柠檬烯和红没药烯的微生物代谢工程
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摘要
柠檬烯和红没药烯均为植物天然产物,分别属于单萜类和倍半萜类化合物,能够预防和治疗癌症等多种疾病。以其作为前体物,还可以转化合成多种具有高附加值的工业产品,例如药品、保健品、化妆品及生物燃料等。目前柠檬烯和红没药烯的工业生产主要是通过植物提取法实现的,但从植物组织中提取柠檬烯和红没药烯存在着产物含量低和分离纯化困难等缺点。微生物代谢工程的快速发展为这些植物天然产物的生产提供了一条更具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些有价值的植物天然产物的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。文中系统综述了近年来代谢工程技术在微生物合成柠檬烯和红没药烯过程中的应用进展,包括所涉及的宿主菌株、关键酶、代谢途径及其改造等,并探讨了其未来发展方向。
Limonene(C_(10)H_(16)) and bisabolene(C_(15)H_(24)) are both naturally occurring terpenes in plants. Depending on thenumber of C_5 units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.
Limonene(C_(10)H_(16)) and bisabolene(C_(15)H_(24)) are both naturally occurring terpenes in plants. Depending on thenumber of C_5 units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.
引文
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[2]Du FL.Construction and optimization of(-)-limonene biosynthesis pathways in Escherichia coli[D].Shanghai:East China University of Science and Technology,2014(in Chinese).杜府亮.大肠杆菌MEP途径的改造及用于(-)-柠檬烯生物合成的研究[D].上海:华东理工大学,2014.
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[10]Ciriminna R,Lomeli-Rodriguez M,CaràPD,et al.Limonene:a versatile chemical of the bioeconomy.Chem Commun,2014,50(97):15288–15296.
[11]Jongedijk E,Cankar K,Buchhaupt M,et al.Biotechnological production of limonene in microorganisms.Appl Microbiol Biotechnol,2016,100(7):2927–2938.
[12]Inouye S,Takizawa T,Yamaguchi H.Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact.J Antimicrob Chemother,2001,47(5):565–573.
[13]Miller JA,Thompson PA,Hakim IA,et al.d-Limonene:a bioactive food component from citrus and evidence for a potential role in breast cancer prevention and treatment.Oncol Rev,2011,5(1):31–42.
[14]Tracy NI,Chen DC,Crunkleton DW,et al.Hydrogenated monoterpenes as diesel fuel additives.Fuel,2009,88(11):2238–2240.
[15]Leonard E,Ajikumar PK,Thayer K,et al.Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control.Proc Natl Acad Sci USA,2010,107(31):13654–13659.
[16]Peralta-Yahya PP,Ouellet M,Chan R,et al.Identification and microbial production of a terpene-based advanced biofuel.Nat Commun,2011,2:483.
[17]Davies FK,Work VH,Beliaev AS,et al.Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp.PCC 7002.Front Bioeng Biotechnol,2014,2:21.
[18]Kitaoka N,Lu X,Yang B,et al.The application of synthetic biology to elucidation of plant mono-,sesqui-,and diterpenoid metabolism.Mol Plant,2015,8(1):6–16.
[19]Colby SM,Alonso WR,Katahira EJ,et al.4S-limonene synthase from the oil glands of spearmint(Mentha spicata).c DNA isolation,characterization,and bacterial expression of the catalytically active monoterpene cyclase.J Biol Chem,1993,268(31):23016–23024.
[20]Yuba A,Yazaki K,Tabata M,et al.c DNA cloning,characterization,and functional expression of4S-(-)-limonene synthase from Perilla frutescens.Arch Biochem Biophys,1996,332(2):280–287.
[21]Bohlmann J,Steele CL,Croteau R.Monoterpene synthases from grand fir(Abies grandis).c DNA isolation,characterization,and functional expression of myrcene synthase,(-)-(4S)-limonene synthase,and(-)-(1S,5S)-pinene synthase.J Biol Chem,1997,272(35):21784–21792.
[22]Maruyama T,Ito M,Kiuchi F,et al.Molecular cloning,functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia.Biol Pharm Bull,2001,24(4):373–377.
[23]Lücker J,El Tamer MK,Schwab W,et al.Monoterpene biosynthesis in lemon(Citrus limon):c DNA isolation and functional analysis of four monoterpene synthases.Eur J Biochem,2002,269(13):3160–3171.
