产蒎烯人工酵母细胞的构建
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摘要
蒎烯可衍生为高能量密度燃料,但在酿酒酵母中的全生物合成却未见报道。酿酒酵母由于拥有强大的蛋白表达和翻译后修饰系统以及完整的内膜系统,相比于大肠杆菌等原核生物更适于P450等蛋白的表达,因此将酿酒酵母作为宿主细胞,对于蒎烯或者其他物质实现如"疯狂碳环"的高能量化是至关重要的。本研究在酿酒酵母底盘中表达内源焦磷酸香叶酯合成酶(ERG20)的突变体ERG20ww和火炬松来源的蒎烯合酶(PtPS)构建了蒎烯的合成路径。通过截短PtPS N端2～51位氨基酸残基(tPtPS),蒎烯产量较初始产量(0.329 mg·L~(-1))提高了2.23倍。在过表达异戊二烯焦磷酸异构酶(IDI1)和RNA聚合酶Ш负调控因子(MAF1)的基础上,表达ERG20ww和tPtPS的融合蛋白,蒎烯产量进一步提高了5.16倍。通过将内源基因ERG20启动子原位替换为弱启动子HXT1,下调ERG20的转录,蒎烯的产量提高了26.0%。最终通过调节发酵过程中的培养基pH使蒎烯产量达11.7 mg·L~(-1),较初始产量提高了34.5倍。本研究在酿酒酵母中实现蒎烯的从头合成,并获得已知蒎烯摇瓶水平的最高产量。
The derivatives of pinene can be employed as high energy density fuels. However, the biosynthesis of pinene from single carbon sources has not been realized in yeast. As Saccharomyces cerevisiae has stronger ability onprotein expression and post-translational modification as well as processes a maturity endomembrane system, it ismore suitable for expressing complex proteins(e.g. cytochrome P450) by yeast than by prokaryotic hosts such asEscherichia coli. Therefore, it is crucial to engineer S. cerevisiae as the host cell to produce high energy density fuels(like"Crazy Carbon Ring") based on the derivatization of pinene or other terpenes compounds. Here, in order toachieve pinene synthesis in yeast, endogenous farnesyl diphosphate synthase(ERG20) mutant ERG20 wwand Pinus taeda pinene synthase(PtPS) were expressed in Saccharomyces cerevisiae strain, obtaining an initial pinene titer of0.329 mg·L~(-1). N-terminus truncation(from 2A to 51P) of PtPS(obtaining tPtPS) improve the pinene production by2.23-fold. The pinene titer was further enhanced by 5.16-fold by expression the fusion of ERG20ww/tPtPS on the basis of overexpression of isoprene pyrophosphate isomerase(IDI1) and the repressor of RNA polymerase III(MAF1). Replacing the promoter of gene ERG20 by a weaker promoter HXT1 down-regulated the transcription ofERG20. And correspondingly the pinene output was increased by 26.0%. Eventually, adjusting the pH of thefermentation medium further increased the pinene production by 42.2%(to 11.7 mg·L~(-1)). This titer was 34.5-foldhigher than the initial one. This study is the first to achieve de novo synthesis of terpenes in Saccharomyces cerevisiae and to obtain the highest yield of known terpene shake flasks.
The derivatives of pinene can be employed as high energy density fuels. However, the biosynthesis of pinene from single carbon sources has not been realized in yeast. As Saccharomyces cerevisiae has stronger ability onprotein expression and post-translational modification as well as processes a maturity endomembrane system, it ismore suitable for expressing complex proteins(e.g. cytochrome P450) by yeast than by prokaryotic hosts such asEscherichia coli. Therefore, it is crucial to engineer S. cerevisiae as the host cell to produce high energy density fuels(like"Crazy Carbon Ring") based on the derivatization of pinene or other terpenes compounds. Here, in order toachieve pinene synthesis in yeast, endogenous farnesyl diphosphate synthase(ERG20) mutant ERG20 wwand Pinus taeda pinene synthase(PtPS) were expressed in Saccharomyces cerevisiae strain, obtaining an initial pinene titer of0.329 mg·L~(-1). N-terminus truncation(from 2A to 51P) of PtPS(obtaining tPtPS) improve the pinene production by2.23-fold. The pinene titer was further enhanced by 5.16-fold by expression the fusion of ERG20ww/tPtPS on the basis of overexpression of isoprene pyrophosphate isomerase(IDI1) and the repressor of RNA polymerase III(MAF1). Replacing the promoter of gene ERG20 by a weaker promoter HXT1 down-regulated the transcription ofERG20. And correspondingly the pinene output was increased by 26.0%. Eventually, adjusting the pH of thefermentation medium further increased the pinene production by 42.2%(to 11.7 mg·L~(-1)). This titer was 34.5-foldhigher than the initial one. This study is the first to achieve de novo synthesis of terpenes in Saccharomyces cerevisiae and to obtain the highest yield of known terpene shake flasks.
