在大肠杆菌中拓展合成途径合成异胡椒醇
作者:
基金项目:

福建省自然科学基金(2022J01094)


Expanding the pathway to synthesize isopiperitenol in Escherichia coli
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [17]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    天然异胡椒醇作为一种植物来源香料,具有很高的经济价值。目前国内外还未见利用大肠杆菌工程菌株转化生产异胡椒醇的报道。【目的】构建工程菌,采用生物合成方法获得天然香料异胡椒醇。【方法】对来自胡椒薄荷的细胞色素P450家族的柠檬烯-3-羟化酶(LIM3H)基因PM2进行改造,截短LIM3H的N端疏水区,分别添加17α短肽或2B1短肽增加其亲水性,采用CO差示光谱法检测LIM3H活性;将N端疏水区截短后的来自拟南芥(Arabidopsis thaliana)的NADPH-细胞色素P450还原酶基因(trATR)和修饰后LIM3H基因(Modi-LIM3H)融合后表达,最终以柠檬烯为底物进行全细胞转化。【结果】3种N端修饰LIM3H基因均检测到LIM3H表达和异胡椒醇生成,其中17α短肽修饰后的蛋白表达量最高,异胡椒醇产量达到1.94 mg/L。【结论】本研究在大肠杆菌中增加外源LIM3H与ATR,成功催化柠檬烯生成异胡椒醇,为天然异胡椒醇的生产提供了新的方法。

    Abstract:

    As a plant-derived aromatic compound, isopiperitenol has a high economic value. Little is known about the production of isopiperitenol in Escherichia coli engineering strains. [Objective] To construct the engineering strains of E. coli for the biosynthesis of natural isopiperitenol. [Methods] We modified the limonene-3-hydroxylase (LIM3H) gene PM2 of cytochrome P450 family from Mentha×piperita by truncating the N-terminal hydrophobic region or adding 17α short peptide or 2B1 short peptide of LIM3H to increase its hydrophilicity. The activity of LIM3H was detected by CO differential spectroscopy. The NADPH-cytochrome P450 reductase gene with truncated N-terminal hydrophobic region (trATR) from Arabidopsis thaliana was fused with the modified LIM3H (Modi-LIM3H) gene and the fusion gene was expressed in E. coli. The obtained fusion protein was used to catalyze the production of isopiperitenol from limonene. [Results] LIM3H expression and isopiperitenol production were detected in all the strains with the LIM3H N-terminus modified in three different ways. The LIM3H modified by addition of 17α short peptide showed the highest protein level, and the strain carrying this modified gene had the highest yield (1.94 mg/L) of isopiperitenol.[Conclusion] In this study, exogenous LIM3H and ATR were introduced into E. coli and the fusion protein catalyzed the production of isopiperitenol from limonene, which provided a new method for the production of natural isopiperitenol.

