利用Golden Gate “One-POT”技术组装运动发酵单胞菌转录单元
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摘要
为研究运动发酵单胞菌合成生物学及代谢工程,评估运动发酵单胞菌内源启动子及终止子,需要建立一种方便有效的多基因代谢途径组装方法.通过Golden Gate方法构建了一个含有运动发酵单胞菌乙醇脱氢酶2编码基因启动子区(P_(adh2))、丙酮酸脱羧酶编码基因终止子区(T_(pdc))和绿色荧光蛋白开放阅读框(gfp)的人工转录单元.将含有IIS型限制内切酶Bbs I识别和酶切序列的特异性接头添加到P_(adh2)、T_(pdc)和gfp序列两侧,从而保证在一个反应体系中完成上述3个生物元件的酶切和连接,因此该技术被称作"One-POT".插入上述人工转录单元的穿梭质粒导入大肠杆菌和运动发酵单胞菌后可检测到发出绿色荧光的细胞.结果显示通过Golden Gate方法可有效组装转录单元所需DNA生物元件.本研究不仅可为运动发酵单胞菌合成生物学和代谢工程研究,尤其是设计多基因代谢途径组装方面提供技术工具,也可为评估运动发酵单胞菌内源启动子和终止子提供一种有效方法.
In order to study synthetic biology and metabolic engineering in Zymomonas mobilis, besides evaluating its native promoters and terminators, a convenient and effective multigene metabolic pathway assembly method needs to be established.In this study, an artificial transcriptional unit(TU), which comprised the promoter region of ethanol dehydrogenase 2-encoding gene(P_(adh2)) and the terminator region of pyruvate decarboxylase-encoding gene(T_(pdc)) of Z. mobilis and an open reading frame(ORF) of green fluorescent protein(gfp), was assembled via the Golden Gate method. Specific adapters containing the sequence of type IIS restriction endonuclease Bbs I flanked the above three biological parts to allow the digestion and assembly of the Promoter, ORF, and Terminator in one single reaction, hence the names "One-POT". The shuttle plasmid embedding the artificial TU was introduced into Escherichia coli and Z. mobilis, and the cells with green fluorescence were detected. The results demonstrate that the biological parts of TU were efficiently assembled in the defined order via the Golden Gate method.This study not only provides tools for the research on synthetic biology and metabolic engineering, especially on the assembly of multigene metabolic pathway in Z. mobilis, but also offers an effective means to evaluate native promoters and terminators of Z. mobilis.
In order to study synthetic biology and metabolic engineering in Zymomonas mobilis, besides evaluating its native promoters and terminators, a convenient and effective multigene metabolic pathway assembly method needs to be established.In this study, an artificial transcriptional unit(TU), which comprised the promoter region of ethanol dehydrogenase 2-encoding gene(P_(adh2)) and the terminator region of pyruvate decarboxylase-encoding gene(T_(pdc)) of Z. mobilis and an open reading frame(ORF) of green fluorescent protein(gfp), was assembled via the Golden Gate method. Specific adapters containing the sequence of type IIS restriction endonuclease Bbs I flanked the above three biological parts to allow the digestion and assembly of the Promoter, ORF, and Terminator in one single reaction, hence the names "One-POT". The shuttle plasmid embedding the artificial TU was introduced into Escherichia coli and Z. mobilis, and the cells with green fluorescence were detected. The results demonstrate that the biological parts of TU were efficiently assembled in the defined order via the Golden Gate method.This study not only provides tools for the research on synthetic biology and metabolic engineering, especially on the assembly of multigene metabolic pathway in Z. mobilis, but also offers an effective means to evaluate native promoters and terminators of Z. mobilis.
