Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

Abstract

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.

FIG. 1.

Biotransformation of eugenol to vanillin.

FIG. 2.

Production of ferulic acid from eugenol by E. coli XL1-Blue(pSKvaomPcalAmcalB). Cells were grown in 50 ml of liquid TB medium in the presence of tetracycline, ampicillin, and IPTG overnight at 30°C. Portions (25 μl) of eugenol (final concentration, 0.05% [vol/vol]) were added to the culture hourly. Samples were taken, and ferulic acid concentrations were determined by HPLC. Data are means for three independent experiments. The variance is indicated by error bars. —, total amount of added eugenol; ♦, ferulic acid concentration.

FIG. 3.

Fed-batch fermentation of E. coli XL1-Blue(pSKvaomPcalAmcalB) on the 30-liter scale in TB medium. The cultivation was done in a 42-liter stirred-tank reactor, and the cultivation parameters were pH 7.2, aeration at 0.8 to 1.0 volume per volume per minute, and agitation at 200 to 400 rpm. Aeration and agitation were adjusted according to the oxygen demand of the culture. The temperature was decreased from 30 to 28°C at the beginning of the biotransformation process. Eugenol (100 ml) was added to the culture continuously over 2 h (↔), maintaining a final concentration of approximately 0.1% (vol/vol) in the medium. Subsequently, eugenol was added in pulses of 30 ml to the medium (↓).