萜类合成酶定向进化的新思路
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摘要
萜类化合物是天然产物中种类最多且主要存在于植物和微生物体内的一类化合物。随着越来越多具有应用价值的萜类化合物被挖掘,其应用前景引起了人们的关注,但由于含量低、提取成本高等缺点,因此制约了萜类化合物的广泛应用。合成生物学的兴起,为异源合成具有应用价值的萜类化合物提供了新思路,使构建定向、高效的微生物细胞工厂成为现实。萜类合成酶常作为萜类化合物异源合成代谢调控的靶酶,但天然的萜类合成酶存在催化效率低、底物专一性差、立体/区域选择性差、稳定性差等问题,严重影响萜类化合物的产量。萜类合成酶的定向进化可以有效地解决上述问题,为实现微生物细胞工厂异源、高效合成萜类化合物奠定基础。本文综述了近年来酶的定向进化技术的最新进展及应用,并提出了萜类合成酶定向进化的策略。
Terpenoids are mostly existing compounds in natural products like plants and microorganisms. The application prospect of terpenoids attracts much attention owing to more and more valuable terpenoids discovered. However, limited yield and high extraction cost of terpenoids restrict their wide applications. The rise of synthetic biology has provided new ideas for biosynthesis of valuable terpenoids using targeted and high efficient microbial cell factories. Although terpenoid synthases are widely used as target enzymes in metabolic regulation of terpenoids biosynthesis, many natural terpenoid synthases have some disadvantages, such as insufficient catalytic activity, poor substrate specificity, poor regio-or stereoselectivity, poor stability and so on,which unfavorably affect the yield of terpenoids. To solve above problems, directed evolution of terpenoid synthases has been applied, which will have profound impact on biosynthesis of terpenoids by microbial cell factories. This review summarizes recent advances and their applications in directed evolution of enzymes.Meanwhile, the strategies for directed evolution of terpenoid synthases are proposed.
Terpenoids are mostly existing compounds in natural products like plants and microorganisms. The application prospect of terpenoids attracts much attention owing to more and more valuable terpenoids discovered. However, limited yield and high extraction cost of terpenoids restrict their wide applications. The rise of synthetic biology has provided new ideas for biosynthesis of valuable terpenoids using targeted and high efficient microbial cell factories. Although terpenoid synthases are widely used as target enzymes in metabolic regulation of terpenoids biosynthesis, many natural terpenoid synthases have some disadvantages, such as insufficient catalytic activity, poor substrate specificity, poor regio-or stereoselectivity, poor stability and so on,which unfavorably affect the yield of terpenoids. To solve above problems, directed evolution of terpenoid synthases has been applied, which will have profound impact on biosynthesis of terpenoids by microbial cell factories. This review summarizes recent advances and their applications in directed evolution of enzymes.Meanwhile, the strategies for directed evolution of terpenoid synthases are proposed.
引文
[1]Sun LC,Li SY,Wang FZ,Xin FJ.Research progresses in the synthetic biology of terpenoids.Biotechnology Bulletin,2017,33(1):64-75.(in Chinese)孙丽超,李淑英,王凤忠,辛凤姣.萜类化合物的合成生物学研究进展.生物技术通报,2017,33(1):64-75.
[2]Baunach M,Franke J,Hertweck C.Terpenoid biosynthesis off the beaten track:unconventional cyclases and their impact on biomimetic synthesis.Angewandte Chemie International Edition,2015,54(9):2604-2626.
[3]Hu ZH,Chen BX,Yu AQ,Xiao DG.Strategies of metabolic engineering Saccharomyces cerevisiae to produce plant-derived D-Limonene.Acta Microbiologica Sinica,2018,58(9):1542-1550.(in Chinese)胡智慧,谌柄旭,于爱群,肖冬光.代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略.微生物学报,2018,58(9):1542-1550.
[4]Qu G,Zhao J,Zheng P,Sun JB,Sun ZT.Recent advances in directed evolution.Chinese Journal of Biotechnology,2018,34(1):1-11.(in Chinese)曲戈,赵晶,郑平,孙际宾,孙周通.定向进化技术的最新进展.生物工程学报,2018,34(1):1-11.
[5]Christianson DW.Structural and chemical biology of terpenoidcyclases.Chemical Reviews,2017,117(17):11570.
[6]Oldfield E,Lin FY.Terpene biosynthesis:modularity rules.Angewandte Chemie,2012,51(5):1124-1137.
[7]Jennewein S,Croteau R.Taxol:biosynthesis,molecular genetics,and biotechnological applications.ApplMicrobiolBiotechnol,2001,57(1-2):13-19.
