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Production of ρ-Hydroxyacetophenone by Engineered Escherichia coli Heterologously Expressing 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase

  • Wenmei Wu (Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine) ;
  • Xiwei Yuan (Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine) ;
  • Xin Gao (Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine) ;
  • Chaoyang Tan (Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine) ;
  • Shunxiang Li (Hunan Engineering Technology Research Center for Bioactive Substance Discovery of Chinese Medicine) ;
  • Dehong Xu (Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine)
  • Received : 2023.10.13
  • Accepted : 2023.11.24
  • Published : 2024.02.28

Abstract

ρ-Hydroxyacetophenone is an important and versatile compound that has been widely used in medicine, cosmetics, new materials, and other fields. At present, there are two ways to obtain ρ-hydroxyacetophenone. One is to extract it from plants, such as Artemisia capillaris Thunb and Cynanchum otophyllum Schneid, and the other is to synthesize it by using chemical methods. Of these two methods, the second is the main one, although it has problems, such as flammable and explosive reagents, difficult separation of by-products, and harsh reaction conditions. To solve these issues, we adopted genetic engineering in this study to construct engineered Escherichia coli containing Hped gene or EbA309 gene. Whole-cell biotransformation was conducted under the same conditions to select the engineered E. coli with the higher activity. Orthogonal tests were conducted to determine the optimal biotransformation condition of the engineered E. coli. The results showed that the optimal condition was as follows: substrate concentration of 40 mmol/l, IPTG concentration of 0.1 mmol/l, an induction temperature of 25℃, and a transformation temperature of 35℃. Under this condition, the effects of transformation time on the ρ-hydroxyacetophenone concentration and cell growth were further studied. We found that as the transformation time extended, the ρ-hydroxyacetophenone concentration showed a gradually increasing trend. However, when the ρ-hydroxyacetophenone concentration increased to 1583.19 ± 44.34 mg/l in 24 h, cell growth was inhibited and then entered a plateau. In this research, we realized the synthesis of ρ-hydroxyacetophenone by biotransformation, and our findings lay a preliminary foundation for further improving and developing this method.

Keywords

Acknowledgement

This work was supported by the National Natural Science Foundation of China (82104324), the Key Discipline of Biological Engineering in the Fourteenth Five-year Plan of Hunan University of Chinese Medicine [XiaoXingFaGuiZi-2023-No.2], and the School-level Postgraduates Innovative Project of Hunan University of Chinese Medicine in 2023 (2023CX138).