引文
[1] Harvey B G, Wright M E, Quintana R L. High-density renewable fuels based on the selective dimerization of pinenes[J]. Energy&Fuels, 2010, 24(1):267-273.
[2]邹吉军,张香文,王莅,等.高密度液体碳氢燃料合成及应用进展[J].含能材料, 2007,(4):411-415.Zou J J, Zhang X W, Wang L, et al. Progress on the synthesis and application of high-density liquid hydrocarbon fuels[J]. Chinese Journal of Energetic Materials, 2007,(4):411-415.
[3]冯红茹,杨建明,秦利,等.β-蒎烯合成酶(QH6)在大肠杆菌中的表达及其产β-蒎烯的研究[J].生物加工过程, 2015, 13(1):28-34.Feng H R, Yang J M, Qin L, et al. Expression ofβ-pinene synthase(QH6)in Escherichia coli for the biosynthesis ofβ-pinene[J]. Chinese Journal of Bioprocess Engineering, 2015, 13(1):28-34.
[4] Clomburg J M, Gonzalez R. Biofuel production in Escherichia coli:the role of metabolic engineering and synthetic biology[J]. Appl.Microbiol. Biotechnol., 2010, 86(2):419-434.
[5] Leonard E, Lim K H, Saw P N, et al. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli[J]. Appl. Environ. Microbiol., 2007, 73(12):3877-3886.
[6] Schmidt-Dannert C, Umeno D, Arnold F H. Molecular breeding of carotenoid biosynthetic pathways[J]. Nat. Biotechnol., 2000, 18(7):750-753.
[7] Zhang L, Xiao W H, Wang Y, et al. Chassis and key enzymes engineering for monoterpenes production[J]. Biotechnol. Adv.,2017, 35(8):1022-1031.
[8] Niu F, Lu Q, Bu Y, et al. Metabolic engineering for the microbial production of isoprenoids:carotenoids and isoprenoid-based biofuels[J]. Synthetic and Systems Biotechnology, 2017, 2(3):167-175.
[9] Kang M, Eom J, Kim Y, et al. Biosynthesis of pinene from glucose using metabolically-engineered[J]. Biotechnology Letters, 2014,36(10):2069-2077.
[10] Sarria S, Wong B, García Martín H, et al. Microbial synthesis of pinene[J]. ACS Synthetic Biology, 2014, 3(7):466-475.
[11] Yang J, Nie Q, Ren M, et al. Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene[J]. Biotechnol. Biofuels,2013, 6(1):60.
[12] Zhang X, Mcvay R C, Huang D D, et al. Formation and evolution of molecular products in alpha-pinene secondary organic aerosol[J]. Proc. Natl. Acad. Sci. USA, 2015, 112(46):14168-14173.
[13]邓云祥,王玉平,王力昌,等.α-蒎烯选择性二聚合及低聚反应研究(Ⅱ):二聚异构体研究[J].高分子学报, 1995,(2):170-175.Deng Y X, Wang Y P, Wang L C, et al. Studies on selective dimerization and oligomerization ofα-pinene(Ⅱ):Studies of the dimeric isomers[J]. Acta Polymerica Sinica, 1995,(2):170-175.
[14] Chen K, Huang X, Kan S, et al. Enzymatic construction of highly strained carbocycles[J]. Science, 2018, 360(6384):71-75.