[8]S?rensen PM,Iacob RE,Fritzsche M,Engen JR,Brieher WM.Charras G,Eggert US.The natural product cucurbitacin E inhibits depolymerization of actin filaments.ACS Chemical Biology,2012,7(9):1502-1508.
[9]Ukiya M,Akihisa T,Yasukawa K,Tokuda H,Toriumi M,Koike K,Kimura Y,Nikaido T,AoiW NH,Takido M.Anti-inflammatory and anti-tumor promoting effects of cucurbitane glycosides from the roots of Bryoniadioica.Journal of Natural Products,2002,65:179-183.
[10]Yan X,Fan Y,Wei W,Wang P,Liu Q,Wei Y,Zhang L,Zhao G,Yue J,Zhou Z.Production of bioactive ginsenoside compound K in metabolically engineered yeast.Cell Research,2014,24:770-773.
[11]Kirsh VA,Hayes RB,Mayne ST,Chatterjee N,Subar AF,Dixon LB,Albanes D,Andriole GL,Urban DA,Peters U.Supplemental and dietary Vitamin E,β-carotene,and Vitamin C intakes and prostate cancer risk.JNCI-Journal of the National Cancer Institute,2006,98(4):245-254.
[12]Martin VJ,Pitera DJ,Withers ST,Newman JD,Keasling JD.Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nature Biotechnology,2003,21:796-802.
[13]Aharoni A,Jongsma MA,Bouwmeester HJ.Volatilescience?Metabolic engineering of terpenoids in plants.Trends in Plant Science,2005,10:594-602.
[14]Pichersky E,Gershenzon J.The formation and function of plant volatiles:perfumes for pollinator attraction and defense.Current Opinion in Plant Biology,2002,5(3):237-243.
[15]Alonsogutierrez J,Chan R,Batth TS,Adams PD,Keasling JD,Petzold CJ,Lee TS.Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.Metabolic Engineering,2013,19(5):33-41.
[16]Wang C,Yoon SH,Jang HJ,Chung YR,Kim JY,Choi ES,Kim SW.Metabolic engineering of Escherichia coli forα-farnesene production.Metabolic Engineering,2011,13(6):648-655.
[17]Phelan RM,Sekurova ON,Keasling JD,Zotchev SB.Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene.ACS Synthetic Biology,2015,4(4):393-399.
[18]Sheldon RA,Pereira PC.Biocatalysis engineering:the big picture.Chemical Society Reviews,2017,46(10):2678-2691.
[19]Kwan DH,Sun YH,Schulz F,Hui H,Popovic B,Sim-Stark JC,Haydock SF,Leadlay PF.Prediction and manipulation of the stereochemistry of enoylreduction in modular polyketide synthases.Chemistry&Biology,2008,15(11):1231-1240.
[20]Reetz MT,Sheng W.Greatly reduced amino acid alphabets in directed evolution:Making the right choice for saturation mutagenesis at homologous enzyme positions.Chemical Communications,2008,43(43):5499-5501.
[21]Reetz MT,Torre C,Eipper A,Lohmer R,Hermes M,Brunner B,Maichele A,Bocola M,Arand M,Cronin A,Genze Y,Archelas A,Furstoss R.Enhancing the enantioselectivity of an epoxide hydrolase by directed evolution.Organic Letters,2004,6(2):177-80.
[22]Coco WM,Levinson WE,Crist MJ,Hektor HJ,Darzins A,Pienkos PT,Squires CH,Monticello DJ.DNA shuffling method for generating highly recombined genes and evolved enzymes.Nature Biotechnology,2001,19(4):354.
[23]Wong TS,Tee KL,Hauer B,Schwaneberg U.Sequence saturation mutagenesis(SeSaM):a novel method for directed evolution.Nucleic Acids Research,2004,32(3):e26.
[24]Lin YR,Koga N,Tatsumi-Koga R,Liu GH,Clouser AF,Montelione GT,Baker D.Control over overall shape and size in de novo designed proteins.Proceedings of the National Academy of Sciences of the United States of America,2015,112(40):5478-85.
[25]Chen F,Gaucher EA,Leal NA,Huttera D,Havemanna SA,Govindarajand S,Ortlunde EA,and Benner SA.Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNPdetection.Proceedings of the National Academy of Sciences of the United States of America,2010,107(5):1948-1953.