[24]Shimada T,Endo T,Fujii H,et al.Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc.Plant Sci,2004,166(1):49–58.
[25]Shimada T,Endo T,Fujii H,et al.Isolation and characterization of a new d-limonene synthase gene with a different expression pattern in Citrus unshiuMarc.Sci Hortic,2005,105(4):507–512.
[26]Bohlmann J,Crock J,Jetter R,et al.Terpenoid-based defenses in conifers:c DNA cloning,characterization,and functional expression of wound-inducible(E)-α-bisabolene synthase from grand fir(Abies grandis).Proc Natl Acad Sci USA,1998,95(12):6756–6761.
[27]Martin DM,F?ldt J,Bohlmann J.Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.Plant Physiol,2004,135(4):1908–1927.
[28]Parveen I,Wang M,Zhao JP,et al.Investigating sesquiterpene biosynthesis in Ginkgo biloba:molecular cloning and functional characterization of(E,E)-farnesol andα-bisabolene synthases.Plant Mol Biol,2015,89(4):451–462.
[29]Fujisawa M,Harada H,Kenmoku H,et al.Cloning and characterization of a novel gene that encodes(S)-beta-bisabolene synthase from ginger,Zingiber officinale.Planta,2010,232(1):121–130.
[30]Jones CG,Moniodis J,Zulak KG,et al.Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase(TPS)-a and TPS-b subfamilies,including santalene synthases.J Biol Chem,2011,286(20):17445–17454.
[31]Srivastava PL,Daramwar PP,Krithika R,et al.Functional characterization of novel sesquiterpene synthases from Indian sandalwood,Santalum album.Sci Rep,2015,5:10095.
[32]Huber DPW,Philippe RN,Godard KA,et al.Characterization of four terpene synthase c DNAs from methyl jasmonate-induced Douglas-fir,Pseudotsuga menziesii.Phytochemistry,2005,66(12):1427–1439.
[33]Ro DK,Ehlting J,Keeling CI,et al.Microarray expression profiling and functional characterization of At TPS genes:duplicated Arabidopsis thalianasesquiterpene synthase genes At4g13280 and At4g13300 encode root-specific and wound-inducible(Z)-gamma-bisabolene synthases.Arch Biochem Biophys,2006,448(1/2):104–116.
[34]Aschenbrenner AK,Kwon M,Conrad J,et al.Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower(Helianthus annuus L.,Asteraceae).Phytochemistry,2016,124:29–37.
[35]Alonso-Gutierrez J,Chan R,Batth TS,et al.Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.Metab Eng,2013,19:33–41.
[36]Jongedijk E,Cankar K,Ranzijn J,et al.Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae.Yeast,2014,32(1):159–171.
[37]Jiang GZ,Yao MD,Wang Y,et al.Manipulation of GESand ERG20 for geraniol overproduction in Saccharomyces cerevisiae.Metab Eng,2017,41:57–66.
[38]Blazeck J,Liu LQ,Redden H,et al.Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach.Appl Environ Microbiol,2011,77(22):7905–7914.
[39]Ajikumar PK,Xiao WH,Tyo KE,et al.Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli.Science,2010,330(6000):70–74.
[40]Ro DK,Paradise EM,Ouellet M,et al.Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,2006,440(7086):940–943.
[41]Dueber JE,Wu GC,Malmirchegini GR,et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat Biotechnol,2009,27(8):753–759.
[42]Lv XM,Wang F,Zhou PP,et al.Dual regulation of cytoplasmic and mitochondrial acetyl-Co A utilization for improved isoprene production in Saccharomyces cerevisiae.Nat Commun,2016,7:12851.
[43]Chen FJ,Zhou JW,Shi ZP,et al.Effect of acetyl-Co A synthase gene overexpression on physiological function of Saccharomyces cerevisiae.Acta Microbiol Sin,2010,50(9):1172–1179(in Chinese).陈孚江,周景文,史仲平,等.乙酰辅酶A合成代谢对酿酒酵母生理功能的影响.微生物学报,2010,50(9):1172–1179.
[44]Lian JZ,Zhao HM.Functional reconstitution of a pyruvate dehydrogenase in the cytosol of Saccharomyces cerevisiae through lipoylation machinery engineering.ACS Synth Biol,2016,5(7):689–697.
[45]Kozak BU,van Rossum HM,Luttik MAH,et al.Engineering acetyl coenzyme A supply:functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.m Bio,2014,5(5):e01696–14.
