbial consortium“Saccharomyces cerevisiae-Ketogulonigenium vulgare”based on systems biology analysis.In the consortium,K.vulgare was the 2-KLG pro-ducing strain,and S.cerevisiae acted as the helper strain.Comparative transcriptomic analysis was performed on an engineered S.cerevisiae(VTC2)and a wild-type S.cerevisiae BY4741.The results showed that the up-regulated genes in VTC2,compared with BY4741,were mainly involved in glycolysis,TCA cycle,purine metabolism,and biosynthesis of amino acids,B vitamins,and antioxidant proteases,all of which play important roles in pro-moting the growth of K.vulgare.Furthermore,Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG.Therefore,VTC2 would be of great advantage in working with K.vulgare.Thus,the synthetic microbial consortium"VTC2-K.vulgare"was constructed based on transcriptomics analyses,and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K.vulgare,reaching 13.2±0.52 g/L.In addition,the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes(sod,cat and gpd)in K.vulgare.The results indicated that the oxida-tive stress in the synthetic microbial consortium was efficiently reduced.Thus,systems analysis confirmed a favorable symbiotic relationship between microorganisms,providing guidance for further engineering synthetic consortia.展开更多
Naturally occurring structural variations(SVs)are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes.Controllable methods aimed at introducing the complex SVs and their rela...Naturally occurring structural variations(SVs)are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes.Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched.In this study,an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution(SCRaMbLE)technology with two synthetic chromosomes,namely synV and synX.The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries.Our findings showed that complex SVs,including a pericentric inversion and a trans-chromosome translocation between synV and synX,resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin.Also,genetic targets were mapped,which resulted in a higher astaxanthin yield,thus demonstrating the SVs’ability to reorganize genetic information along the chromosomes.The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena.Collectively,this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.展开更多
The emerging field of synthetic biology has gained great attractions in recent years, both in China and around the world. As a result, the number of publications in this field has grown exponentially in the past decad...The emerging field of synthetic biology has gained great attractions in recent years, both in China and around the world. As a result, the number of publications in this field has grown exponentially in the past decade. Analysis of this trend can be found in the article ＂Bibliometrics analysis of synthetic biology based on Web of Science＂ [1], which is authored in 2015 by researchers from Peking Union Medical College/Chinese Academy of Medical Sciences with support from the Consulting Project of Chinese Academy of Engineedng. Just using ＂synthetic biology＂ as the ＂topic＂ in Web of Science Core Collection, we can get a coarse estimate of the speed of its growth （Fig. 1）. Only 56 papers were published in 1998 globally and in 2016 the number reached more than 1000. In China, only 10 papers were published in 2007 and the numbers reached over 150 each year in 2015 and 2016.展开更多
Androgen receptor(AR)mutation is closely associated with prostate cancer(PCa)and is one of the mechanisms of resistance to PCa therapies such as AR antagonists.Although sequencing technologies like next-generation seq...Androgen receptor(AR)mutation is closely associated with prostate cancer(PCa)and is one of the mechanisms of resistance to PCa therapies such as AR antagonists.Although sequencing technologies like next-generation sequencing(NGS)contributes to the high-throughput and precise detection of AR mutations carried by PCa patients,the lack of interpretations of these clinical genetic variants has still been a roadblock for PCa-targeted precision medicine.Here,we established a designer yeast reporter assay to simulate natural androgen receptor(AR)selection using AR antagonists.Yeast HIS3 gene transactivation was associated with the ligand-induced recruitment of steroid receptor coactivator-1(SRC-1)by AR mutants,where yeast growth in histidine-free medium was determined as the outcome.This assay is applicable to determine a wide range of clinical AR mutants including those with loss of function relating to androgen insensitivity syndrome(AIS),and those associated with PCa conferring resistance to AR antagonists such as enzalutamide(ENZ),bicalutamide(BIC),and cyproterone acetate(CPA).One clinical AR mutant previously reported to confer ENZ-resistance,F877L,was found to confer partial resistance to CPA as well using designer yeast.Our simple and efficient assay can enable precise one-pot screening of AR mutants,providing a reference for tailored medicine.展开更多
The applications of supramolecular metal-peptide assemblies as catalyst or catalyst precursor have recent attracted increasing attentions.In this work,a fragment of the amyloid β-peptide,NH_(2)-KLVFF-COOH,was assembl...The applications of supramolecular metal-peptide assemblies as catalyst or catalyst precursor have recent attracted increasing attentions.In this work,a fragment of the amyloid β-peptide,NH_(2)-KLVFF-COOH,was assembled into nanofilms with encapsulated Pd,Pt and Au nanoparticles(NPs)via a one-step room temperature electron induction method.The effects of building block,intermolecular interaction,driving force and side-chain on the assembly were investigated.The assembly mechanism was thereby proposed.The crosslinking of peptide monomers results in mainly random and unordered structures.The obtained metal-peptide assemblies are extremely stable in water at neutral pH for long term.However,the metal NPs are able to be responsively released under basic and reductive conditions.The released NPs show a high activity to catalyze the reduction of 4-nitrophenol.The present studies on assembly mechanism and responsive release will be helpful for the design of organic skeletons and also for the future development of peptide stabilized metallic NPs with applications beyond catalysts.展开更多
基金National Program on Key Basic Research Project by the Ministry of Science and Technology of China(2014CB745100)the National Natural Science Foundation of China(21676190 and 21621004)the Innovative Talents and Platform Program of Tianjin(16PTGCCX00140 and 16PTSYJC00050).