引文
1 He MX,Wu B,Qin H,Ruan ZY,Tan FR,Wang JL,Shui ZX,Dai LC,Zhu QL,Pan K,Tang XY,Wang WG,Hu QC.Zymomonas mobilis:a novel platform for future biorefineries[J].Biotechnol Biofuels,2014,7(1):101
2 Yang SH,Fei Q,Zhang YP,Contreras LM,Utturkar SM,Brown SD,Himmel ME,Zhang M.Zymomonas mobilis as a model system for production of biofuels and biochemicals[J].Microbial Biotechnol,2016,9(6):699-717
3 Yang SH,Mohagheghi A,Franden MA,Chou YC,Chen XW,Dowe N,Himmel ME,Zhang M,Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars[J].Biotechnol Biofuels,2016,9(1):189
4税宗霞,王景丽,秦晗,吴波,谭芙蓉,何明雄.产丁二酸运动发酵单胞菌的构建和发酵性能[J].应用与环境生物学报,2015,21(4):657-664[Shui ZX,Wang JL,Qin H,Wu B,Tan FR,He MX.Construction and preliminary fermentation of succinate-producing recombinant ethanologenic Zymomonas mobilis[J].Chin J Appl Environ Biol,2015,21(4):657-664]
5 Hippel PH.An integrated model of the transcription complex in elongation,termination,and editing[J].Science,1998,281(5377):660-665
6 Bourdichon F,Casaregola S,Farrokh C,Frisvad JC,Gerds ML,Hammes WP,Harnett J,Huys G,Laulund S,Ouwehand A,Powell IB,Prajapati JB,Seto Y,Ter SE,Van BA,Vankerckhoven V,Zgoda A,Tuijtelaars S,Hansen EB.Food fermentations:microorganisms with technological beneficial use[J].Int J Food Microbiol,2012,154(3):87-97
7 Reynen M,Reipen I,Sahm H,Sprenger GA.Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis[J].Mol Gen Genet,1990,223:335-341
8 He MX,Feng H,Zhang YZ.Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif[J].Biotechnol Lett,2008,30(12):2111-7.
9 Li ZJ,Cai L,Wu Q,Chen GQ.Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate)production[J].Appl Microbiol Biotechnol,2009,83(5):939-947
10 Chung A,Liu Q,Ouyang SP,Wu Q,Chen GQ.Microbial production of3-hydroxydodecanoic acid by pha operon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene[J].Appl Microbiol Biotechnol,2009,83(3):513-519
11 Liu Q,Ouyang SP,Chung A,Wu Q,Chen GQ.Microbial production of R-3-hydroxybutyric acid by recombinant E.coli harboring genes of phbA,phbB,and tesB[J].Appl Microbiol Biotechnol,2007,76(4):811-818
12 Peng H,Fu B,Mao Z,Shao W.Electrotransformation of Thermoanaerobacter ethanolicus JW200[J].Biotechnol Letters,2006,28(23):1913-1917
13 Wang GJ,Wang ZS,Zhang YW,Zhang YZ.Cloning and expression of amyE gene from Bacillus subtilis in Zymomonas mobilis and direct production of ethanol from soluble starch[J].Biotechnold Bioprocess Eng,2012,17(4):780-786
14 Marsischky G,La BJ.Many paths to many clones:a comparative look at high-throughput cloning methods[J].Genome Res,2004,14(10B):2020-2028
15 Anderson JC,Dueber JE,Leguia M,Wu GC,Goler JA,Arkin AP,Keasling JD.BglBricks:a flexible standard for biological part assembly[J].J Biological Eng 2010,4(1):1
16 Engler C,Kandzia R,Marillonnet S.A one pot,one step,precision cloning method with high throughput capability[J].PLoS One,2008,3(11):e3647
17 Engler C,Gruetzner R,Kandzia R,Marillonnet S.Golden gate shuffling:a one-pot DNA shuffling method based on type IIs restriction enzymes[J].PLoS ONE,2009,4(5):e5553
