title: Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (-)-pinoresinol via kinetic resolution.
abstract: BACKGROUND
  Pinoresinol is a high-value plant-derived lignan with multiple health supporting effects. Enantiomerically pure pinoresinol can be isolated from natural sources, but with low efficiency. Most chemical and biocatalytic approaches that have been described for the synthesis of pinoresinol furnish the racemic mixture. In this study we devised a three-step biocatalytic cascade for the production of enantiomerically pure pinoresinol from the cheap compound eugenol. Two consecutive oxidations of eugenol through vanillyl-alcohol oxidase and laccase are followed by kinetic resolution of racemic pinoresinol by enantiospecific pinoresinol reductases.
  
  
  RESULTS
  The addition of the enantiospecific pinoresinol reductase from Arabidopsis thaliana for kinetic resolution of (±)-pinoresinol to an in vitro cascade involving the vanillyl-alcohol oxidase from Penicillium simplicissimum and the bacterial laccase CgL1 from Corynebacterium glutamicum resulted in increasing ee values for (+)-pinoresinol; however, an ee value of 34% was achieved in the best case. The ee value could be increased up to ≥ 99% by applying Escherichia coli-based whole-cell biocatalysts. The optimized process operated in a one-pot "two-cell" sequential mode and yielded 876 µM (+)-pinoresinol with an ee value of 98%. Switching the reductase to the enantiospecific pinoresinol lariciresinol reductase from Forsythia intermedia enabled the production of 610 µM (-)-pinoresinol with an ee value of 97%.
  
  
  CONCLUSIONS
  A new approach for the synthesis of enantiomerically pure (+)- and (-)-pinoresinol is described that combines three biotransformation steps in one pot. By switching the reductase in the last step, the whole-cell biocatalysts can be directed to produce either (+)- or (-)-pinoresinol. The products of the reductases' activity, (-)-lariciresinol and (-)-secoisolariciresinol, are valuable precursors that can also be applied for the synthesis of further lignans.
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Esther
authors.initials: E
authors.lastname: Ricklefs
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Girhard
authors.affiliation: Institute of Biochemistry, Heinrich-Heine University, Universitätsstraße 1, 40225, Düsseldorf, Germany. vlada.urlacher@uni-duesseldorf.de.
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D048230:Corynebacterium glutamicum / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D005663:Furans / Q000032:analysis / Q000737:chemistry; D007700:Kinetics; D042845:Laccase / Q000235:genetics / Q000378:metabolism; D017705:Lignans / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D005663:Furans; D017705:Lignans; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C084969:pinoresinol reductase; C076865:vanillyl-alcohol oxidase; D042845:Laccase; C103298:pinoresinol
keywords: Biocatalysis; Coniferyl alcohol; Eugenol; Kinetic resolution; Laccase; Lignan; Pinoresinol; Pinoresinol lariciresinol reductase; Pinoresinol reductase; Vanillyl-alcohol oxidase