[26]Fox RJ,Davis SC,Mundorff EC,NewmanLM,Gavrilovic V,Ma SK,Chung LM,Ching C,Tam S,Muley S,Grate J,Gruber J,Whitman JC,Sheldon RA,Huisman GW.Improving catalytic function by ProSAR-driven enzyme evolution.Nature Biotechnology,2007,25(3):338-344.
[27]Cheng F,Zhu LL,Schwaneberg U.Directed evolution 2.0:improving and deciphering enzyme properties.Chemical Communications,2015,51(48):9760-9772.
[28]Gutierrez EA,Mundhada H,Meier T,Duefel H,Bocola M,Schwaneberg U.Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.Biosensors&Bioelectronics,2013,50(4):84-90.
[29]Sun ZT,Wikmark Y,B?ckvall JE,Reetz MT.New concepts for increasing the efficiency in directed evolution of stereoselective enzymes.Chemistry,2016,22(15):5046-5054.
[30]Sun ZT,Lonsdale R,Kong XD,Xu JH,Zhou J,Reetz MT.Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity:use of smallest amino acid alphabets in directed evolution.Angewandte Chemie,2015,54(42):12410-12415.
[31]Sun Z,Lonsdale R,Li GY,Reetz MT.Comparing different strategies in directed evolution of enzyme stereoselectivity:single versus double code saturation mutagenesis.Chembiochem,2016,17(19):1865-1872.
[32]Sun ZT,Lonsdale R,Wu L,Li GY,Li AT,Wang JB,Zhou JH,Reetz MT.Structure-guided triple-code saturation mutagenesis:efficient tuning of the stereoselectivity of an epoxide hydrolase.ACS Catalysis,2016,6(3):1590-1597.
[33]Li AT,Ilie A,Sun ZT,Lonsdale R,Xu JH,Reetz MT.Whole-cell-catalyzed multiple regio-and stereo selective function alizations in cascade reactions enabled by directed evolution.AngewandteChemie International Edition,2016,55(39):12026-12029.
[34]Leferink NGH,Ranaghan K,Karrupiah V,Currin A,Kamp MVD,Mulholland AJ,Scrutton NS.Experiment and simulation reveal how mutations in functional plasticity regions guide plant monoterpene synthase product outcome.ACS Catalysis,2018,8(5).
[35]Furubayashi M,Ikezumi M,Kajiwara J,Iwasaki M,Fujii A,Li L,Saito K,Umeno D.A high throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.PLoS One,2014,9(3):e93317.
[36]Tashiro M,Kiyota H,Kawai-Noma S,Saito K,Ikeuchi M,Iijima Y,Umeno D.Bacterial production of pinene by a laboratory-evolved pinene synthase.ACS Synthetic Biology,2016,5(9):10-11.
[37]Jiang GZ,Yao MD,Wang Y,Zhou L,Song TQ,Liu H,Xiao WH,Xuan YJ.Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.Metabolic Engineering,2017,41:57-66.
[38]Xie WP,Lv XM,Ye LD,Zhou PP,Yu HW.Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.Metabolic Engineering,2015,30:69-78.
[39]Sangeetha VL,Jiang JY,Zakharian T,Cane DE,Christianson DW.Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis:probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-motif.Archives of Biochemistry&Biophysics,2008,469(2):184-194.
[40]Xu JK,Ai Y,Wang JH,Xu JW,Zhang YK,Yang D.Converting s-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.Phytochemistry,2017,137:34-41.
[41]Lutz S.Beyond directed evolutionsemi-rationalprotein engineering and design.Current Opinion in Biotechnology,2010,21(6):734-743.
[42]Chica RA,Doucet N,Pelletier JN.Semi-rational approaches to engineering enzyme activity:combining the benefits of directed evolution and rational design.Current Opinion in Biotechnology,2005,16(4):378-384.
[43]Denard CA,Ren HQ,Zhao HM.Improving and repurposing biocatalysts via directed evolution.Current Opinion in Chemical Biology,2015,25:55-64.
[2]Baunach M,Franke J,Hertweck C.Terpenoid biosynthesis off the beaten track:unconventional cyclases and their impact on biomimetic synthesis.Angewandte Chemie International Edition,2015,54(9):2604-2626.
[3]Hu ZH,Chen BX,Yu AQ,Xiao DG.Strategies of metabolic engineering Saccharomyces cerevisiae to produce plant-derived D-Limonene.Acta Microbiologica Sinica,2018,58(9):1542-1550.(in Chinese)胡智慧,谌柄旭,于爱群,肖冬光.代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略.微生物学报,2018,58(9):1542-1550.