[15] Kan S, Huang X, Gumulya Y, et al. Genetically programmed chiral organoborane synthesis[J]. Nature, 2017, 552(7683):132-136.
[16] Ajikumar P K, Xiao W H, Tyo K E, et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli[J]. Science, 2010, 330(6000):70-74.
[17] Jiang G, Yao M, Wang Y, et al. Manipulation of GES and ERG20for geraniol overproduction in Saccharomyces cerevisiae[J].Metabolic Engineering, 2017, 41:57-66.
[18]李博,梁楠,刘夺,等.合成8-二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建[J].中国生物工程杂志, 2017,(9):71-81.Li B, Liang N, Liu D, et al. Metabolic engineering of Saccharomyces cerevisiae for production of 8-dimenthylally naringenin[J]. China Biotechnology, 2017,(9):71-81.
[19] Gietz R D. Yeast transformation by the LiAc/SS carrier DNA/PEG method[J]. Methods Mol. Biol., 2014, 1205:1-12.
[20] Mathiasen D P, Lisby M. Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae[J]. FEMS Microbiol.Rev., 2014, 38(2):172-184.
[21] Peck R F, Dassarma S, Krebs M P. Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker[J]. Mol. Microbiol., 2000, 35(3):667-676.
[22] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods, 2001, 25(4):402-408.
[23] Szkopinska A, Plochocka D. Farnesyl diphosphate synthase;regulation of product specificity[J]. Acta Biochim. Pol., 2005, 52(1):45-55.
[24] Grabinska K, Palamarczyk G. Dolichol biosynthesis in the yeast Saccharomyces cerevisiae:an insight into the regulatory role of farnesyl diphosphate synthase[J]. FEMS Yeast Res., 2002, 2(3):259-265.
[25] Ignea C, Pontini M, Maffei M E, et al. Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase[J]. ACS Synth. Biol., 2014, 3(5):298-306.
[26] Turner G, Gershenzon J, Nielson E E, et al. Limonene synthase,the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells[J]. Plant Physiol., 1999, 120(3):879-886.
[27] Bohlmann J, Meyer-Gauen G, Croteau R. Plant terpenoid synthases:molecular biology and phylogenetic analysis[J]. Proc.Natl. Acad. Sci. USA, 1998, 95(8):4126-4133.
[28] K?llner T G, Schnee C, Gershenzon J, et al. The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes[J]. The Plant Cell,2004, 16(5):1115-1131.
[29] Cao Y, Zhang H, Liu H, et al. Biosynthesis and production of sabinene:current state and perspectives[J]. Applied Microbiology and Biotechnology, 2018, 102(4):1535-1544.
[30] Liu J, Zhang W, Du G, et al. Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae[J].Journal of Biotechnology, 2013, 168(4):446-451.
[31] Pluta K, Lefebvre O, Martin N C, et al. Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae[J]. Molecular and Cellular Biology, 2001, 21(15):5031-5040.
[32] Clastre M, Bantignies B, Feron G, et al. Purification and characterization of geranyl diphosphate synthase from Vitis vinifera L. cv Muscat de Frontignan cell cultures[J]. Plant Physiol., 1993, 102(1):205-211.
[33] Dunlop M J, Dossani Z Y, Szmidt H L, et al. Engineering microbial biofuel tolerance and export using efflux pumps[J].Molecular Systems Biology, 2011, 7(1):487.
[34] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes[J]. Appl. Environ. Microbiol.,2011, 77(3):1033-1040.
[35] Ohto C, Muramatsu M, Obata S, et al. Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae[J]. Appl.Microbiol. Biotechnol., 2010, 87(4):1327-1334.
[36] Zhao J, Li C, Zhang Y, et al. Dynamic control of ERG20expression combined with minimized endogenous downstream metabolism contributes to the improvement of geraniol production in Saccharomyces cerevisiae[J]. Microbial Cell Factories, 2017, 16(1):17.
[37] Xie W, Ye L, Lv X, et al. Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2015, 28:8-18.