18 Fromme T,Klingenspor M.Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase[J].JBiological Eng,2007,1(1):7
19 Guo Y,Dong J,Zhou T,Auxillos J,Li T,Zhang W,Wang L,Shen Y,Luo Y,Zheng Y,Lin J,Chen GQ,Wu Q,Cai Y,Dai J.YeastFab:the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae[J].Nucleic Acids Res,2015,43(13):e88
20 Agmon N,Mitchell LA,Cai Y,Ikushima S,Chuang J,Zheng A,Choi WJ,Martin JA,Caravelli K,Stracquadanio G,Boeke JD.Yeast Golden Gate(yGG)for the efficient assembly of S.cerevisiae transcription units[J].ACS Synthetic Biol,2015,4(7):853-859
21 Celinska E,Ledesma-Amaro R,Larroude M,Rossignol T,Pauthenier C,Nicaud JM.Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica[J].Microb Biotechnol,2017,10(2):450-455
22 Terfruchte M,Joehnk B,Fajardo SR,Braus GH,Riquelme M,Schipper K,Feldbrugge M.Establishing a versatile Golden Gate cloning system for genetic engineering in fungi[J].Fungal Genet Biol,2014,62:1-10
23 Kakui Y,Sunaga T,Arai K,Dodgson J,Ji L,Csikasz NA,Carazo SR,Sato M.Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe[J].Open Biol,2015,5(6):150054
24 Mitchell LA,Chuang J,Agmon N,Khunsriraksakul C,Phillips NA,Cai Y,Truong DM,Veerakumar A,Wang Y,Mayorga M,Blomquist P,Sadda P,Trueheart J,Boeke JD.Versatile genetic assembly system(VEGAS)to assemble pathways for expression in S.cerevisiae[J].Nucleic Acids Res,2015,43(13):6620-6630
25 Iverson SV,Haddock TL,Beal J,Densmore DM.CIDAR MoClo:improved MoClo assembly standard and new E.coli part library enable rapid combinatorial design for synthetic and traditional biology[J].ACSSynth Biol,2016,5(1):99-103
26 Moore SJ,Lai HE,Kelwick RJ,Chee SM,Bell DJ,Polizzi KM,Freemont PS.EcoFlex:a multifunctional MoClo kit for E.coli synthetic biology[J].ACS Synth Biol,2016,5(10):1059-1069
27 Engler C,Youles M,Gruetzner R,Ehnert TM,Werner S,Jones JD,Patron NJ,Marillonnet S.A golden gate modular cloning toolbox for plants[J].ACS Synth Biol,2014,3(11):839-843
28 Sambrook J,Russell DW.Molecular Cloning-A Laboratory Manual[M].3rd ed.New York:Cold Spring Harbor Laboratory Press,2001:292-446
29 Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,Smith HO.Enzymatic assembly of DNA molecules up to several hundred kilobases[J].Nat Methods,2009,6(5):343-347
30何明雄.运动发酵单胞菌直接发酵甘薯淀粉为乙醇的研究[D].成都:四川大学,2008[He MX.Direct fermentation of raw sweet potato starch to ethanol by Zymomonas mobilis[D].Chengdu:Sichuan University,2008]
31吴波.运动发酵单孢菌的限制修饰系统基因突变及α-淀粉酶基因的分泌表达[D].成都:四川大学,2011[Wu B.Mutagenesis of restriction-modification systems and secretion expression ofα-amylase in Zymomonas mobilis[D].Chengdu:Sichuan University,2011]
32 Yang TT,Cheng L,Kain SR.Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein[J].Nucleic Acids Res,1996,24(22):4592-4593
33 Seo JS,Chong H,Park HS,Yoon KO,Jung C,Kim JJ,Hong JH,Kim H,Kim JH,Kil JI,Park CJ,Oh HM,Lee JS,Jin SJ,Um HW,Lee HJ,Oh SJ,Kim JY,Kang HL,Lee SY,Lee KJ,Kang HS.The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4[J].Nat Biotechnol,2005,23(1):63-68
34 Wu T,Ye LJ,Zhao DD,Li SW,Li QY,Zhang BL,Bi CH,Zhang XL.Membrane engineering-a novel strategy to enhance the production and accumulation ofβ-carotene in Escherichia coli[J].Metabolic Eng,2017,43:85-91