[4]Qu G,Zhao J,Zheng P,Sun JB,Sun ZT.Recent advances in directed evolution.Chinese Journal of Biotechnology,2018,34(1):1-11.(in Chinese)曲戈,赵晶,郑平,孙际宾,孙周通.定向进化技术的最新进展.生物工程学报,2018,34(1):1-11.
[5]Christianson DW.Structural and chemical biology of terpenoidcyclases.Chemical Reviews,2017,117(17):11570.
[6]Oldfield E,Lin FY.Terpene biosynthesis:modularity rules.Angewandte Chemie,2012,51(5):1124-1137.
[7]Jennewein S,Croteau R.Taxol:biosynthesis,molecular genetics,and biotechnological applications.ApplMicrobiolBiotechnol,2001,57(1-2):13-19.
[8]S?rensen PM,Iacob RE,Fritzsche M,Engen JR,Brieher WM.Charras G,Eggert US.The natural product cucurbitacin E inhibits depolymerization of actin filaments.ACS Chemical Biology,2012,7(9):1502-1508.
[9]Ukiya M,Akihisa T,Yasukawa K,Tokuda H,Toriumi M,Koike K,Kimura Y,Nikaido T,AoiW NH,Takido M.Anti-inflammatory and anti-tumor promoting effects of cucurbitane glycosides from the roots of Bryoniadioica.Journal of Natural Products,2002,65:179-183.
[10]Yan X,Fan Y,Wei W,Wang P,Liu Q,Wei Y,Zhang L,Zhao G,Yue J,Zhou Z.Production of bioactive ginsenoside compound K in metabolically engineered yeast.Cell Research,2014,24:770-773.
[11]Kirsh VA,Hayes RB,Mayne ST,Chatterjee N,Subar AF,Dixon LB,Albanes D,Andriole GL,Urban DA,Peters U.Supplemental and dietary Vitamin E,β-carotene,and Vitamin C intakes and prostate cancer risk.JNCI-Journal of the National Cancer Institute,2006,98(4):245-254.
[12]Martin VJ,Pitera DJ,Withers ST,Newman JD,Keasling JD.Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nature Biotechnology,2003,21:796-802.
[13]Aharoni A,Jongsma MA,Bouwmeester HJ.Volatilescience?Metabolic engineering of terpenoids in plants.Trends in Plant Science,2005,10:594-602.
[14]Pichersky E,Gershenzon J.The formation and function of plant volatiles:perfumes for pollinator attraction and defense.Current Opinion in Plant Biology,2002,5(3):237-243.
[15]Alonsogutierrez J,Chan R,Batth TS,Adams PD,Keasling JD,Petzold CJ,Lee TS.Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.Metabolic Engineering,2013,19(5):33-41.
[16]Wang C,Yoon SH,Jang HJ,Chung YR,Kim JY,Choi ES,Kim SW.Metabolic engineering of Escherichia coli forα-farnesene production.Metabolic Engineering,2011,13(6):648-655.
[17]Phelan RM,Sekurova ON,Keasling JD,Zotchev SB.Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene.ACS Synthetic Biology,2015,4(4):393-399.
[18]Sheldon RA,Pereira PC.Biocatalysis engineering:the big picture.Chemical Society Reviews,2017,46(10):2678-2691.
[19]Kwan DH,Sun YH,Schulz F,Hui H,Popovic B,Sim-Stark JC,Haydock SF,Leadlay PF.Prediction and manipulation of the stereochemistry of enoylreduction in modular polyketide synthases.Chemistry&Biology,2008,15(11):1231-1240.
[20]Reetz MT,Sheng W.Greatly reduced amino acid alphabets in directed evolution:Making the right choice for saturation mutagenesis at homologous enzyme positions.Chemical Communications,2008,43(43):5499-5501.
[21]Reetz MT,Torre C,Eipper A,Lohmer R,Hermes M,Brunner B,Maichele A,Bocola M,Arand M,Cronin A,Genze Y,Archelas A,Furstoss R.Enhancing the enantioselectivity of an epoxide hydrolase by directed evolution.Organic Letters,2004,6(2):177-80.
[22]Coco WM,Levinson WE,Crist MJ,Hektor HJ,Darzins A,Pienkos PT,Squires CH,Monticello DJ.DNA shuffling method for generating highly recombined genes and evolved enzymes.Nature Biotechnology,2001,19(4):354.
[23]Wong TS,Tee KL,Hauer B,Schwaneberg U.Sequence saturation mutagenesis(SeSaM):a novel method for directed evolution.Nucleic Acids Research,2004,32(3):e26.
