Redox self-sufficient biocatalyst system for conversion of 3,4-Dihydroxyphenyl-l-alanine into (R)- or (S)-3,4-Dihydroxyphenyllactic acid

  • Tianzhen Xiong
  • Jing Jiang
  • Yajun Bai
  • Tai-Ping Fan
  • Ye Zhao
  • Xiaohui Zheng
  • Yujie CaiEmail author
Metabolic Engineering and Synthetic Biology - Original Paper


We developed an efficient multi-enzyme cascade reaction to produce (R)- or (S)-3,4-Dihydroxyphenyllactic acid [(R)- or (S)-Danshensu, (R)- or (S)-DSS] from 3,4-Dihydroxyphenyl-l-alanine (l-DOPA) in Escherichia coli by introducing tyrosine aminotransferase (tyrB), glutamate dehydrogenase (cdgdh) and d-aromatic lactate dehydrogenase (csldhD) or l-aromatic lactate dehydrogenase (tcldhL). First, the genes in the pathway were overexpressed and fine-tuned for (R)- or (S)-DSS production. The resulting strain, E. coli TGL 2.1 and E. coli TGL 2.2, which overexpressed tyrB with the stronger T7 promoter and cdgdh, csldhD or tcldhL with the weaker Trc promoter, E. coli TGL 2.1 yielded 57% increase in (R)-DSS production: 59.8 ± 2.9 mM. Meanwhile, E. coli TGL 2.2 yielded 54% increase in (S)-DSS production: 52.2 ± 2.4 mM. The optimal concentration of L-glutamate was found to be 20 mM for production of (R)- or (S)-DSS. Finally, l-DOPA were transformed into (R)- or (S)-DSS with an excellent enantiopure form (enantiomeric excess > 99.99%) and productivity of 6.61 mM/h and 4.48 mM/h, respectively.


(R)-3,4-Dihydroxyphenyllactic acid (S)-3,4-Dihydroxyphenyllactic acid 3,4-Dihydroxyphenyl-l-alanine Self-sufficient Whole-cell biotransformation 



We thank the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), national first-class discipline program of Light Industry Technology and Engineering (LITE2108-04), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1841), the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (IRT_15R55), the seventh group of Hundred-Talent Program of Shanxi Province (2015), and The Key Project of Research and Development Plan of Shaanxi (2017ZDCXL-SF-01-02-01) for financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10295_2019_2200_MOESM1_ESM.docx (494 kb)
Supplementary material 1 (DOCX 494 kb)


