络塞是一种抗高原反应药物的主要活性成分,可通过尿苷二磷酸糖基转移酶UGT73C5催化肉桂醇糖基化反应合成。然而UGT73C5在大肠杆菌宿主中可溶性表达极差,严重限制了其工业应用。该研究分别通过与分子伴侣共表达和与助溶蛋白标签融合表达的方式,提高UGT73C5在Escherichia coli BL21(DE3)中的可溶性表达。结果显示,除质粒pKJE7外,与分子伴侣质粒pG-KJE8、pGro7和pG-TF2共表达后均可提高UGT73C5酶活力,分别为原始酶活力(18.56 U/g细胞)的1.27、1.18、1.37倍。此外,利用硫氧还蛋白(thioredoxin,Trx)和谷胱甘肽巯基转移蛋白(glutathione S-transferase,GST)两种助溶蛋白标签,构建的融合酶Trx-UGT73C5 和GST-UGT73C5的酶活力为原始酶活力的1.45、2.54倍,且纯化后的比酶活力为原始酶的80%~90%。最后,在2 mmol/L肉桂醇合成络塞的反应中,相同浓度细胞破碎液(粗酶)融合酶GST-UGT73C5比原始酶催化效率更高,4 h后转化率可以达到90%以上,最终转化率达到93.6%。因此,助溶蛋白标签融合表达法可有效提高UGT73C5在大肠杆菌中的可溶性表达,且融合酶GST-UGT73C5在络塞的酶法合成中具有更大的应用潜力。
The uridine diphosphate glycosyltransferase UGT73C5 can catalyze the glycosylation of cinnamyl alcohol for the synthesis of rosin, which is one of the main active ingredients of anti-altitude sickness drugs. However, the poor soluble expression of UGT73C5 in Escherichia coli severely limits its industrial application. Here, the soluble expression of glycosyltransferase UGT73C5 was improved by co-expression of molecular chaperone protein and fusion expression with solubilizing-tag, respectively. Results showed that besides plasmid pKJE7, pET28a-UGT73C5 co-expressed with chaperone plasmids pG-KJE8, pGro7 and pG-TF2 could increase the enzymatic activities to 1.27-, 1.18- and 1.37-fold of that of UGT73C5 (18.56 U/g cells). Two solubilizing protein tags, thioredoxin (Trx) and glutathione S-transferase (GST), were fused with UGT73C5. The activities of fusion enzymes Trx-UGT73C5 and GST-UGT73C5 were 1.45- and 2.54-fold of that of the UGT73C5, respectively. After purification, the specific activity of the fusion enzymes possessed 80%-90% of that of the UGT73C5. Finally, rosin synthesis from 2 mmol/L cinnamyl alcohol was performed based on crude enzymes (disruption of same concentration of cells), the fusion enzyme GST-UGT73C5 led to higher catalytic efficiency than UGT73C5. The conversion ratio of cinnamyl alcohol catalyzed by GST-UGT73C5 reached more than 90% at 4 h, and finally reached 93.6%. Therefore, the fusion expression approach is more effective for the enhanced soluble expression of UGT73C5 in E. coli, and the fusion enzyme GST-UGT73C5 is more potential in the enzymatic production of rosin.
[1] NA L, LI R, CHEN X.Recent progress in synthesis of carbohydrates with sugar nucleotide-dependent glycosyltransferases[J].Current Opinion in Chemical Biology, 2021, 61:81-95.
[2] TIWARI P, SANGWAN R S, SANGWAN N S.Plant secondary metabolism linked glycosyltransferases:An update on expanding knowledge and scopes[J].Biotechnology Advances, 2016, 34(5):714-739.
[3] DE BRUYN F, MAERTENS J, BEAUPREZ J, et al.Biotechnological advances in UDP-sugar based glycosylation of small molecules[J].Biotechnology Advances, 2015, 33(2):288-302.
[4] LIU S K, LYU Y B, YU S Q, et al.Efficient production of orientin and vitexin from luteolin and apigenin using coupled catalysis of glycosyltransferase and sucrose synthase[J].Journal of Agricultural and Food Chemistry, 2021, 69(23):6 578-6 587.
[5] CHEN T, CHEN Z, WANG N, et al.Highly regioselective and efficient biosynthesis of polydatin by an engineered UGTBL 1-At SuSy cascade reaction[J].Journal of Agricultural and Food Chemistry, 2021.69(31):8 695-8 702.
[6] POPPENBERGER B, FUJIOKA S, SOENO K, et al.The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids[J].Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(42):15 253-15 258.
[7] ZHOU W, BI H P, ZHUANG Y B, et al.Production of cinnamyl alcohol glucoside from glucose in Escherichia coli[J].Journal of Agricultural and Food Chemistry, 2017, 65(10):2 129-2 135.