文摘Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB29050100,XDB29050500,XDA24020102)to X.E.Zhang,C.Liu and C.Gao,respectivelythe National Natural Science Foundation of China(31725002,31861143017,32022044,62050152 and 32071428)to J.Dai,Y.Yuan,C.You,and X.Wang,respectivelythe National Key Research and Development Program of China(2020YFA0907700,2018YFA0901600,2019YFA09004500)to Y.Feng and P.Wei。
文摘Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology,including synthesis and assembly of a genome,DNA storage,gene editing,molecular evolution and de novo design of function proteins,cell and gene circuit engineering,cell-free synthetic biology,artificial intelligence(AI)-aided synthetic biology,as well as biofoundries.We also introduce the concept of quantitative synthetic biology,which is guiding synthetic biology towards increased accuracy and predictability or the real rational design.We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.
基金the Ministry of Science and Technology of China (2014CB745100)the National Natural Science Foundation of China (21390203 and 21706186).
文摘Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.
基金This work was supported by the International S＆T Cooperation Program of China （2015DFA00960）, the National Natural Science Foundation of China （Grant Nos. 31600052 and 21676192） and Innovative Talents and Platform Program of Tianjin （16PTSYJC00050）.
文摘The conversion of r-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps ofketolation are catalyzed by β-carotene hydroxylase （CrtZ） and β-carotene ketolase （CrtW） respectively. Here, astaxanthin biosynthesis path- way was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high r-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE Scl 18060 with CrtW from Brevundimonas vesicu- laris DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Scl 18060 was replaced from FBAlp to TEFlp, the astaxanthin yield was increased by 30.4% （from 3.4 to 4.5 mg/g DCW）. In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtWwere observed at early exponential phase in medium with 4% （w/v） glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% （w/v） glucose, the transcription level of both crtZ and crtW were actually decreased during the whole timecourse, consequently leading to no significant improve- ment on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance lation by employing the key desired compounds accumu- enzyme（s） in microbes.
基金supported by the National Natural Science Foundation of China (21621004, 21750001 and 21676192)Young Elite Scientist Sponsorship Program by CAST (YESS) (2018QNRC001)
文摘Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE,different levels of loss of heterozygosity(LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome Ⅹ, whole chromosome LOH of synthetic chromosome Ⅹ, and duplication of chromosome Ⅷ(trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.
基金supported by Major Program of the National Natural Science Foundation of China(21621004)the Natural Science Foundation of Tianjin City(19JCQNJC09200)the Young Elite Scientists Sponsorship Program by Tianjin(TJSQNTJ-2018-16).
文摘Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.
基金the National Basic Research Program of China(“973”Program:2014CB745100)the National Natural Science Foundation of China(No.21576197)Tianjin Research Program of Application Foundation and Advanced Technology(No.14JCQNJC06700).
文摘The rapid development of synthetic biology has conferred almost perfect modification on single cells,and provided methodological support for synthesizing microbial consortia,which have a much wider application potential than synthetic single cells.Co-cultivating multiple cell populations with rational strategies based on interacting relationships within natural microbial consortia provides theoretical as well as experimental support for the successful obtaining of synthetic microbial consortia,promoting it into extensive research on both industrial applications in plenty of areas and also better understanding of natural microbial consortia.According to their composition complexity,synthetic microbial consortia are summarized in three aspects in this reviewand are discussed in principles of design and construction,insights and methods for analysis,and applications in energy,healthcare,etc.