  1. 1.
    Anderson BM, Anderson CD, Vantassell RL, Lyerly DM, Wilkins TD (1993) Purification and characterization of Clostridium difficile glutamate dehydrogenase. Arch Biochem Biophys 300(483–488):4. Google Scholar
  2. 2.
    Bai Y, Zhang Q, Jia P, Yang L, Sun Y, Nan Y, Wang S, Meng X, Wu Y, Qin F, Sun Z, Gao X, Liu P, Luo K, Zhang Y, Zhao X, Xiao C, Liao S, Liu J, Wang C, Fang J, Wang X, Wang J, Gao R, An X, Zhang X, Zheng X (2014) Improved process for pilot-scale synthesis of danshensu ((±)-DSS) and Its enantiomer derivatives. Org Process Res Dev 18(1667–1673):16. Google Scholar
  3. 3.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefGoogle Scholar
  4. 4.
    Chen L, Bai Y, Fan T-P, Zheng X, Cai Y (2017) Characterization of a d-Lactate dehydrogenase from lactobacillus fermentum JN248 with high phenylpyruvate reductive activity. J Food Sci 82:2269–2275. CrossRefGoogle Scholar
  5. 5.
    De Felice A, Bader A, Leone A, Sosa S, Della Loggia R, Tubaro A, De Tommasi N (2006) New polyhydroxylated triterpenes and anti-inflammatory activity of Salvia hierosolymitana. Planta Med 72:643–649. CrossRefGoogle Scholar
  6. 6.
    Dong C, Wang Y, Zhu Y (2009) Asymmetric synthesis and biological evaluation of danshensu derivatives as anti-myocardial ischemia drug candidates. Bioorg Med Chem 17:3499–3507. CrossRefGoogle Scholar
  7. 7.
    Findrik Z, Poljanac M, Vasic-Racki D (2005) Modelling and optimization of the (R)-(+)-3,4-dihydroxyphenyllactic acid production catalyzed with d-lactate dehydrogenase from Lactobacillus leishmannii using genetic algorithm. Chem Biochem Eng Q 19:351–358Google Scholar
  8. 8.
    Huo M, Wang Z, Wu D, Zhang Y, Qiao Y (2017) Using coexpression protein interaction network analysis to identify mechanisms of danshensu affecting patients with coronary heart disease. Int J Mol Sci 18:1298. CrossRefGoogle Scholar
  9. 9.
    Hwang JY, Park J, Seo JH, Cha M, Cho BK, Kim J, Kim BG (2009) Simultaneous synthesis of 2-phenylethanol and l-homophenylalanine using aromatic transaminase with yeast Ehrlich pathway. Biotechnol Bioeng 102:1323–1329. CrossRefGoogle Scholar
  10. 10.
    Janet TP, John FM (1978) Role of the Escherichia coli Aromatic amino acid aminotransferase in leucine biosynthesis. J Bacteriol 136:1–4Google Scholar
  11. 11.
    Jean M, DeMoss RD (1968) Indolelactate dehydrogenase from Clostridium sporogenes. Can J Microbiol 14:429–435. CrossRefGoogle Scholar
  12. 12.
    Lam FF, Yeung JH, Chan KM, Or PM (2007) Relaxant effects of danshen aqueous extract and its constituent danshensu on rat coronary artery are mediated by inhibition of calcium channels. Vascul Pharmacol 46:271–277. CrossRefGoogle Scholar
  13. 13.
    Lim HN, Lee Y, Hussein R (2011) Fundamental relationship between operon organization and gene expression. P Natl Acad Sci Usa 108:10626–10631. CrossRefGoogle Scholar
  14. 14.
    Lu H, Bai Y, T-p Fan, Zhao Y, Zheng X, Cai Y (2018) Identification of a L-Lactate dehydrogenase with 3,4-dihydroxyphenylpyruvic reduction activity for l-Danshensu production. Process Biochem 72:119–123. CrossRefGoogle Scholar
  15. 15.
    Luna-Velasco A, Field JA, Cobo-Curiel A, Sierra-Alvarez R (2011) Inorganic nanoparticles enhance the production of reactive oxygen species (ROS) during the autoxidation of l-3,4-dihydroxyphenylalanine (l-dopa). Chemosphere 85:19–25. CrossRefGoogle Scholar
  16. 16.
    Lyerly DM, Barroso LA, Wilkins TD (1991) Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase. J Clin Microbiol 29:2639–2642Google Scholar
  17. 17.
    Montemartini M, Santome JA, Cazzulo JJ, Nowicki C (1994) Purification and partial structural and kinetic characterization of an aromatic l-α-hydroxyacid dehydrogenase from epimastigotes of Trypanosoma cruzi. Mol Biochem Parasit 68:15–23. CrossRefGoogle Scholar
  18. 18.
    O’Reilly E, Iglesias C, Ghislieri D, Hopwood J, Galman JL, Lloyd RC, Turner NJ (2014) A regio- and stereoselective omega-transaminase/monoamine oxidase cascade for the synthesis of chiral 2,5-disubstituted pyrrolidines. Angew Chem Int Ed Engl 53:2447–2450. CrossRefGoogle Scholar
  19. 19.
    Patel RN (2008) Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coordin Chem Rev 252:659–701. CrossRefGoogle Scholar
  20. 20.
    Sehl T, Hailes HC, Ward JM, Wardenga R, von Lieres E, Offermann H, Westphal R, Pohl M, Rother D (2013) Two steps in one pot: enzyme cascade for the synthesis of nor(pseudo)ephedrine from inexpensive starting materials. Angew Chem Int Ed Engl 52:6772–6775. CrossRefGoogle Scholar
  21. 21.
    Sun H, Zhang H, Ang EL, Zhao H (2017) Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates. Bioorgan Med Chem 26:1252–1274. Google Scholar
  22. 22.
    Tang Y, Wang M, Le X, Meng J, Huang L, Yu P, Chen J, Wu P (2011) Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats. Phytomedicine 18:1024–1030. CrossRefGoogle Scholar
  23. 23.
    Wang Y, Bai Y, Fan T, Zheng X, Cai Y (2018) Reducing 3,4-dihydroxyphenylpyruvic acid to d-3,4-dihydroxyphenyllactic acid via a coenzyme nonspecific d-lactate dehydrogenase from Lactobacillus reuteri. J Appl Microbiol 125:1739–1748. CrossRefGoogle Scholar
  24. 24.
    Weber N, Gorwa-Grauslund M, Carlquist M (2014) Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols. Microb Cell Fact 13:118. CrossRefGoogle Scholar
  25. 25.
    Wu S, Liu J, Li Z (2017) Biocatalytic formal Anti-Markovnikov hydroamination and hydration of aryl alkenes. Acs Catal 7:5225–5233. CrossRefGoogle Scholar
  26. 26.
    Yao Y, Wang C, Qiao J, Zhao G (2013) Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway. Metab Eng 19:79–87. CrossRefGoogle Scholar
  27. 27.
    Yun H, Yang YH, Cho BK, Hwang BY, Kim BG (2003) Simultaneous synthesis of enantiomerically pure (R)-1-phenylethanol and (R)-alpha-methylbenzylamine from racemic alpha-methylbenzylamine using omega-transaminase/alcohol dehydrogenase/glucose dehydrogenase coupling reaction. Biotechnol Lett 25:809–814. CrossRefGoogle Scholar
  28. 28.
    Zhang L, Chen L, Lu Y, Wu J, Xu B, Sun Z, Zheng S, Wang A (2010) Danshensu has anti-tumor activity in B16F10 melanoma by inhibiting angiogenesis and tumor cell invasion. Eur J Pharmacol 643:195–201. CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  • Tianzhen Xiong
    • 1
  • Jing Jiang
    • 1
  • Yajun Bai
    • 2
  • Tai-Ping Fan
    • 3
  • Ye Zhao
    • 2
  • Xiaohui Zheng
    • 2
  • Yujie Cai
    • 1
    Email author
  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.College of Life SciencesNorthwest UniversityXi’anChina
  3. 3.Department of PharmacologyUniversity of CambridgeCambridgeUK

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