One-Pot Biocatalytic Synthesis of rac-Syringaresinol from a Lignin-Derived Phenol

Abstract

The drive for a circular bioeconomy has resulted in a great demand for renewable, biobased chemicals. We present a one-pot biocatalytic cascade reaction for the production of racemic syringaresinol, a lignan with applications as a nutraceutical and in polymer chemistry. The process consumes dihydrosinapyl alcohol, which can be produced renewably from the lignocellulosic material. To achieve this, a variant of eugenol oxidase was engineered for the oxidation of dihydrosinapyl alcohol into sinapyl alcohol with good conversion and chemoselectivity. The crystal structure of the engineered oxidase revealed the molecular basis of the influence of the mutations on the chemoselectivity of the oxidation of dihydrosinapyl alcohol. By using horseradish peroxidase, the subsequent oxidative dimerization of sinapyl alcohol into syringaresinol was achieved. Conditions for the one-pot, two-enzyme synthesis were optimized, and a high yield of syringaresinol was achieved by cascading the oxidase and peroxidase steps in a stepwise fashion. This study demonstrates the efficient production of syringaresinol from a compound that can be renewed by reductive catalytic fractionation of lignocellulose, providing a biocatalytic route for generating a valuable compound from lignin.

Figure 1

Scheme of the biocatalytic synthesis of syringaresinol from lignin-derived phenolic compounds.

Figure 2

Mutant screening for enzymatic conversion of dihydroconiferyl alcohol and dihydrosinapyl alcohol. Dihydroconiferyl alcohol/dihydrosinapyl alcohol, dehydrogenation products coniferyl alcohol/sinapyl alcohol, and the corresponding oxygenation products (alcohol/ketones) are shown together in cyan, red, and orange, respectively. Mutants of interest are marked with blue arrows. (A) 24 h conversion of dihydroconiferyl alcohol by EUGO single mutants using cell-free extracts; (B) 72 h conversion of dihydrosinapyl alcohol by EUGO single mutants using cell-free extracts. Conversions of dihydroconiferyl alcohol or dihydrosinapyl alcohol (5 mM) were carried out in KPi buffer (50 mM, pH 7.5).

Figure 3

Screening of EUGO 5X single, double, and triple mutants for the conversion of dihydrosinapyl alcohol and dihydroconiferyl alcohol. Dihydrosinapyl alcohol/dihydroconiferyl alcohol, sinapyl alcohol/coniferyl alcohol, and their corresponding alcohols and ketones together are shown in cyan, red, and orange, respectively. (A) Conversions of dihydrosinapyl alcohol (5 mM) were carried out in the presence of purified enzymes (10 μM) in a KPi buffer (50 mM, pH 7) with DMSO (10%) and analyzed by HPLC. (B) Evolution of eugenol oxidase for chemoselective oxidation of dihydrosinapyl alcohol. Using the 24 h conversion of dihydrosinapyl alcohol by EUGO10X as reference, the relative activity and selectivity for substrate oxidation were compared among wild-type and a few mutants. (C) Conversions of dihydroconiferyl alcohol (5 mM) were carried out in the presence of purified enzymes (10 μM) in KPi buffer (50 mM, pH 7.5) with DMSO (10%) and analyzed by HPLC.

Figure 4

X-ray crystal structure of the 8-fold mutant, EUGO8X. (A) Backbone of EUGO8X with its mutation sites (spheres). The active-site mutations I427V and L381Q are in magenta. The FAD is shown in yellow sticks. (B) Comparison of active sites of EUGO8X (magenta) and wild-type EUGO (cyan; PDB code 5FXD). The 2Fo-Fc electron density map of sinapyl alcohol is contoured at a 1.2 σ level.

Figure 5

X-ray structure of EUGO10X. (A) Backbone of EUGO10X with its mutation sites shown as spheres. The two additional chemoselectivity-affording mutations are represented with purple spheres (D151E and Q425S). (B) 2Fo-Fc electron density map of dihydrosinapyl alcohol in the active site. The map is contoured at a 1.0 σ level. (C) Comparison between EUGO8X (orange) and EUGO10X (green). The bound ligands are shown in wheat (EUGO8X) and green (EUGO10X).

Figure 6

Time-course monitoring of one-pot conversion of dihydrosinapyl alcohol to syringaresinol by an oxidase-HRP cascade reaction. Dihydrosinapyl alcohol, sinapyl alcohol, and syringaresinol are displayed in the cyan, orange, and violet lines, respectively. The conversion of dihydroconiferyl alcohol (5 mM) was carried out in the presence of EUGO10X (10 μM) and HRP (10 μM) in KPi (50 mM, pH 7.5) with DMSO (10%), 25 °C.

Figure 7

Effects of temperature and pH on conversion of dihydrosinapyl alcohol to syringaresinol by a EUGO10X-HRP cascade reaction. The substrate, dihydrosinapyl alcohol, and final product, syringaresinol, are displayed in cyan and violet, respectively. Conversions of dihydrosinapyl alcohol (5 mM) were carried out using EUGO10X (10 μM) and HRP (10 μM) in KPi buffer (50 mM, pH 7.5) with DMSO (10% v/v) at the indicated temperatures over (A) 3 and (B) 24 h. Conversions of dihydroconiferyl alcohol (5 mM) were carried out using EUGO10X (10 μM) and HRP (10 μM) in KPi buffer (50 mM, pH 6, 7 and 7.5) or Tris (pH 8 and 9) with DMSO (10%) at 25 °C over (C) 3 and (D) 24 h. The theoretical maximum yield of syringaresinol is 2.5 mM.

Figure 8

Effect of amount of HRP in the formation of syringaresinol in a EUGO10X-HRP cascade reaction starting from dihydrosinapyl alcohol. The one-pot conversions with no HRP (purple) and 10 μM HRP (blue), as well as a delayed addition of 10 μM HRP (orange) are shown.