基金by the National Key Research and Development Program of China (2019YFA0706900)National Natural Science Foundation of China (22278310).
文摘Environmental sustainability is an increasingly important issue in industry.As an environmentally friendly and sustainable way,constructing microbial cell factories to produce all kinds of valuable products has attracted more and more attention.In the process of constructing microbial cell factories,systems biology plays a crucial role.This review summarizes the recent applications of systems biology in the design and construction of microbial cell factories from four perspectives,including functional genes/enzymes discovery,bottleneck pathways identification,strains tolerance improvement and design and construction of synthetic microbial consortia.Systems biology tools can be employed to identify functional genes/enzymes involved in the biosynthetic pathways of products.These discovered genes are introduced into appropriate chassis strains to build engineering microorganisms capable of producing products.Subsequently,systems biology tools are used to identify bottleneck pathways,improve strains tolerance and guide design and construction of synthetic microbial consortia,resulting in increasing the yield of engineered strains and constructing microbial cell factories successfully.
基金Supported by the National Basic Research Program of China (‘‘973’’ Program:2014CB745100)the National Natural Science Foundation of China (No.21576201 and No.21176183)
基金supported by grants from the National Key Research and Development Program of China (2019YFA0903800)the National Natural Science Foundation of China (31800719 and 21621004)。
文摘Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.
基金supported by the National Key R&D Program of China(2021YFC2102500)National Natural Science Foundation of China(31971351).
文摘Varied environmental stress can affect cell growth and activity of the cellular catalyst.Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype,meanwhile,it is a challenge to dissect the underlying genetic mechanism.Here,using SCRaMbLE,a genome scale tool to generate random structural variations,a total of 222 evolved yeast strains with enhanced environmental tolerances were obtained in haploid or diploid yeasts containing six synthetic chromosomes.Whole genome sequencing of the evolved strains revealed that these strains generated different structural variants.Notably,by phenotypic-genotypic analysis of the SCRaMbLEd strains,we find that a deletion of gene YFR009W(GCN20)can improve salt tolerance of Saccharomyces cerevisiae,and a deletion of gene YER056C can improve 5-flucytosine tolerance of Saccharomyces cerevisiae.This study shows applications of SCRaMbLE to accelerate phenotypic evolution for varied environmental stress and to explore relationships between structural variations and evolved phenotypes.
基金The authors declare no competing financial interest. This work was funded by the National Basic Research Program of China （973 Program, Grant No. 2014CB745100） and the National High Technology Research and Development Program of China （863 Program, Grant No. 2012AA02AT01）, the International S＆T Cooperation Program of China （2015DFA00960）, and the National Natural Science Foundation of China （Major Program, Grant No. 21390203）.
文摘Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the main gene targets in the upstream pathways to investigate their influences on the downstream resver- atrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.144-3.74 mg.L-1, 2.27 times higher than the initial production in YPD medium （4% glucose）. The newly discovered gene targets TRP1 expressing phosphoribosy- lanthranilate isomerase, AR03 expressing 3-deoxy-D- arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.
基金the National Program on Key Basic Research Project of China (2014CB745100)the National Natural Science Foundation of China (21750001and 21621004)for funding.
基金the National Key Research and Development Program of China(2018YFA0902100)National Natural Science Foundation of China(21676190).
文摘Currently,the establishment of synthetic microbial consortia with rational strategies has gained extensive attention,becoming one of the important frontiers of synthetic biology.Systems biology can offer insights into the design and construction of synthetic microbial consortia.Taking the high-efficiency production of 2-keto-L-gulonic acid(2-KLG)as an example,we constructed a synthetic microbial consortium“Saccharomyces cerevisiae-Ketogulonigenium vulgare”based on systems biology analysis.In the consortium,K.vulgare was the 2-KLG pro-ducing strain,and S.cerevisiae acted as the helper strain.Comparative transcriptomic analysis was performed on an engineered S.cerevisiae(VTC2)and a wild-type S.cerevisiae BY4741.The results showed that the up-regulated genes in VTC2,compared with BY4741,were mainly involved in glycolysis,TCA cycle,purine metabolism,and biosynthesis of amino acids,B vitamins,and antioxidant proteases,all of which play important roles in pro-moting the growth of K.vulgare.Furthermore,Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG.Therefore,VTC2 would be of great advantage in working with K.vulgare.Thus,the synthetic microbial consortium"VTC2-K.vulgare"was constructed based on transcriptomics analyses,and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K.vulgare,reaching 13.2±0.52 g/L.In addition,the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes(sod,cat and gpd)in K.vulgare.The results indicated that the oxida-tive stress in the synthetic microbial consortium was efficiently reduced.Thus,systems analysis confirmed a favorable symbiotic relationship between microorganisms,providing guidance for further engineering synthetic consortia.
