大肠杆菌高密度发酵表达4-羟基苯乙酸酯3-羟化酶及咖啡酸的高效生物合成
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广东省自然科学基金(2114050001492);江苏省高校自然科学基金(19KJA430013)


High-density fermentation of Escherichia coli to express 4-hydroxyphenylacetate 3-hydroxylase and efficient biosynthesis of caffeic acid
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    摘要:

    来源于大肠杆菌的4-羟基苯乙酸酯3-羟化酶(4-hydroxyphenylacetate 3-hydroxylase,4HPA3H)可以催化对香豆酸生物合成咖啡酸。为了实现4HPA3H的扩大生产和咖啡酸的高效生物合成,首先构建过表达4HPA3H的大肠杆菌工程菌,其次使用5 L发酵罐进行高密度发酵生产4HPA3H,再而优化采用工程菌株进行全细胞催化产咖啡酸的条件。最终实现了在5 L发酵罐中发酵,工程菌株生物量达到干重34.80 g/L。通过使用5 L发酵罐作为生物反应器进行全细胞催化,经过6 h的催化可产生18.74 g/L (0.85 g/(L·OD600))咖啡酸,摩尔转化率为78.81%,是目前文献报道4HPA3H以对香豆酸为底物合成咖啡酸的最高水平。初步实现了高密度培养大肠杆菌表达4HPA3H并高效生物合成咖啡酸,为工业化生产奠定了基础。

    Abstract:

    The 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H), originated from Escherichia coli, converts p-coumaric acid to caffeic acid. In order to improve the efficiency of caffeic acid biosynthesis, we engineered E. coli for overexpression of 4HPA3H. The high-density fermentation of the engineered E. coli was conducted in a 5 L bioreactor. Subsequently, the conditions for whole-cell biocatalysis were optimized. The dry cell weight of the 4HPA3H-expressed strain reached 34.80 g/L. After incubated in the bioreactor for 6 h, 18.74 g/L (0.85 g/(L·OD600)) of caffeic acid was obtained, with a conversion rate of 78.81% achieved. To the best of our knowledge, the titer of caffeic acid is the highest reported to date. The high-density fermentation of E. coli for overexpression of 4HPA3H and the efficient biosynthesis of caffeic acid may facilitate future large-scale production of caffeic acid.