[46]Jiang JP,Wu XR,Chen YJ.Strategy to solve cofactor issues in oxidoreductase catalyzed biocatalytic applications.Chin J Biotech,2012,28(4):410–419(in Chinese).江金鹏,吴旭日,陈依军.解决氧化还原酶反应体系中辅酶问题的策略及其应用.生物工程学报,2012,28(4):410-419.
[2]Du FL.Construction and optimization of(-)-limonene biosynthesis pathways in Escherichia coli[D].Shanghai:East China University of Science and Technology,2014(in Chinese).杜府亮.大肠杆菌MEP途径的改造及用于(-)-柠檬烯生物合成的研究[D].上海:华东理工大学,2014.
[3]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/319222027.
[4]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/249928896.
[5]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/319078015.
[6]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/319297517.
[7]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/319220468.
[8]National Center for Biotechnology Information.Pub Chem Substance Database[EB/OL].[2017-06-05].https://pubchem.ncbi.nlm.nih.gov/substance/316964431.
[9]Johnson TJ,Jahandideh A,Johnson MD,et al.Producing next-generation biofuels from filamentous cyanobacteria:an economic feasibility analysis.Algal Res,2016,20:218–228.
[10]Ciriminna R,Lomeli-Rodriguez M,CaràPD,et al.Limonene:a versatile chemical of the bioeconomy.Chem Commun,2014,50(97):15288–15296.
[11]Jongedijk E,Cankar K,Buchhaupt M,et al.Biotechnological production of limonene in microorganisms.Appl Microbiol Biotechnol,2016,100(7):2927–2938.
[12]Inouye S,Takizawa T,Yamaguchi H.Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact.J Antimicrob Chemother,2001,47(5):565–573.
[13]Miller JA,Thompson PA,Hakim IA,et al.d-Limonene:a bioactive food component from citrus and evidence for a potential role in breast cancer prevention and treatment.Oncol Rev,2011,5(1):31–42.
[14]Tracy NI,Chen DC,Crunkleton DW,et al.Hydrogenated monoterpenes as diesel fuel additives.Fuel,2009,88(11):2238–2240.
[15]Leonard E,Ajikumar PK,Thayer K,et al.Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control.Proc Natl Acad Sci USA,2010,107(31):13654–13659.
[16]Peralta-Yahya PP,Ouellet M,Chan R,et al.Identification and microbial production of a terpene-based advanced biofuel.Nat Commun,2011,2:483.
[17]Davies FK,Work VH,Beliaev AS,et al.Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp.PCC 7002.Front Bioeng Biotechnol,2014,2:21.
[18]Kitaoka N,Lu X,Yang B,et al.The application of synthetic biology to elucidation of plant mono-,sesqui-,and diterpenoid metabolism.Mol Plant,2015,8(1):6–16.
[19]Colby SM,Alonso WR,Katahira EJ,et al.4S-limonene synthase from the oil glands of spearmint(Mentha spicata).c DNA isolation,characterization,and bacterial expression of the catalytically active monoterpene cyclase.J Biol Chem,1993,268(31):23016–23024.
[20]Yuba A,Yazaki K,Tabata M,et al.c DNA cloning,characterization,and functional expression of4S-(-)-limonene synthase from Perilla frutescens.Arch Biochem Biophys,1996,332(2):280–287.
[21]Bohlmann J,Steele CL,Croteau R.Monoterpene synthases from grand fir(Abies grandis).c DNA isolation,characterization,and functional expression of myrcene synthase,(-)-(4S)-limonene synthase,and(-)-(1S,5S)-pinene synthase.J Biol Chem,1997,272(35):21784–21792.
[22]Maruyama T,Ito M,Kiuchi F,et al.Molecular cloning,functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia.Biol Pharm Bull,2001,24(4):373–377.
[23]Lücker J,El Tamer MK,Schwab W,et al.Monoterpene biosynthesis in lemon(Citrus limon):c DNA isolation and functional analysis of four monoterpene synthases.Eur J Biochem,2002,269(13):3160–3171.
[24]Shimada T,Endo T,Fujii H,et al.Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc.Plant Sci,2004,166(1):49–58.
[25]Shimada T,Endo T,Fujii H,et al.Isolation and characterization of a new d-limonene synthase gene with a different expression pattern in Citrus unshiuMarc.Sci Hortic,2005,105(4):507–512.