基金This work was supported by the Ministry of Science and Technology,the National Key Research and Development Program of China(2021YFC2100800)the National Natural Science Foundation of China(31800719,31861143017,21621004).
文摘Naturally occurring structural variations(SVs)are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes.Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched.In this study,an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution(SCRaMbLE)technology with two synthetic chromosomes,namely synV and synX.The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries.Our findings showed that complex SVs,including a pericentric inversion and a trans-chromosome translocation between synV and synX,resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin.Also,genetic targets were mapped,which resulted in a higher astaxanthin yield,thus demonstrating the SVs’ability to reorganize genetic information along the chromosomes.The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena.Collectively,this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.
文摘The emerging field of synthetic biology has gained great attractions in recent years, both in China and around the world. As a result, the number of publications in this field has grown exponentially in the past decade. Analysis of this trend can be found in the article ＂Bibliometrics analysis of synthetic biology based on Web of Science＂ [1], which is authored in 2015 by researchers from Peking Union Medical College/Chinese Academy of Medical Sciences with support from the Consulting Project of Chinese Academy of Engineedng. Just using ＂synthetic biology＂ as the ＂topic＂ in Web of Science Core Collection, we can get a coarse estimate of the speed of its growth （Fig. 1）. Only 56 papers were published in 1998 globally and in 2016 the number reached more than 1000. In China, only 10 papers were published in 2007 and the numbers reached over 150 each year in 2015 and 2016.
基金supported by the National Basic Research Program of China(‘‘973’’Program,No.2013CB733601)the National High Technology Research and Development Program of China(‘‘863’’Program,No.2012AA02A701)+1 种基金the National Natural Science Foundation of China(No.21390203)the Tianjin Municipal Science and Technology Committee(No.13RCGFSY19800)
基金This work was supported by National Natural Science Foundation of China under the grants 21621004 and 31861143017 to Y.Y.
文摘Androgen receptor(AR)mutation is closely associated with prostate cancer(PCa)and is one of the mechanisms of resistance to PCa therapies such as AR antagonists.Although sequencing technologies like next-generation sequencing(NGS)contributes to the high-throughput and precise detection of AR mutations carried by PCa patients,the lack of interpretations of these clinical genetic variants has still been a roadblock for PCa-targeted precision medicine.Here,we established a designer yeast reporter assay to simulate natural androgen receptor(AR)selection using AR antagonists.Yeast HIS3 gene transactivation was associated with the ligand-induced recruitment of steroid receptor coactivator-1(SRC-1)by AR mutants,where yeast growth in histidine-free medium was determined as the outcome.This assay is applicable to determine a wide range of clinical AR mutants including those with loss of function relating to androgen insensitivity syndrome(AIS),and those associated with PCa conferring resistance to AR antagonists such as enzalutamide(ENZ),bicalutamide(BIC),and cyproterone acetate(CPA).One clinical AR mutant previously reported to confer ENZ-resistance,F877L,was found to confer partial resistance to CPA as well using designer yeast.Our simple and efficient assay can enable precise one-pot screening of AR mutants,providing a reference for tailored medicine.
文摘The applications of supramolecular metal-peptide assemblies as catalyst or catalyst precursor have recent attracted increasing attentions.In this work,a fragment of the amyloid β-peptide,NH_(2)-KLVFF-COOH,was assembled into nanofilms with encapsulated Pd,Pt and Au nanoparticles(NPs)via a one-step room temperature electron induction method.The effects of building block,intermolecular interaction,driving force and side-chain on the assembly were investigated.The assembly mechanism was thereby proposed.The crosslinking of peptide monomers results in mainly random and unordered structures.The obtained metal-peptide assemblies are extremely stable in water at neutral pH for long term.However,the metal NPs are able to be responsively released under basic and reductive conditions.The released NPs show a high activity to catalyze the reduction of 4-nitrophenol.The present studies on assembly mechanism and responsive release will be helpful for the design of organic skeletons and also for the future development of peptide stabilized metallic NPs with applications beyond catalysts.