2 Yang SH,Fei Q,Zhang YP,Contreras LM,Utturkar SM,Brown SD,Himmel ME,Zhang M.Zymomonas mobilis as a model system for production of biofuels and biochemicals[J].Microbial Biotechnol,2016,9(6):699-717
3 Yang SH,Mohagheghi A,Franden MA,Chou YC,Chen XW,Dowe N,Himmel ME,Zhang M,Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars[J].Biotechnol Biofuels,2016,9(1):189
4税宗霞,王景丽,秦晗,吴波,谭芙蓉,何明雄.产丁二酸运动发酵单胞菌的构建和发酵性能[J].应用与环境生物学报,2015,21(4):657-664[Shui ZX,Wang JL,Qin H,Wu B,Tan FR,He MX.Construction and preliminary fermentation of succinate-producing recombinant ethanologenic Zymomonas mobilis[J].Chin J Appl Environ Biol,2015,21(4):657-664]
5 Hippel PH.An integrated model of the transcription complex in elongation,termination,and editing[J].Science,1998,281(5377):660-665
6 Bourdichon F,Casaregola S,Farrokh C,Frisvad JC,Gerds ML,Hammes WP,Harnett J,Huys G,Laulund S,Ouwehand A,Powell IB,Prajapati JB,Seto Y,Ter SE,Van BA,Vankerckhoven V,Zgoda A,Tuijtelaars S,Hansen EB.Food fermentations:microorganisms with technological beneficial use[J].Int J Food Microbiol,2012,154(3):87-97
7 Reynen M,Reipen I,Sahm H,Sprenger GA.Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis[J].Mol Gen Genet,1990,223:335-341
8 He MX,Feng H,Zhang YZ.Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif[J].Biotechnol Lett,2008,30(12):2111-7.
9 Li ZJ,Cai L,Wu Q,Chen GQ.Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate)production[J].Appl Microbiol Biotechnol,2009,83(5):939-947
10 Chung A,Liu Q,Ouyang SP,Wu Q,Chen GQ.Microbial production of3-hydroxydodecanoic acid by pha operon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene[J].Appl Microbiol Biotechnol,2009,83(3):513-519
11 Liu Q,Ouyang SP,Chung A,Wu Q,Chen GQ.Microbial production of R-3-hydroxybutyric acid by recombinant E.coli harboring genes of phbA,phbB,and tesB[J].Appl Microbiol Biotechnol,2007,76(4):811-818
12 Peng H,Fu B,Mao Z,Shao W.Electrotransformation of Thermoanaerobacter ethanolicus JW200[J].Biotechnol Letters,2006,28(23):1913-1917
13 Wang GJ,Wang ZS,Zhang YW,Zhang YZ.Cloning and expression of amyE gene from Bacillus subtilis in Zymomonas mobilis and direct production of ethanol from soluble starch[J].Biotechnold Bioprocess Eng,2012,17(4):780-786
14 Marsischky G,La BJ.Many paths to many clones:a comparative look at high-throughput cloning methods[J].Genome Res,2004,14(10B):2020-2028
15 Anderson JC,Dueber JE,Leguia M,Wu GC,Goler JA,Arkin AP,Keasling JD.BglBricks:a flexible standard for biological part assembly[J].J Biological Eng 2010,4(1):1
16 Engler C,Kandzia R,Marillonnet S.A one pot,one step,precision cloning method with high throughput capability[J].PLoS One,2008,3(11):e3647
17 Engler C,Gruetzner R,Kandzia R,Marillonnet S.Golden gate shuffling:a one-pot DNA shuffling method based on type IIs restriction enzymes[J].PLoS ONE,2009,4(5):e5553
18 Fromme T,Klingenspor M.Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase[J].JBiological Eng,2007,1(1):7