References

  1. Vosmanska M, Sykora D, Fahnrich J, Kovarova M, Volka K. 2005. Extraction of p-hydroxyacetophenone and catechin from Norway spruce needles. Comparison of different extraction solvents. Anal. Bioanal. Chem. 382: 1135-1140.  https://doi.org/10.1007/s00216-005-3229-x
  2. Wang ZW, Gao J, Tan XJ, Ma TT, Chen XH, Bi KS. 2009. RP-HPLC simultaneous determination of chlorogenic acid, caffeic acid and 4-hydroxy acetophenone in herb of Artemisia capillaris Thunb. under UV-vis wavelengths. Chin. J. Pharm. Anal. 29: 919-922. 
  3. Zan K, Guo LN, Zheng J, Ma SC. 2016. Chemical constituents of ethnic medicine Cynanchum otophyllum. Zhongguo Zhong Yao Za Zhi 41: 101-105. 
  4. Hunan Institute of Pharmaceutical Industry. 1972. Preliminary pharmacology of ρ-hydroxyacetophenone, a choleretic active ingredient of Artemisia annua. J. Chin. Herb. Med. 5: 25-27. 
  5. Infectious disease laboratory of traditional Chinese medicine research institute. 1972. Clinical summary of ρ-hydroxyacetophenone. J. Chin. Herb. Med. 5: 27. 
  6. Ghiaci M, Aghaei H, Oroojeni M, Aghabarari B, Rives V, Vicente MA, et al. 2009. Synthesis of paracetamol by liquid phase Beckmann rearrangement of 4-hydroxyacetophenone oxime over H3PO4/Al-MCM-41. Catal. Commun. 10: 1486-1492.  https://doi.org/10.1016/j.catcom.2009.03.025
  7. Song SL, Lian CX, Chen LP, Huang LJ. 2020. Synthesis of the impurity F of salbutamol. J. Asian Nat. Prod. Res. 22: 956-965.  https://doi.org/10.1080/10286020.2020.1771700
  8. Chen T. 2020. Study on the local toxicities of p-hydroxyacetophenone for cosmetics. Flavour Fragr. Cosmet. 3: 56-60. 
  9. Wang XH, Xu XM, Shen YW, Huang XH. 2018. Study on bacteriostasis of preservatives in cosmetic water. Hangzhou Chem. Ind. 48: 11-14. 
  10. Mullemwar SY, Zade GD, Kalyani NT, Dhoble SJ. 2016. Blue light emitting P-hydroxy DPQ phosphor for OLEDs. Optik 127: 10546-10553.  https://doi.org/10.1016/j.ijleo.2016.08.077
  11. Wang YH, Liu X, Liu ZN, Yu Q, Tu YB. 2022. Research progress on synthesis of p-hydroxyacetophenone via Fries rearrangement. Ind. Catal. 30: 19-29. 
  12. Zhang Z, Yang MH, Duan YF. 2010. Research on synthesis of p-hydroxyacetophenone. Chem. Eng. 24: 55-56. 
  13. Lu XG. 2002. Improvement of the synthesis reaction of ρ-hydroxyacetophenone. J. Acad. Military Med. Sci. 2: 152-160. 
  14. Westbrook AW, Miscevic D, Kilpatrick S, Bruder MR, Moo-Young M, Chou CP. 2019. Strain engineering for microbial production of value-added chemicals and fuels from glycerol. Biotechnol. Adv. 37: 538-568.  https://doi.org/10.1016/j.biotechadv.2018.10.006
  15. Cravens A, Payne J, Smolke CD. 2019. Synthetic biology strategies for microbial biosynthesis of plant natural products. Nat. Commun. 10: 2142-2154.  https://doi.org/10.1038/s41467-019-09848-w
  16. Rabus R, Widdel F. 1995. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch. Microbiol. 163: 96-103.  https://doi.org/10.1007/BF00381782
  17. Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, et al. 2005. The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EBN1. Arch. Microbiol. 183: 27-36.  https://doi.org/10.1007/s00203-004-0742-9
  18. Rabus R, Kube M, Beck A, Widdel F, Reinhardt R. 2002. Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium, strain EBN1. Arch. Microbiol. 178: 506-516.  https://doi.org/10.1007/s00203-002-0487-2
  19. Wohlbrand L, Wilkes H, Halder T, Rabus R. 2008. Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins. J. Bacteriol. 190: 5699-5709.  https://doi.org/10.1128/JB.00409-08
  20. Muhr E, Schuhle K, Clermont L, Sunwoldt K, Kleinsorge D, Seyhan D, et al. 2015. Enzymes of anaerobic ethylbenzene and p-ethylphenol catabolism in 'aromatoleum aromaticum': differentiation and differential induction. Arch. Microbiol. 197: 1051-1062.  https://doi.org/10.1007/s00203-015-1142-z
  21. Busing I, Hoffken HW, Breuer M, Wohlbrand L, Hauer B, Rabus R. 2015. Molecular genetic and crystal structural analysis of 1-(4-hydroxyphenyl)-ethanol dehydrogenase from 'aromatoleum aromaticum' EBN1. J. Mol. Microbiol. Biotechnol. 25: 327-339.  https://doi.org/10.1159/000439113
  22. Sahdev S, Khattar SK, Saini KS. 2008. Production of active eukaryotic proteins through bacterial expression systems: A review of the existing biotechnology strategies. Mol. Cell Biochem. 307: 249-264.  https://doi.org/10.1007/s11010-007-9603-6
  23. Tataruch M, Illeova V, Milaczewska A, Borowski T, Mihal' M, Polakovic M. 2023. Inactivation and aggregation of R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase from Aromatoleum aromaticum. Int. J. Biol. Macromol, 234: 123772. 
  24. Spyrou G, Haggard-Ljungquist E, Krook M, Jornvall H, Nilsson E, Reichard P. 1991. Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme. J. Bacteriol. 173: 3673-3679.  https://doi.org/10.1128/jb.173.12.3673-3679.1991
  25. Tannous C, Booz GW, Altara R, Muhieddine DH, Mericskay M, Refaat MM, et al. 2021. Nicotinamide adenine dinucleotide: biosynthesis, consumption and therapeutic role in cardiac diseases. Acta Physiol. (Oxf). 231: e13551. 
  26. Lu L, Wang X, Zhou L, Liu Q, Zhang G, Xue B, et al. 2023. Establishing a biosynthetic pathway for the production of p-hydroxyacetophenone and its glucoside in Escherichia coli. Metab. Eng. 76: 110-119. 
  27. Chaudhary RG, Chauke PB, Ingale VN .2015. Solvent free synthesis of p-hydroxyacetophenone in a situ using eco-friendly catalyst in Fries rearrangement. J. Chem. Pharm. Res. 7: 727-731.