[8] HU Y M, XUE J, MIU J, et al.Biocatalytic synthesis of ginsenoside Rh2 using Arabidopsis thaliana glucosyltransferase-catalyzed coupled reactions[J].Journal of Biotechnology, 2020, 309:107-112.
[9] POPPENBERGER B, BERTHILLER F, LUCYSHYN D, et al.Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana[J].Journal of Biological Chemistry, 2003, 278(48):47905.
[10] 黄庶识, 卢明倩, 李冰,等.重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J].中国激光, 2014, 41(12):257-265.
HUANG S S, LU M Q, LI B, et al.Real time detection on the expression of soluble protein and inclusion body in the recombinant Escherichia coli with laser tweezers Raman spectroscopy[J].Chinese Journal of Lasers, 2014, 41(12):257-265.
[11] 张磊, 唐永凯, 李红霞, 等.促进原核表达蛋白可溶性的研究进展[J].中国生物工程杂志, 2021, 41(S1):138-149.
ZHANG L, TANG Y K, LI H X, et al.Advances in promoting solubility of prokaryotic expressed proteins[J].China Biotechnology, 2021, 41(S1):138-149.
[12] 朱红裕, 李强.外源蛋白在大肠杆菌中的可溶性表达策略[J].过程工程学报, 2006, 6(1):150-155.
ZHU H Y, LI Q.Strategies for expression of soluble heterologous proteins in Escherichia coli[J]. The Chinese Journal of Process Engineering, 2006, 6(1):150-155.
[13] LASKEY R A, HONDA B M, MILLS A D, et al.Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA[J].Nature, 1978, 275(5 679):416-420.
[14] ROBIN S, TOGASHI D M, RYDER A G, et al.Trigger factor from the Psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone[J].Journal of Bacteriology, 2009, 191(4):1 162-1 168.
[15] DE MARCO A, DEUERLING E, MOGK A, et al.Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E.coli[J].BMC Biotechnology, 2007, 7(1):32.
[16] BUKAU B, DEUERLING E, PFUND C, et al.Getting newly synthesized proteins into shape[J].Cell, 2000, 101(2):119-122.
[17] YAO D, FAN J, HAN R, et al.Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones[J].Protein Expression and Purification, 2020, 169:105571.
[18] DUAN X G, ZHANG X Y, SHEN Z Y, et al.Efficient production of aggregation prone 4-α-glucanotransferase by combined use of molecular chaperones and chemical chaperones in Escherichia coli[J].Journal of Biotechnology, 2019, 292:68-75.
[19] YOUNG C L, BRITTON Z T, ROBINSON A S.Recombinant protein expression and purification:A comprehensive review of affinity tags and microbial applications[J].Biotechnology Journal, 2012, 7(5):620-634.
[20] SMITH D B, JOHNSON K S.Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase[J].Gene, 1988, 67(1):31-40.
[21] DŬMMLER A, LAWRENCE A M DE, MARCO A.Simplified screening for the detection of soluble fusion constructs expressed in E.coli using a modular set of vectors[J].Microbial Cell Factories, 2005, 4(1):34-34.
[22] 沈加斌, 徐攀, 李鑫雨, 等.人胰岛素原基因在大肠杆菌中的可溶性表达及分离纯化研究[J].食品与生物技术学报, 2016, 35(7):721-727.
SHEN J B, XU P, LI X Y, et al.Soluble expression and purification of human proinsulin in Escherichia coli[J].Journal of Food Science and Biotechnology, 2016, 35(7):721-727.
[23] RABHI-ESSAFI I,SADOK A,KHALAF N,et al.A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E.coli[J].Protein Engineering Design and Selection Peds, 2007, 20(5):201-209.
[24] LANGER T, PFEIFER G, MARTIN J, et al.Chaperonin-mediated protein folding:GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity[J].The EMBO Journal, 1992, 11(13):4 757-4 765.
[25] YOON H, HONG J, RYU S.Effects of chaperones on mRNA stability and gene expression in Escherichia coli[J].Journal of Microbiology and Biotechnology, 2008, 18(2):228-233.
[26] CALLONI G, CHEN T, SCHERMANN S, et al.DnaK functions as a central hub in the E.coli chaperone network[J].Cell Reports, 2012, 1(3):251-264.
[27] GRAGEROV A, NUDLER E, KOMISSAROVA N, et al.Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli[J].Proceedings of the National Academy of Sciences of the United States of America, 1992, 89(21):10 341-10 344.
[28] PATZELT H, RŬDIGER S, BREHMER D, et al.Binding specificity of Escherichia coli trigger factor[J].Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(25):14 244-14 249.