[38] Wang Y, Lim L, Diguistini S, et al. A specialized ABC efflux transporter GcABC-G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle-associated fungal pathogen of pine trees[J]. New Phytol., 2013, 197(3):886-898.
[39] Piper P W. The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap[J]. FEMS Microbiol. Lett., 1995, 134(2/3):121-127.
[40] Jiang X, Zhang H, Yang J, et al. Induction of gene expression in bacteria at optimal growth temperatures[J]. Appl. Microbiol.Biotechnol., 2013, 97(12):5423-5431.
[2]邹吉军,张香文,王莅,等.高密度液体碳氢燃料合成及应用进展[J].含能材料, 2007,(4):411-415.Zou J J, Zhang X W, Wang L, et al. Progress on the synthesis and application of high-density liquid hydrocarbon fuels[J]. Chinese Journal of Energetic Materials, 2007,(4):411-415.
[3]冯红茹,杨建明,秦利,等.β-蒎烯合成酶(QH6)在大肠杆菌中的表达及其产β-蒎烯的研究[J].生物加工过程, 2015, 13(1):28-34.Feng H R, Yang J M, Qin L, et al. Expression ofβ-pinene synthase(QH6)in Escherichia coli for the biosynthesis ofβ-pinene[J]. Chinese Journal of Bioprocess Engineering, 2015, 13(1):28-34.
[4] Clomburg J M, Gonzalez R. Biofuel production in Escherichia coli:the role of metabolic engineering and synthetic biology[J]. Appl.Microbiol. Biotechnol., 2010, 86(2):419-434.
[5] Leonard E, Lim K H, Saw P N, et al. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli[J]. Appl. Environ. Microbiol., 2007, 73(12):3877-3886.
[6] Schmidt-Dannert C, Umeno D, Arnold F H. Molecular breeding of carotenoid biosynthetic pathways[J]. Nat. Biotechnol., 2000, 18(7):750-753.
[7] Zhang L, Xiao W H, Wang Y, et al. Chassis and key enzymes engineering for monoterpenes production[J]. Biotechnol. Adv.,2017, 35(8):1022-1031.
[8] Niu F, Lu Q, Bu Y, et al. Metabolic engineering for the microbial production of isoprenoids:carotenoids and isoprenoid-based biofuels[J]. Synthetic and Systems Biotechnology, 2017, 2(3):167-175.
[9] Kang M, Eom J, Kim Y, et al. Biosynthesis of pinene from glucose using metabolically-engineered[J]. Biotechnology Letters, 2014,36(10):2069-2077.
[10] Sarria S, Wong B, García Martín H, et al. Microbial synthesis of pinene[J]. ACS Synthetic Biology, 2014, 3(7):466-475.
[11] Yang J, Nie Q, Ren M, et al. Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene[J]. Biotechnol. Biofuels,2013, 6(1):60.
[12] Zhang X, Mcvay R C, Huang D D, et al. Formation and evolution of molecular products in alpha-pinene secondary organic aerosol[J]. Proc. Natl. Acad. Sci. USA, 2015, 112(46):14168-14173.
[13]邓云祥,王玉平,王力昌,等.α-蒎烯选择性二聚合及低聚反应研究(Ⅱ):二聚异构体研究[J].高分子学报, 1995,(2):170-175.Deng Y X, Wang Y P, Wang L C, et al. Studies on selective dimerization and oligomerization ofα-pinene(Ⅱ):Studies of the dimeric isomers[J]. Acta Polymerica Sinica, 1995,(2):170-175.
[14] Chen K, Huang X, Kan S, et al. Enzymatic construction of highly strained carbocycles[J]. Science, 2018, 360(6384):71-75.
[15] Kan S, Huang X, Gumulya Y, et al. Genetically programmed chiral organoborane synthesis[J]. Nature, 2017, 552(7683):132-136.
[16] Ajikumar P K, Xiao W H, Tyo K E, et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli[J]. Science, 2010, 330(6000):70-74.
[17] Jiang G, Yao M, Wang Y, et al. Manipulation of GES and ERG20for geraniol overproduction in Saccharomyces cerevisiae[J].Metabolic Engineering, 2017, 41:57-66.
