A high-throughput screening method for improved R-2-(4-hydroxyphenoxy)propionic acid biosynthesis

  • Hai-Yan Zhou
  • Yi-Zuo Li
  • Rui Jiang
  • Hai-Feng Hu
  • Yuan-Shan Wang
  • Zhi-Qiang Liu
  • Ya-Ping XueEmail author
  • Yu-Guo Zheng
Research Paper


R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key intermediate of the enantiomerically pure phenoxypropionic acid herbicides. R-HPPA could be biosynthesized through selective introduction of a hydroxyl group (–OH) into the substrate R-2-phenoxypropionic acid (R-PPA) at C-4 position, facilitated by microorganisms with hydroxylases. In this study, an efficient high-throughput screening method for improved R-HPPA biosynthesis through microbial hydroxylation was developed. As a hydroxylated aromatic product, R-HPPA could be oxidized by oxidant potassium dichromate to form brown-colored quinone-type compound. The concentration of R-HPPA can be quantified according to the absorbance of the colored compound at a suitable wavelength of 570 nm; and the R-HPPA biosynthetic capability of microorganism strains could also be rapidly evaluated. After optimization of the assay conditions, the high-throughput screening method was successfully used in identification of Beauveria bassiana mutants with enhanced R-HPPA biosynthesis capacity. A positive mutant C-7 with high tolerance to 20 g/L R-PPA was rapidly selected from 1920 mutants. The biomass and R-HPPA titer were 12.5- and 38.19-fold higher compared with the original strain at 20 g/L R-PPA. This high-throughput screening method developed in this work could also be a potential tool for screening strains producing other important phenolic compounds.


High-throughput screening R-2-(4-hydroxyphenoxy)propionic acid R-2-phenoxypropionic acid Hydroxylation Potassium dichromate 



