基于重组毕赤酵母的fusaruside生物合成
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摘要
目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。
Objective: A strain of Pichia pastoris producing fusaruside was constructed to solve the source problem of fusaruside,a selective immunosuppressive molecule. Method: Two related biosynthetic genes coding delta 3(E)-sphingolipid desaturase [Δ3(E)-SD]and delta 10(E)-sphingolipid desaturase [Δ10(E)-SD]were amplified from fungus Fusarium graminearum PH-1. 2 A peptide based strategy was used to construct the coexpression vector. Then the 2A polyprotein construct was transformed into Pichia pastoris GS115 for induction.Finally,P. pastoris cells were extracted with methanol and dichloromethane,and the extract was detected by high performance liquid chromatography mass spectrometer(HPLC-MS). Result: The two desaturases were successfully co-expressed in P. pastoris. SDS-PAGE showed that the molecular weight of Δ3(E)-SD and Δ10(E)-SD were about 48 kDa and 65 kDa,respectively. HPLC-MS indicated that fusaruside could be produced by the recombinant yeast. Conclusion: Compared with fusarium which producing fusaruside,the engineered yeast had shorter fermentation time and higher yield,laying a foundation for further development and application of fusaruside.
Objective: A strain of Pichia pastoris producing fusaruside was constructed to solve the source problem of fusaruside,a selective immunosuppressive molecule. Method: Two related biosynthetic genes coding delta 3(E)-sphingolipid desaturase [Δ3(E)-SD]and delta 10(E)-sphingolipid desaturase [Δ10(E)-SD]were amplified from fungus Fusarium graminearum PH-1. 2 A peptide based strategy was used to construct the coexpression vector. Then the 2A polyprotein construct was transformed into Pichia pastoris GS115 for induction.Finally,P. pastoris cells were extracted with methanol and dichloromethane,and the extract was detected by high performance liquid chromatography mass spectrometer(HPLC-MS). Result: The two desaturases were successfully co-expressed in P. pastoris. SDS-PAGE showed that the molecular weight of Δ3(E)-SD and Δ10(E)-SD were about 48 kDa and 65 kDa,respectively. HPLC-MS indicated that fusaruside could be produced by the recombinant yeast. Conclusion: Compared with fusarium which producing fusaruside,the engineered yeast had shorter fermentation time and higher yield,laying a foundation for further development and application of fusaruside.
引文
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[13]Zaüner S,Zahringer U,Lindner B,et al.Identification and functional characterization of the 2-hydroxy fatty N-acyl-Delta3(E)-desaturase from Fusarium graminearum.J Biol Chem,2008,283(52):36734-36742.
[14]张欢,黄思超,蔡绍晖.基于2A肽策略构建多基因表达载体的研究进展.中国生物工程杂志,2013,33(1):104-108.Zhang H,Huang S C,Cai S H.Development of 2A peptide-based strategies for constructing multicistronic expression vectors.China Biotechnology,2013,33(1):104-108.
[15]Brazier-Hicks M,Edwards R.Metabolic engineering of the flavoneC-glycoside pathway using poly protein technology.Metab Eng,2013,16:11-20.
[16]Beekwilder J,van Rossum H M,Koopman F,et al.Polycistronic expression of aβ-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled toβ-ionone production.J Biotechno,2014,192(Part B):383-392.
[17]Geier M,Fauland P,Vogl T,et al.Compact multi-enzyme pathways in P.pastoris.Chem Commun,2015,51(9):1643-1646.
[2]O’Shea J J,Pesu M,Borie D C,et al.A new modality for immunosuppression:targeting the JAK/STAT pathway.Nat Rev Drug Discov,2004,3(7):555-564.
[3]Wu X F,Wu X X,Guo W J,et al.Cerebroside D,a glycoceramide compound,improves experimental colitis in mice with multiple targets against activated T lymphocytes.Toxicol Appl Pharm,2012,263(3):296-302.
[4]Wu X X,Sun Y,Guo W J,et al.Rebuilding the balance of STAT1and STAT3 signalings by fusaruside,a cerebroside compound,for the treatment of T-cell-mediated fulminant hepatitis in mice.Biochem Pharmacol,2012,84(9):1164-1173.
[5]Li J,Ferris R L.PD-1/SHP-2 negatively regulate Tc1/Th1phenotypic responses and activation of T cells in the tumor microenvironment.J Immunother Cancer,2014,2(S3):221.
[6]Wu X X,Guo W J,Wu L M.et al.Selective sequestration of STAT1 in the cytoplasm via phosphorylated SHP-2 ameliorates murine experimental colitis.J Immunol,2012,189(7):3497-3507.
[7]Shu R G,Wang F W,Yang Y M,et al.Antibacterial and xanthine oxidase inhibitory cerebrosides from Fusarium sp.IF-121,an endophytic fungus in Quercus variabilis.Lipids,2004,39(7):667-673.
[8]Black F J,Kocienski P.Synthesis of phalluside-1 and Sch II using1,2-metallate rearrangements.Org Biomol Chem,2010,8(5):1188-1193.
[9]Ro D K,Paradise E M,Ouellet M,et al.Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,2006,440(7086):940-943.
[10]Farhi M,Marhevka E,Ben-Ari J,et al.Generation of thepotent anti-malarial drug artemisinin in tobacco.Nat Biotechnol,2011,29(12):1072-1074.
[11]Ternes P,Wobbe T,Schwarz M,et al.Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris.J Biol Chem,2011,286(13):11401-11414.
[12]Tian Y,Zhao G Y,Fang W,et al.Δ10(E)-Sphingolipid desaturase involved in fusaruside mycosynthesis and stress adaptation in Fusarium graminearum.Sci Rep,2015,5:10486.
[13]Zaüner S,Zahringer U,Lindner B,et al.Identification and functional characterization of the 2-hydroxy fatty N-acyl-Delta3(E)-desaturase from Fusarium graminearum.J Biol Chem,2008,283(52):36734-36742.
[14]张欢,黄思超,蔡绍晖.基于2A肽策略构建多基因表达载体的研究进展.中国生物工程杂志,2013,33(1):104-108.Zhang H,Huang S C,Cai S H.Development of 2A peptide-based strategies for constructing multicistronic expression vectors.China Biotechnology,2013,33(1):104-108.
[15]Brazier-Hicks M,Edwards R.Metabolic engineering of the flavoneC-glycoside pathway using poly protein technology.Metab Eng,2013,16:11-20.
[16]Beekwilder J,van Rossum H M,Koopman F,et al.Polycistronic expression of aβ-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled toβ-ionone production.J Biotechno,2014,192(Part B):383-392.
[17]Geier M,Fauland P,Vogl T,et al.Compact multi-enzyme pathways in P.pastoris.Chem Commun,2015,51(9):1643-1646.