    参考文献
    [1] 陈吟, 亓希武, 徐东北, 陈泽群, 房海灵, 梁呈元. 薄荷柠檬烯-6-羟化酶基因(MhL6OH)的克隆与表达分析[J]. 植物资源与环境学报, 2019, 28(2):25-31. CHEN Y, QI XW, XU DB, CHEN ZQ, FANG HL, LIANG CY. Cloning and expression analysis on limonene-6-hydroxylase gene (MhL6OH) of Mentha haplocalyx[J]. Journal of Plant Resources and Environment, 2019, 28(2):25-31(in Chinese).
    [2] NÍ CA, MANSELL DAVID J, TOOGOOD HELEN S, SHIRLEY T, ANTONIOS L, SCRUTTON NIGEL S, GARDINER JOHN M. Chemoenzymatic synthesis of the intermediates in the peppermint monoterpenoid biosynthetic pathway[J]. Journal of Natural Products, 2018, 81(7):1546-1552.
    [3] 薛劲松, 李鸿雁, 傅强, 马世平. 左旋薄荷酮抗抑郁作用及机制研究[J]. 药学与临床研究, 2015, 23(3):238-241. XUE JS, LI HY, FU Q, MA SP. Antidepressant-like effects of l-menthone in depressed mice and its possible mechanisms[J]. Pharmaceutical and Clinical Research, 2015, 23(3):238-241(in Chinese).
    [4] van DYK M, van RENSBURG E, MOLELEKI N. Hydroxylation of () limonene, (−) α-pinene and (−) β-pinene by a Hormonema sp.[J]. Biotechnology Letters, 1998, 20(4):431-436.
    [5] HAUDENSCHILD C, SCHALK M, KARP F, CROTEAU R. Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and Saccharomyces cerevisiae[J]. Archives of Biochemistry and Biophysics, 2000, 379(1):127-136.
    [6] SCHEMPP FLORENCE M, INGMAR S, ETSCHMANN MARIA MW, ELENA B, JOHANNES P, HENDRIK S, JENS S, MARKUS B. Identification of fungal limonene-3-hydroxylase for biotechnological menthol production[J]. Applied and Environmental Microbiology, 2021, 87(10):e02873-20.
    [7] OMURA T, SATO R. The carbon monoxide-binding pigment of liver microsomes[J]. Journal of Biological Chemistry, 1964, 239(7):2370-2378.
    [8] HUSSAIN R, AHMED M, KHAN TA, AKHTER Y. Fungal P450 monooxygenases-the diversity in catalysis and their promising roles in biocontrol activity[J]. Applied Microbiology and Biotechnology, 2020, 104(3):989-999.
    [9] LI JH, TIAN CF, XIA YH, MUTANDA I, WANG KB, WANG Y. Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine[J]. Metabolic Engineering, 2019, 52:124-133.
    [10] ICHINOSE H, HATAKEYAMA M, YAMAUCHI Y. Sequence modifications and heterologous expression of eukaryotic cytochromes P450 in Escherichia coli[J]. Journal of Bioscience and Bioengineering, 2015, 120(3):268-274.
    [11] WILLIAMS PA, COSME J, SRIDHAR V, JOHNSON EF, McREE DE. Mammalian microsomal cytochrome P450 monooxygenase[J]. Molecular Cell, 2000, 5(1):121-131.
    [12] 麦婉莹, 洪葵. 真菌细胞色素P450在大肠杆菌中的表达[J]. 微生物学通报, 2019, 46(5):1092-1099. MAI WY, HONG K. Heterologous expression of a fungal cytochrome P450 in Escherichia coli[J]. Microbiology China, 2019, 46(5):1092-1099(in Chinese).
    [13] BARNES HJ, ARLOTTO MP, WATERMAN MR. Expression and enzymatic activity of recombinant cytochrome P45017 alpha-hydroxylase in Escherichia coli[J]. PNAS, 1991, 88(13):5597-5601.
    [14] EFFENDI L, KOFFAS MATTHEOS AG. Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli[J]. Applied and Environmental Microbiology, 2007, 73(22):7246-7251.
    [15] MORRONE D, CHEN XM, COATES RM, PETERS RJ. Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis[J]. The Biochemical Journal, 2010, 431(3):337-344.
    [16] MUNRO AW, GIRVAN HM, MCLEAN KJ. Cytochrome P450-redox partner fusion enzymes[J]. Biochimica et Biophysica Acta-General Subjects, 2007, 1770(3):345-359.
    [17] WÜST M, LITTLE DB, SCHALK M, CROTEAU R. Hydroxylation of limonene enantiomers and analogs by recombinant (−)-limonene 3-and 6-hydroxylases from mint (Mentha) species:evidence for catalysis within sterically constrained active sites[J]. Archives of Biochemistry and Biophysics, 2001, 387(1):125-136.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

马文晓,陈弼致,孟春,王航. 在大肠杆菌中拓展合成途径合成异胡椒醇[J]. 微生物学报, 2023, 63(3): 1243-1253

复制
分享
文章指标
  • 点击次数:298
  • 下载次数: 965
  • HTML阅读次数: 1468
  • 引用次数: 0
历史
  • 收稿日期:2022-08-01
  • 录用日期:2022-10-09
  • 在线发布日期: 2023-03-08
  • 出版日期: 2023-03-04
文章二维码