The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 31500031) and the Postgraduate Teaching Reform Project of Zhejiang University of Technology (No. 2018114).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Brown RB, Kruse JA, Counts GW, Russell JA, Christou NV, Sands ML, the Endotracheal Tobramycin Study Group (1990) Double-blind study of endotracheal tobramycin in the treatment of gram-negative bacterial pneumonia. Antimicrob Agents Chemother 34:269–272CrossRefGoogle Scholar
  2. 2.
    Ishige T, Honda K, Shimizu S (2005) Whole organism biocatalysis. Curr Opin Chem Biol 9:174–180CrossRefGoogle Scholar
  3. 3.
    Ullrich R, Hofrichter M (2007) Enzymatic hydroxylation of aromatic compounds. Cell Mol Life Sci 64:271–293CrossRefGoogle Scholar
  4. 4.
    Kinne M, Ullrich R, Hammel KE, Scheibner K, Hofrichter M (2008) Regioselective preparation of (R)-2-(4-hydroxyphenoxy)propionic acid with a fungal peroxygenase. Tetrahedron Lett 49:5950–5953CrossRefGoogle Scholar
  5. 5.
    Shoji O, Wiese C, Fujishiro T, Shirataki C, Wunsch B, Watanabe Y (2010) Aromatic C-H bond hydroxylation by P450 peroxygenases: a facile chromogenic assay for monooxygenation activities of enzymes based on Russig’s blue formation. J Biol Inorg Chem 15:1109–1115CrossRefGoogle Scholar
  6. 6.
    Boersma MG, Primus J-L, Koerts J, Veeger C, Rietjens IMCM (2000) Heme-(hydro)peroxide mediated O- and N-dealkylation. Eur J Biochem 267:6673–6678CrossRefGoogle Scholar
  7. 7.
    Yarman A, Badalyan A, Gajovic-Eichelmann N, Wollenberger U, Scheller FW (2011) Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11. Biosens Bioelectron 30:320–323CrossRefGoogle Scholar
  8. 8.
    Peng L, Wollenberger U, Kinne M, Hofrichter M, Ullrich R, Scheibner K, Fischer A (2010) Scheller FW, Peroxygenase based sensor for aromatic compounds. Biosens Bioelectron 26:1432–1436CrossRefGoogle Scholar
  9. 9.
    Kluge M, Ullrich R, Dolge C, Scheibner K, Hofrichter M (2009) Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation. Appl Microbiol Biotechnol 81:1071–1076CrossRefGoogle Scholar
  10. 10.
    Dingler C, Ladner W, Krei GA, Cooper B, Hauer B (1996) Preparation of (R)-2-(4-hydroxyphenoxy) propionic acid by biotransformation. Pestic Sci 46:33–35CrossRefGoogle Scholar
  11. 11.
    Ladner W, Staudenmaier HR, Hauer B, Mueller U, Pressler U, Meyer J, Siegel H, Process for the hydroxylation of aromatic acids using strains of the fungus Beauveria, United States Patent, No. 5928912, 1999-07-27Google Scholar
  12. 12.
    Holland HL, Morris TA, Nava PJ, Zabic M (1999) A new paradigm for biohydroxylation by Beauveria bassiana ATCC 7159. Tetrahedron 55:7441–7460CrossRefGoogle Scholar
  13. 13.
    Cooper B, Ladner W, Hauer B, Siegel H, Preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation, United States Patent, No. 5296363, 1994-3-22Google Scholar
  14. 14.
    Tang XL, Suo H, Zheng RC, Zheng YG (2018) An efficient chromogenic high-throughput screening method for synthetic activity of tyrosine phenol-lyase. Anal Biochem 560:7–11CrossRefGoogle Scholar
  15. 15.
    Xue YP, Zhang YQ, Wang W, Wang YJ, Liu ZQ, Zou SP, Zheng YG, Shen YC (2013) Highly enantioselective oxidation of alpha-hydroxyacids bearing a substituent with an aryl group: co-production of optically active alpha-hydroxyacids and alpha-ketoacids. Bioresour Technol 132:391–394CrossRefGoogle Scholar
  16. 16.
    Xue YP, Yang YK, Lv SZ, Liu ZQ, Zheng YG (2016) High-throughput screening methods for nitrilases. Appl Microbiol Biotechnol 100:3421–3432CrossRefGoogle Scholar
  17. 17.
    Xue YP, Wang W, Wang YJ, Liu ZQ, Zheng YG, Shen YC (2012) Isolation of enantioselective alpha-hydroxyacid dehydrogenases based on a high-throughput screening method. Bioprocess Biosyst Eng 35:1515–1522CrossRefGoogle Scholar
  18. 18.
    Chen LY, Cheng CW, Liang JY (2015) Effect of esterification condensation on the Folin–Ciocalteu method for the quantitative measurement of total phenols. Food Chem 170:10–15CrossRefGoogle Scholar
  19. 19.
    Nezhad MRH, Alimohammadi M, Tashkhourian J, Razavian SM (2008) Optical detection of phenolic compounds based on the surface plasmon resonance band of Au nanoparticles. Spectrochim Acta, Part A 71:199–203CrossRefGoogle Scholar
  20. 20.
    Rodríguez JV, Grubešić RJ, Kremer D, Kokot V (2016) Quality assessment of two spectrophotometric procedures for polyphenol determination and application in Moltkia petraea species. J Chin Chem Soc 63:677–687CrossRefGoogle Scholar
  21. 21.
    Chen H, Zhang Q, Wang X, Yang J, Wang Q (2011) Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MSn. Phytochem Anal 22:247–257CrossRefGoogle Scholar
  22. 22.
    Meenu M, Sharma A, Guha P, Mishra S (2016) A rapid high-performance liquid chromatography photodiode array detection method to determine phenolic compounds in mung bean (Vigna radiata L.). Int J Food Prop 19:2223–2237CrossRefGoogle Scholar