[24]Lin YR,Koga N,Tatsumi-Koga R,Liu GH,Clouser AF,Montelione GT,Baker D.Control over overall shape and size in de novo designed proteins.Proceedings of the National Academy of Sciences of the United States of America,2015,112(40):5478-85.
[25]Chen F,Gaucher EA,Leal NA,Huttera D,Havemanna SA,Govindarajand S,Ortlunde EA,and Benner SA.Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNPdetection.Proceedings of the National Academy of Sciences of the United States of America,2010,107(5):1948-1953.
[26]Fox RJ,Davis SC,Mundorff EC,NewmanLM,Gavrilovic V,Ma SK,Chung LM,Ching C,Tam S,Muley S,Grate J,Gruber J,Whitman JC,Sheldon RA,Huisman GW.Improving catalytic function by ProSAR-driven enzyme evolution.Nature Biotechnology,2007,25(3):338-344.
[27]Cheng F,Zhu LL,Schwaneberg U.Directed evolution 2.0:improving and deciphering enzyme properties.Chemical Communications,2015,51(48):9760-9772.
[28]Gutierrez EA,Mundhada H,Meier T,Duefel H,Bocola M,Schwaneberg U.Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.Biosensors&Bioelectronics,2013,50(4):84-90.
[29]Sun ZT,Wikmark Y,B?ckvall JE,Reetz MT.New concepts for increasing the efficiency in directed evolution of stereoselective enzymes.Chemistry,2016,22(15):5046-5054.
[30]Sun ZT,Lonsdale R,Kong XD,Xu JH,Zhou J,Reetz MT.Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity:use of smallest amino acid alphabets in directed evolution.Angewandte Chemie,2015,54(42):12410-12415.
[31]Sun Z,Lonsdale R,Li GY,Reetz MT.Comparing different strategies in directed evolution of enzyme stereoselectivity:single versus double code saturation mutagenesis.Chembiochem,2016,17(19):1865-1872.
[32]Sun ZT,Lonsdale R,Wu L,Li GY,Li AT,Wang JB,Zhou JH,Reetz MT.Structure-guided triple-code saturation mutagenesis:efficient tuning of the stereoselectivity of an epoxide hydrolase.ACS Catalysis,2016,6(3):1590-1597.
[33]Li AT,Ilie A,Sun ZT,Lonsdale R,Xu JH,Reetz MT.Whole-cell-catalyzed multiple regio-and stereo selective function alizations in cascade reactions enabled by directed evolution.AngewandteChemie International Edition,2016,55(39):12026-12029.
[34]Leferink NGH,Ranaghan K,Karrupiah V,Currin A,Kamp MVD,Mulholland AJ,Scrutton NS.Experiment and simulation reveal how mutations in functional plasticity regions guide plant monoterpene synthase product outcome.ACS Catalysis,2018,8(5).
[35]Furubayashi M,Ikezumi M,Kajiwara J,Iwasaki M,Fujii A,Li L,Saito K,Umeno D.A high throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.PLoS One,2014,9(3):e93317.
[36]Tashiro M,Kiyota H,Kawai-Noma S,Saito K,Ikeuchi M,Iijima Y,Umeno D.Bacterial production of pinene by a laboratory-evolved pinene synthase.ACS Synthetic Biology,2016,5(9):10-11.
[37]Jiang GZ,Yao MD,Wang Y,Zhou L,Song TQ,Liu H,Xiao WH,Xuan YJ.Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.Metabolic Engineering,2017,41:57-66.
[38]Xie WP,Lv XM,Ye LD,Zhou PP,Yu HW.Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.Metabolic Engineering,2015,30:69-78.
[39]Sangeetha VL,Jiang JY,Zakharian T,Cane DE,Christianson DW.Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis:probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-motif.Archives of Biochemistry&Biophysics,2008,469(2):184-194.
[40]Xu JK,Ai Y,Wang JH,Xu JW,Zhang YK,Yang D.Converting s-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.Phytochemistry,2017,137:34-41.
[41]Lutz S.Beyond directed evolutionsemi-rationalprotein engineering and design.Current Opinion in Biotechnology,2010,21(6):734-743.
[42]Chica RA,Doucet N,Pelletier JN.Semi-rational approaches to engineering enzyme activity:combining the benefits of directed evolution and rational design.Current Opinion in Biotechnology,2005,16(4):378-384.
[43]Denard CA,Ren HQ,Zhao HM.Improving and repurposing biocatalysts via directed evolution.Current Opinion in Chemical Biology,2015,25:55-64.