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.

Investigation of the Amycolatopsis sp. Strain ATCC 39116 Vanillin Dehydrogenase and Its Impact on the Biotechnical Production of Vanillin

“…The bioconversion of ferulic acid into vanillin was demonstrated previously for various microorganisms, including bacteria like Pseudomonas and Rhodococcus (5)(6)(7)(8) and fungi such as the basidiomycete Pycnoporus cinnabarinus (9). Additionally, several genes encoding the responsible enzymes in some of these organisms were heterologously expressed in Escherichia coli and enabled the corresponding strains to convert ferulic acid into vanillin (10,11). However, high concentrations of vanillin are attended by serious problems for the cells due to the toxicity of this highly reactive aromatic aldehyde, which is detoxified in the course of further catabolism or leads to cell death.…”

Obtenção de vanilina: oportunidade biotecnológica

“…HR199, produzindo as enzimas álcool coniferílico desidrogenase e aldeído coniferílico desidrogenase. Na segunda etapa, o recombinante Escherichia coli (pSKechE/Hfcs) fez a conversão do ácido ferúlico em vanilina, com rendimento de 0,3 g/L 35 . Streptomyces setonii acumulou vanilina até uma concentração de 6,4 g/L e um rendimento molar de 68%.…”

Recovery and Utilization of Lignin Monomers as Part of the Biorefinery Approach

“…Chemicals produced from lignin-derived substrates or pure compounds known to be present in lignin are listed in Table 5. Vanillin can be produced by a number of specialized microorganisms from aromatic molecules such as eugenol [151][152][153][154][155][156][157], isoeugenol [157][158][159][160][161][162][163][164][165][166][167][168], ferulic acid [154,162,[169][170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186], vanillic acid [172,174], and green coconut husk [187]. However, the low titers of vanillin and degradation of vanillin by the microorganisms are problematic [107].…”