    参考文献
    [1] Del Rio D, Rodriguez-Mateos A, Spencer JPE, et al. Dietary (poly)phenolics in human health:structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid Redox Signal, 2013, 18(14):1818-1892.
    [2] 杨九凌, 祝晓玲, 李成文, 等. 咖啡酸及其衍生物咖啡酸苯乙酯药理作用研究进展. 中国药学杂志, 2013, 48(8):577-582. Yang JL, Zhu XL, Li CW, et al. Research progress on pharmacological action of caffeic acid and its derivative phenethyl caffeic acid. Chin Pharm J, 2013, 48(8):577-582(in Chinese).
    [3] Mirzaei S, Gholami MH, Zabolian A, et al. Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways:new hope in the fight against cancer. Pharmacol Res, 2021, 171:105759.
    [4] Andrade S, Loureiro JA, Pereira MC. Caffeic acid for the prevention and treatment of Alzheimer's disease:the effect of lipid membranes on the inhibition of aggregation and disruption of Aβ fibrils. Int J Biol Macromol, 2021, 190:853-861.
    [5] Kassa T, Whalin JG, Richards MP, et al. Caffeic acid:an antioxidant with novel antisickling properties. FEBS Open Bio, 2021, 11(12):3293-3303.
    [6] Sova M, Saso L. Natural sources, pharmacokinetics, biological activities and health benefits of hydroxycinnamic acids and their metabolites. Nutrients, 2020, 12(8):2190.
    [7] Xing Y, Peng HY, Zhang MX, et al. Caffeic acid product from the highly copper-tolerant plant Elsholtzia splendens post-phytoremediation:its extraction, purification, and identification. J Zhejiang Univ Sci B, 2012, 13(6):487-493.
    [8] Flourat AL, Combes J, Bailly-Maitre-Grand C, et al. Accessing p -hydroxycinnamic acids:chemical synthesis, biomass recovery, or engineered microbial production? ChemSusChem, 2021, 14(1):118-129.
    [9] Cao MF, Gao MR, Suástegui M, et al. Building microbial factories for the production of aromatic amino acid pathway derivatives:from commodity chemicals to plant-sourced natural products. Metab Eng, 2020, 58:94-132.
    [10] Hernández-Chávez G, Martinez A, Gosset G. Metabolic engineering strategies for caffeic acid production in Escherichia coli. Electron J Biotechnol, 2019, 38:19-26.
    [11] Zhou PP, Yue CL, Shen B, et al. Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid. Appl Microbiol Biotechnol, 2021, 105(14/15):5809-5819.
    [12] Lin YH, Yan YJ. Biotechnological production of plant-specific hydroxylated phenylpropanoids. Biotechnol Bioeng, 2014, 111(9):1895-1899.
    [13] Prieto MA, Garcia JL. Molecular characterization of 4-hydroxyphenylacetate 3-hydroxylase of Escherichia coli. a two-protein component enzyme. J Biol Chem, 1994, 269(36):22823-22829.
    [14] Huang Q, Lin YH, Yan YJ. Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain. Biotechnol Bioeng, 2013, 110(12):3188-3196.
    [15] Louie TM, Xie XS, Xun LY. Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase. Biochemistry, 2003, 42(24):7509-7517.
    [16] Shen XL, Zhou DY, Lin YH, et al. Structural insights into catalytic versatility of the flavin-dependent hydroxylase (HpaB) from Escherichia coli. Sci Rep, 2019, 9(1):7087.
    [17] Wachtmeister J, Rother D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Curr Opin Biotechnol, 2016, 42:169-177.
    [18] Furuya T, Kino K. Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives. Appl Microbiol Biotechnol, 2014, 98(3):1145-1154.
    [19] 樊祥臣, 陈瑞东, 刘佳, 等. l-谷氨酸氧化酶高密度发酵及催化合成α-酮戊二酸. 过程工程学报, 2016, 16(2):292-297. Fan XC, Chen RD, Liu J, et al. High cell density fermentation of l-glutamate oxidase and its catalysis for synthesis of α-ketoglutaric acid. Chin J Process Eng, 2016, 16(2):292-297(in Chinese).
    [20] 王秀婷. 重组大肠杆菌全细胞转化l-苯丙氨酸合成苯乳酸的研究[D]. 无锡:江南大学, 2018. Wang XT. Study of biosynthesis of phenyllactic acid by recombinant E. coli whole-cell biotransformation of l-phenylalanine[D]. Wuxi:Jiangnan University, 2018(in Chinese).
    [21] 李民, 陈常庆. 重组大肠杆菌高密度发酵研究进展. 生物工程进展, 2000, 20(2):26-31. Li M, Chen CQ. Progress studies of high cell-density culture of recombinant Escherichia coli. Prog Biotechnol, 2000, 20(2):26-31(in Chinese).
    [22] 徐冰冰, 雷庆子, 曾伟主, 等. 高密度发酵产酪氨酸酚裂解酶及催化合成l-DOPA. 食品与发酵工业, 2019, 45(12):7-14. Xu BB, Lei QZ, Zeng WZ, et al. High-density fermentation for preparing tyrosine phenol lyase and application in l-DOPA synthesis. Food Ferment Ind, 2019, 45(12):7-14(in Chinese).
    [23] 陈亮, 任随周, 许玫英, 等. 乳糖替代IPTG诱导脱色酶TpmD基因在大肠杆菌中的高效表达. 微生物学通报, 2009, 36(4):551-556. Chen L, Ren SZ, Xu MY, et al. Over-expression of highly active triphenylmethane dyes decolorization enzyme (TpmD) induced by lactose instead of IPTG in Escherichia coli BL21(DE3). Microbiology, 2009, 36(4):551-556(in Chinese).
    [24] 王岁楼. 微生物比生长速率μ的含义. 郑州轻工业学院学报, 1993, 8(4):6-9. Wang SL. Meaning on specific growth rate of microorganism. J Zhengzhou Inst Light Ind, 1993, 8(4):6-9(in Chinese).
    [25] 迟雷. 基于过程控制优化的重组大肠杆菌高密度发酵研究[D]. 西安:西北大学, 2011. Chi L. Study on high cell density cultivation in recombinant Escherichia coli based on optimization of process control[D]. Xi՚an:Northwest University, 2011(in Chinese).
    [26] Fordjour E, Adipah FK, Zhou SH, et al. Metabolic engineering of Escherichia coli BL21(DE3) for de novo production of l-DOPA from d-glucose. Microb Cell Fact, 2019, 18(1):74.
    [27] 胡铮瑢, 刘玉环, 阮榕生, 等. 阿魏酸、对香豆酸碱法制备及应用研究进展. 食品科学, 2009, 30(21):438-442. Hu ZR, Liu YH, Ruan RS, PENG H, ZHANG JS, LIU CM, et al. Ferulic acid and p -coumaric acid:applications in various fields and preparation. Food Sci, 2009, 30(21):438-442(in Chinese).
    [28] Battistella C, McCallum NC, Gnanasekaran K, et al. Mimicking natural human hair pigmentation with synthetic melanin. ACS Cent Sci, 2020, 6(7):1179-1188.
    [29] Ahn SY, Jang S, Sudheer PDVN, et al. Microbial production of melanin pigments from caffeic acid and l-tyrosine using Streptomyces glaucescens and FCS-ECH-expressing Escherichia coli. Int J Mol Sci, 2021, 22(5):2413.
    [30] Kim SH, Hisano T, Takeda K, et al. Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8. J Biol Chem, 2007, 282(45):33107-33117.
    [31] Furuya T, Arai Y, Kino K. Biotechnological production of caffeic acid by bacterial cytochrome P450 CYP199A2. Appl Environ Microbiol, 2012, 78(17):6087-6094.
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张红,林金连,胡定行,刘贵友,孙磊. 大肠杆菌高密度发酵表达4-羟基苯乙酸酯3-羟化酶及咖啡酸的高效生物合成[J]. 生物工程学报, 2022, 38(9): 3466-3477

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  • 收稿日期:2022-03-18
  • 录用日期:2022-05-06
  • 在线发布日期: 2022-09-24
  • 出版日期: 2022-09-25
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