Whole‐Cell Carboxylate Reduction for the Synthesis of 3‐Hydroxytyrosol

3‐Hydroxytyrosol (3‐HT) is a phenolic antioxidant that has a number of beneficial effects on human health and is a valuable building block in the synthesis of various pharmaceuticals. Herein, we report a new method for the production of 3‐HT through reduction of 3,4‐dihydroxyphenylacetic acid. The reduction was performed in whole Escherichia coli BL21 (DE3) cells overexpressing carboxylic acid reductase from Nocardia and phosphopantetheinyl transferase from E. coli. An endogenous E. coli aldehyde reducing activity turned out to be highly efficient for further reduction of the aldehyde intermediate to the desired alcohol. The influence of different buffer components, cofactors, and cofactor recycling systems was investigated. A very economic combination of glucose, citrate, and air proved sufficient for recycling of the essential cofactors ATP and NAD(P)H. Selected crucial parameters were then further optimized within a “design of experiments” approach. Finally, first preparative‐scale bioreductions resulted in pure 3‐HT.

[1]  F. García-Carmona,et al.  A new process for obtaining hydroxytyrosol using transformed Escherichia coli whole cells with phenol hydroxylase gene from Geobacillus thermoglucosidasius. , 2013, Food chemistry.

[2]  S. White,et al.  The Role of Invariant Amino Acid Residues at the Hydride Transfer Site of Proton-translocating Transhydrogenase* , 2006, Journal of Biological Chemistry.

[3]  K. Tuck,et al.  Synthesis of tritium-labeled hydroxytyrosol, a phenolic compound found in olive Oil. , 2000, Journal of agricultural and food chemistry.

[4]  H. Simon,et al.  Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes. , 1989, European journal of biochemistry.

[5]  H. Sahm,et al.  Reduction of halogenated derivatives of benzoic acid to the corresponding alcohols by Desulfovibrio vulgaris PY1 , 2000 .

[6]  C. Laane,et al.  Bioreduction of carboxylic acids by Pyrococcus furiosus in batch cultures , 1999 .

[7]  A. Fishman,et al.  Improving process conditions of hydroxytyrosol synthesis by toluene-4-monooxygenase , 2012 .

[8]  H. Simon,et al.  Reduktion von 2‐Enoaten und Alkanoaten mit Kohlenmonoxid oder Formiat, Viologenen und Clostridium thermoaceticum zu gesättigten Säuren und ungesättigten bzw. gesättigten Alkoholen , 1987 .

[9]  C. Olsen,et al.  De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae) , 2009, Applied and Environmental Microbiology.

[10]  A. Fishman,et al.  Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol. , 2009 .

[11]  L. Casalot,et al.  Bioconversion of tyrosol into hydroxytyrosol and 3,4-dihydroxyphenylacetic acid under hypersaline conditions by the new Halomonas sp. strain HTB24. , 2007, FEMS microbiology letters.

[12]  M. Zenk,et al.  Reduktion aromatischer Säuren zu Aldehyden und Alkoholen im zellfreien System , 1969 .

[13]  P. Venkitasubramanian,et al.  Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid. , 2008, Enzyme and microbial technology.

[14]  Andreas Liese,et al.  Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes , 2007, Applied Microbiology and Biotechnology.

[15]  H. Simon,et al.  Reduction of 2‐Enoates and Alkanoates with Carbon Monoxide or Formate, Viologens, and Clostridium thermoaceticum to Saturated Acids and Unsaturated and Saturated Alcohols , 1987 .

[16]  P. Venkitasubramanian,et al.  Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme* , 2007, Journal of Biological Chemistry.

[17]  J. Espín,et al.  Synthesis of the antioxidant hydroxytyrosol using tyrosinase as biocatalyst. , 2001, Journal of agricultural and food chemistry.

[18]  S. Manfredini,et al.  Preparation of 3,4-Dihydroxy-1-benzeneethanol: A Reinvestigation , 1983 .

[19]  P. Sacchetta,et al.  Synthesis, biological evaluation, and molecular modeling of oleuropein and its semisynthetic derivatives as cyclooxygenase inhibitors. , 2009, Journal of agricultural and food chemistry.

[20]  Zhizhen Zhang,et al.  Synthesis and biological evaluation of caffeic acid 3,4-dihydroxyphenethyl ester. , 2010, Journal of natural products.

[21]  G. Beltrán,et al.  Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells , 2011, Nutrients.

[22]  J. Rosazza,et al.  Biocatalytic Synthesis of Vanillin , 2000, Applied and Environmental Microbiology.

[23]  J. Mehta,et al.  Review of Contemporary Antiarrhythmic Drug Therapy for Maintenance of Sinus Rhythm in Atrial Fibrillation , 2012, Journal of cardiovascular pharmacology and therapeutics.

[24]  I. Arends,et al.  A biocatalytic hydrogenation of carboxylic acids. , 2012, Chemical communications.

[25]  Sami Sayadi,et al.  Synthesis of hydroxytyrosol, 2-hydroxyphenylacetic acid, and 3-hydroxyphenylacetic acid by differential conversion of tyrosol isomers using Serratia marcescens strain. , 2005, Journal of agricultural and food chemistry.

[26]  B. Nidetzky,et al.  Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH , 2012, Microbial Cell Factories.

[27]  Pär Tufvesson,et al.  Process considerations for the scale-up and implementation of biocatalysis , 2010 .

[28]  Kristy M. Hawkins,et al.  Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. , 2008, Nature chemical biology.

[29]  Carl-Fredrik Mandenius,et al.  Bioprocess optimization using design‐of‐experiments methodology , 2008, Biotechnology progress.

[30]  R. Nucci,et al.  Antioxidant activity of the main bioactive derivatives from oleuropein hydrolysis by hyperthermophilic beta-glycosidase. , 2001, Journal of agricultural and food chemistry.

[31]  M. C. García-Parrilla,et al.  Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review , 2012 .

[32]  Dr David Vauzour,et al.  Hydroxytyrosol inhibits the proliferation of human colon adenocarcinoma cells through inhibition of ERK1/2 and cyclin D1. , 2009, Molecular nutrition & food research.

[33]  V. Urlacher,et al.  Modern biooxidation : enzymes, reactions and applications , 2007 .

[34]  A. Evidente,et al.  A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste waters. , 1999, Journal of agricultural and food chemistry.

[35]  F. Lottspeich,et al.  Purification and some properties of the tungsten-containing carboxylic acid reductase from Clostridium formicoaceticum. , 1991, Biological chemistry Hoppe-Seyler.

[36]  Z. Bouallagui,et al.  Production of high hydroxytyrosol yields via tyrosol conversion by Pseudomonas aeruginosa immobilized resting cells. , 2006, Journal of agricultural and food chemistry.

[37]  Patrik R. Jones,et al.  Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities , 2012, Proceedings of the National Academy of Sciences.

[38]  C. Huber,et al.  Pterin cofactor, substrate specificity, and observations on the kinetics of the reversible tungsten-containing aldehyde oxidoreductase fromClostridium thermoaceticum , 1995, Archives of Microbiology.

[39]  J. Rosazza,et al.  Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 , 1997, Journal of bacteriology.

[40]  S. Levy,et al.  Effects of toluene permeabilization and cell deenergization on tetracycline resistance in Escherichia coli , 1986, Antimicrobial Agents and Chemotherapy.

[41]  V. Farmer,et al.  Reduction of certain aromatic acids to aldehydes and alcohols by Polystictus versicolor. , 1959, Biochimica et biophysica acta.

[42]  L. Daniels,et al.  Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family , 2004, Applied and Environmental Microbiology.