  23. 23.
    Bae IK, Ham HM, Jeong MH, Kim DH, Kim HJ (2015) Simultaneous determination of 15 phenolic compounds and caffeine in teas and mate using RP-HPLC/UV detection: method development and optimization of extraction process. Food Chem 172:469–475CrossRefGoogle Scholar
  24. 24.
    Ding Y, Ayon A, García CD (2007) Electrochemical detection of phenolic compounds using cylindrical carbon-ink electrodes and microchip capillary electrophoresis. Anal Chim Acta 584:244–251CrossRefGoogle Scholar
  25. 25.
    Scampicchio M, Wang J, Mannino S, Chatrathi MP (2005) Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids. J Chromatogr A 1049:189–194CrossRefGoogle Scholar
  26. 26.
    Godoy-Caballero MP, Acedo-Valenzuela MI, Durán-Merás I, Galeano-Díaz T (2012) Development of a non-aqueous capillary electrophoresis method with UV–visible and fluorescence detection for phenolics compounds in olive oil. Anal Bioanal Chem 403:279–290CrossRefGoogle Scholar
  27. 27.
    Lu L, Zhang L, Zhang X, Huan S, Shen G, Yu R (2010) A novel tyrosinase biosensor based on hydroxyapatite-chitosan nanocomposite for the detection of phenolic compounds. Anal Chim Acta 665:146–151CrossRefGoogle Scholar
  28. 28.
    Rodríguez-Delgado MM, Alemán-Nava GS, Rodríguez-Delgado JM, Dieck-Assad G, Martínez-Chapa SO, Barceló D, Parra R (2015) Laccase-based biosensors for detection of phenolic compounds. Trends Anal Chem 74:21–45CrossRefGoogle Scholar
  29. 29.
    Draghi PF, Fernandes JCB (2017) Label-free potentiometric biosensor based on solid-contact for determination of total phenols in honey and propolis. Talanta 164:413–417CrossRefGoogle Scholar
  30. 30.
    Martín M, Salazar P, Campuzano S, Villalonga R, Pingarrón JM, González-Mora JL (2015) Amperometric magnetobiosensors using poly(dopamine)-modified Fe3O4 magnetic nanoparticles for the detection of phenolic compounds. Anal Methods 7:8801–8808CrossRefGoogle Scholar
  31. 31.
    Wu J, Rickert WS, Masters A (2012) An improved high performance liquid chromatography-fluorescence detection method for the analysis of major phenolic compounds in cigarette smoke and smokeless tobacco products. J Chromatogr A 1264:40–47CrossRefGoogle Scholar
  32. 32.
    Pistonesi MF, Di NM, Centurión ME, Palomeque ME, Lista AG, Fernández Band BS (2006) Determination of phenol, resorcinol and hydroquinone in air samples by synchronous fluorescence using partial least-squares (PLS). Talanta 69:1265–1268CrossRefGoogle Scholar
  33. 33.
    Viñas P, López-Erroz C, Marı́n-Hernández JJ, Hernández-Córdoba M (2000) Determination of phenols in wines by liquid chromatography with photodiode array and fluorescence detection. J Chromatogr A 871:85–93CrossRefGoogle Scholar
  34. 34.
    Hu H-F, Zhou H-Y, Wang M-X, Wang Y-S, Xue Y-P, Zheng Y-G (2019) A rapid throughput assay for screening (R)-2-(4-hydroxyphenoxy)propionic acid producing microbes. J Microbiol Methods 158:44–51CrossRefGoogle Scholar
  35. 35.
    Tian X, Shen Y, Zhuang Y, Zhao W, Hang H, Chu J (2018) Kinetic analysis of sodium gluconate production by Aspergillus niger with different inlet oxygen concentrations. Bioprocess Biosyst Eng 41:1697–1706CrossRefGoogle Scholar
  36. 36.
    Tentscher PR, Bourgin M, von Gunten U (2018) Ozonation of para-substituted phenolic compounds yields p-benzoquinones, other cyclic α, β-unsaturated ketones, and substituted catechols. Environ Sci Technol 52:4763–4773CrossRefGoogle Scholar
  37. 37.
    Dai R, Liu J, Yu C, Sun R, Lan Y, Mao JD (2009) A comparative study of oxidation of Cr(III) in aqueous ions, complex ions and insoluble compounds by manganese-bearing mineral (birnessite). Chemosphere 76:536–541CrossRefGoogle Scholar
  38. 38.
    Albrecht M, Schneider O, Schmidt A (2009) Redox active donor-substituted punicin derivatives. Org Biomol Chem 7:1445–1453CrossRefGoogle Scholar
  39. 39.
    Gedawy A, Al-Salami H, Dass CR (2019) Development and validation of a new analytical HPLC method for simultaneous determination of the antidiabetic drugs, metformin and gliclazide. J Food Drug Anal 27:315–322  CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hai-Yan Zhou
    • 1
    • 2
  • Yi-Zuo Li
    • 1
    • 2
  • Rui Jiang
    • 1
    • 2
  • Hai-Feng Hu
    • 1
    • 2
  • Yuan-Shan Wang
    • 1
    • 2
  • Zhi-Qiang Liu
    • 1
    • 2
  • Ya-Ping Xue
    • 1
    • 2
    Email author
  • Yu-Guo Zheng
    • 1
    • 2
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouPeople’s Republic of China
  2. 2.Engineering Research Center of Bioconversion and Biopurification of the Ministry of EducationZhejiang University of TechnologyHangzhouPeople’s Republic of China

Personalised recommendations