19 Guo Y,Dong J,Zhou T,Auxillos J,Li T,Zhang W,Wang L,Shen Y,Luo Y,Zheng Y,Lin J,Chen GQ,Wu Q,Cai Y,Dai J.YeastFab:the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae[J].Nucleic Acids Res,2015,43(13):e88
20 Agmon N,Mitchell LA,Cai Y,Ikushima S,Chuang J,Zheng A,Choi WJ,Martin JA,Caravelli K,Stracquadanio G,Boeke JD.Yeast Golden Gate(yGG)for the efficient assembly of S.cerevisiae transcription units[J].ACS Synthetic Biol,2015,4(7):853-859
21 Celinska E,Ledesma-Amaro R,Larroude M,Rossignol T,Pauthenier C,Nicaud JM.Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica[J].Microb Biotechnol,2017,10(2):450-455
22 Terfruchte M,Joehnk B,Fajardo SR,Braus GH,Riquelme M,Schipper K,Feldbrugge M.Establishing a versatile Golden Gate cloning system for genetic engineering in fungi[J].Fungal Genet Biol,2014,62:1-10
23 Kakui Y,Sunaga T,Arai K,Dodgson J,Ji L,Csikasz NA,Carazo SR,Sato M.Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe[J].Open Biol,2015,5(6):150054
24 Mitchell LA,Chuang J,Agmon N,Khunsriraksakul C,Phillips NA,Cai Y,Truong DM,Veerakumar A,Wang Y,Mayorga M,Blomquist P,Sadda P,Trueheart J,Boeke JD.Versatile genetic assembly system(VEGAS)to assemble pathways for expression in S.cerevisiae[J].Nucleic Acids Res,2015,43(13):6620-6630
25 Iverson SV,Haddock TL,Beal J,Densmore DM.CIDAR MoClo:improved MoClo assembly standard and new E.coli part library enable rapid combinatorial design for synthetic and traditional biology[J].ACSSynth Biol,2016,5(1):99-103
26 Moore SJ,Lai HE,Kelwick RJ,Chee SM,Bell DJ,Polizzi KM,Freemont PS.EcoFlex:a multifunctional MoClo kit for E.coli synthetic biology[J].ACS Synth Biol,2016,5(10):1059-1069
27 Engler C,Youles M,Gruetzner R,Ehnert TM,Werner S,Jones JD,Patron NJ,Marillonnet S.A golden gate modular cloning toolbox for plants[J].ACS Synth Biol,2014,3(11):839-843
28 Sambrook J,Russell DW.Molecular Cloning-A Laboratory Manual[M].3rd ed.New York:Cold Spring Harbor Laboratory Press,2001:292-446
29 Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,Smith HO.Enzymatic assembly of DNA molecules up to several hundred kilobases[J].Nat Methods,2009,6(5):343-347
30何明雄.运动发酵单胞菌直接发酵甘薯淀粉为乙醇的研究[D].成都:四川大学,2008[He MX.Direct fermentation of raw sweet potato starch to ethanol by Zymomonas mobilis[D].Chengdu:Sichuan University,2008]
31吴波.运动发酵单孢菌的限制修饰系统基因突变及α-淀粉酶基因的分泌表达[D].成都:四川大学,2011[Wu B.Mutagenesis of restriction-modification systems and secretion expression ofα-amylase in Zymomonas mobilis[D].Chengdu:Sichuan University,2011]
32 Yang TT,Cheng L,Kain SR.Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein[J].Nucleic Acids Res,1996,24(22):4592-4593
33 Seo JS,Chong H,Park HS,Yoon KO,Jung C,Kim JJ,Hong JH,Kim H,Kim JH,Kil JI,Park CJ,Oh HM,Lee JS,Jin SJ,Um HW,Lee HJ,Oh SJ,Kim JY,Kang HL,Lee SY,Lee KJ,Kang HS.The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4[J].Nat Biotechnol,2005,23(1):63-68
34 Wu T,Ye LJ,Zhao DD,Li SW,Li QY,Zhang BL,Bi CH,Zhang XL.Membrane engineering-a novel strategy to enhance the production and accumulation ofβ-carotene in Escherichia coli[J].Metabolic Eng,2017,43:85-91
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