[26]Bohlmann J,Crock J,Jetter R,et al.Terpenoid-based defenses in conifers:c DNA cloning,characterization,and functional expression of wound-inducible(E)-α-bisabolene synthase from grand fir(Abies grandis).Proc Natl Acad Sci USA,1998,95(12):6756–6761.
[27]Martin DM,F?ldt J,Bohlmann J.Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.Plant Physiol,2004,135(4):1908–1927.
[28]Parveen I,Wang M,Zhao JP,et al.Investigating sesquiterpene biosynthesis in Ginkgo biloba:molecular cloning and functional characterization of(E,E)-farnesol andα-bisabolene synthases.Plant Mol Biol,2015,89(4):451–462.
[29]Fujisawa M,Harada H,Kenmoku H,et al.Cloning and characterization of a novel gene that encodes(S)-beta-bisabolene synthase from ginger,Zingiber officinale.Planta,2010,232(1):121–130.
[30]Jones CG,Moniodis J,Zulak KG,et al.Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase(TPS)-a and TPS-b subfamilies,including santalene synthases.J Biol Chem,2011,286(20):17445–17454.
[31]Srivastava PL,Daramwar PP,Krithika R,et al.Functional characterization of novel sesquiterpene synthases from Indian sandalwood,Santalum album.Sci Rep,2015,5:10095.
[32]Huber DPW,Philippe RN,Godard KA,et al.Characterization of four terpene synthase c DNAs from methyl jasmonate-induced Douglas-fir,Pseudotsuga menziesii.Phytochemistry,2005,66(12):1427–1439.
[33]Ro DK,Ehlting J,Keeling CI,et al.Microarray expression profiling and functional characterization of At TPS genes:duplicated Arabidopsis thalianasesquiterpene synthase genes At4g13280 and At4g13300 encode root-specific and wound-inducible(Z)-gamma-bisabolene synthases.Arch Biochem Biophys,2006,448(1/2):104–116.
[34]Aschenbrenner AK,Kwon M,Conrad J,et al.Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower(Helianthus annuus L.,Asteraceae).Phytochemistry,2016,124:29–37.
[35]Alonso-Gutierrez J,Chan R,Batth TS,et al.Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.Metab Eng,2013,19:33–41.
[36]Jongedijk E,Cankar K,Ranzijn J,et al.Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae.Yeast,2014,32(1):159–171.
[37]Jiang GZ,Yao MD,Wang Y,et al.Manipulation of GESand ERG20 for geraniol overproduction in Saccharomyces cerevisiae.Metab Eng,2017,41:57–66.
[38]Blazeck J,Liu LQ,Redden H,et al.Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach.Appl Environ Microbiol,2011,77(22):7905–7914.
[39]Ajikumar PK,Xiao WH,Tyo KE,et al.Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli.Science,2010,330(6000):70–74.
[40]Ro DK,Paradise EM,Ouellet M,et al.Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,2006,440(7086):940–943.
[41]Dueber JE,Wu GC,Malmirchegini GR,et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat Biotechnol,2009,27(8):753–759.
[42]Lv XM,Wang F,Zhou PP,et al.Dual regulation of cytoplasmic and mitochondrial acetyl-Co A utilization for improved isoprene production in Saccharomyces cerevisiae.Nat Commun,2016,7:12851.
[43]Chen FJ,Zhou JW,Shi ZP,et al.Effect of acetyl-Co A synthase gene overexpression on physiological function of Saccharomyces cerevisiae.Acta Microbiol Sin,2010,50(9):1172–1179(in Chinese).陈孚江,周景文,史仲平,等.乙酰辅酶A合成代谢对酿酒酵母生理功能的影响.微生物学报,2010,50(9):1172–1179.
[44]Lian JZ,Zhao HM.Functional reconstitution of a pyruvate dehydrogenase in the cytosol of Saccharomyces cerevisiae through lipoylation machinery engineering.ACS Synth Biol,2016,5(7):689–697.
[45]Kozak BU,van Rossum HM,Luttik MAH,et al.Engineering acetyl coenzyme A supply:functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.m Bio,2014,5(5):e01696–14.
[46]Jiang JP,Wu XR,Chen YJ.Strategy to solve cofactor issues in oxidoreductase catalyzed biocatalytic applications.Chin J Biotech,2012,28(4):410–419(in Chinese).江金鹏,吴旭日,陈依军.解决氧化还原酶反应体系中辅酶问题的策略及其应用.生物工程学报,2012,28(4):410-419.