[18]李博,梁楠,刘夺,等.合成8-二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建[J].中国生物工程杂志, 2017,(9):71-81.Li B, Liang N, Liu D, et al. Metabolic engineering of Saccharomyces cerevisiae for production of 8-dimenthylally naringenin[J]. China Biotechnology, 2017,(9):71-81.
[19] Gietz R D. Yeast transformation by the LiAc/SS carrier DNA/PEG method[J]. Methods Mol. Biol., 2014, 1205:1-12.
[20] Mathiasen D P, Lisby M. Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae[J]. FEMS Microbiol.Rev., 2014, 38(2):172-184.
[21] Peck R F, Dassarma S, Krebs M P. Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker[J]. Mol. Microbiol., 2000, 35(3):667-676.
[22] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods, 2001, 25(4):402-408.
[23] Szkopinska A, Plochocka D. Farnesyl diphosphate synthase;regulation of product specificity[J]. Acta Biochim. Pol., 2005, 52(1):45-55.
[24] Grabinska K, Palamarczyk G. Dolichol biosynthesis in the yeast Saccharomyces cerevisiae:an insight into the regulatory role of farnesyl diphosphate synthase[J]. FEMS Yeast Res., 2002, 2(3):259-265.
[25] Ignea C, Pontini M, Maffei M E, et al. Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase[J]. ACS Synth. Biol., 2014, 3(5):298-306.
[26] Turner G, Gershenzon J, Nielson E E, et al. Limonene synthase,the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells[J]. Plant Physiol., 1999, 120(3):879-886.
[27] Bohlmann J, Meyer-Gauen G, Croteau R. Plant terpenoid synthases:molecular biology and phylogenetic analysis[J]. Proc.Natl. Acad. Sci. USA, 1998, 95(8):4126-4133.
[28] K?llner T G, Schnee C, Gershenzon J, et al. The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes[J]. The Plant Cell,2004, 16(5):1115-1131.
[29] Cao Y, Zhang H, Liu H, et al. Biosynthesis and production of sabinene:current state and perspectives[J]. Applied Microbiology and Biotechnology, 2018, 102(4):1535-1544.
[30] Liu J, Zhang W, Du G, et al. Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae[J].Journal of Biotechnology, 2013, 168(4):446-451.
[31] Pluta K, Lefebvre O, Martin N C, et al. Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae[J]. Molecular and Cellular Biology, 2001, 21(15):5031-5040.
[32] Clastre M, Bantignies B, Feron G, et al. Purification and characterization of geranyl diphosphate synthase from Vitis vinifera L. cv Muscat de Frontignan cell cultures[J]. Plant Physiol., 1993, 102(1):205-211.
[33] Dunlop M J, Dossani Z Y, Szmidt H L, et al. Engineering microbial biofuel tolerance and export using efflux pumps[J].Molecular Systems Biology, 2011, 7(1):487.
[34] Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes[J]. Appl. Environ. Microbiol.,2011, 77(3):1033-1040.
[35] Ohto C, Muramatsu M, Obata S, et al. Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae[J]. Appl.Microbiol. Biotechnol., 2010, 87(4):1327-1334.
[36] Zhao J, Li C, Zhang Y, et al. Dynamic control of ERG20expression combined with minimized endogenous downstream metabolism contributes to the improvement of geraniol production in Saccharomyces cerevisiae[J]. Microbial Cell Factories, 2017, 16(1):17.
[37] Xie W, Ye L, Lv X, et al. Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2015, 28:8-18.
[38] Wang Y, Lim L, Diguistini S, et al. A specialized ABC efflux transporter GcABC-G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle-associated fungal pathogen of pine trees[J]. New Phytol., 2013, 197(3):886-898.
[39] Piper P W. The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap[J]. FEMS Microbiol. Lett., 1995, 134(2/3):121-127.
[40] Jiang X, Zhang H, Yang J, et al. Induction of gene expression in bacteria at optimal growth temperatures[J]. Appl. Microbiol.Biotechnol., 2013, 97(12):5423-5431.