Bioflavours Natural Vanillin - Bioprocesses Prof. Dr. Jürgen Rabenhorst A collaborative MOOC-Project by: part of the
Characterization of Vanillin • Flavouring profile – Vanilla like, – Sweet and creamy, – Chocolate like
Characterization of Vanillin • Flavouring profile – Vanilla like, – Sweet and creamy, – Chocolate like • Taste qualities – Vanilla like, – Sweet, – Creamy and phenolic
Main Applications of Vanillin • Ice cream • Chocolates • Bakery and dairy products • Sweets and beverages
Chemical Synthesis of Vanillin • Mainly two production processes 1. from Guaiacol 2. from Lignin
Chemical Synthesis of Vanillin • Mainly two production processes 1. from Guaiacol 2. from Lignin • In the past: Eugenol as a starting material
Structure of Vanillin H 3 CO CHO H O
De novo Synthesis from Glucose • Li and Frost (1998) [K. Li, J. W. Frost; J. Am. Chem. Soc. (1998); 120 (40), pp 10545–10546; DOI: 10.1021/ja9817747 ]
De novo Synthesis from Glucose • Li and Frost (1998) – recombinant E. coli strain – Fed‐batch fermentation – 5 g/L Vanillic acid after 48 h [K. Li, J. W. Frost; J. Am. Chem. Soc. (1998); 120 (40), pp 10545–10546; DOI: 10.1021/ja9817747 ]
De novo Synthesis from Glucose • Li and Frost (1998) – recombinant E. coli strain – Fed‐batch fermentation – 5 g/L Vanillic acid after 48 h – Reduced by aldehyde dehydrogenase (Neurospora crassa ) – 92 % conversion rate [K. Li, J. W. Frost; J. Am. Chem. Soc. (1998); 120 (40), pp 10545–10546; DOI: 10.1021/ja9817747 ]
Vanillin synthesis from glucose • Hansen et al. (2009) – Saccharomyces cervisiae and Schizosaccharomyces pombe [E. H. Hansen, B. Lindberg Møller, G. R. Kock, C. M. Bünner, C. Kristensen, O. R. Jensen, F. T. Okkels, C. E. Olsen, M. S. Motawia, J. Hansen; Appl. Environ. Microbiol. (2009); 75 (9); p. 2765–2774; DOI: 10.1128/AEM.02681-08 ]
Vanillin synthesis from glucose • Hansen et al. (2009) – Saccharomyces cervisiae and Schizosaccharomyces pombe – introduce gene 3‐dehydroshikimate dehydratase (3DSD) Podospora pauciseta • from 3‐dehydroshikimate to protocatechuic acid [E. H. Hansen, B. Lindberg Møller, G. R. Kock, C. M. Bünner, C. Kristensen, O. R. Jensen, F. T. Okkels, C. E. Olsen, M. S. Motawia, J. Hansen; Appl. Environ. Microbiol. (2009); 75 (9); p. 2765–2774; DOI: 10.1128/AEM.02681-08 ]
Vanillin synthesis from glucose • Hansen et al. (2009) – Saccharomyces cervisiae and Schizosaccharomyces pombe – introduce gene 3‐dehydroshikimate dehydratase (3DSD) Podospora pauciseta • from 3‐dehydroshikimate to protocatechuic acid – O‐methyltransferase from Homo sapiens – reduction by aromatic carboxylic acid reductase (ACAR) from Nocardia iowensis [E. H. Hansen, B. Lindberg Møller, G. R. Kock, C. M. Bünner, C. Kristensen, O. R. Jensen, F. T. Okkels, C. E. Olsen, M. S. Motawia, J. Hansen; Appl. Environ. Microbiol. (2009); 75 (9); p. 2765–2774; DOI: 10.1128/AEM.02681-08 ]
Vanillin production by Biotransformation • Manifold opportunities • Numerous natural aromatic precursors (Curcumin, Eugenol, Ferulic acid, Guaiacol, Isoeugenol, Lignin …)
From Curcumin to Vanillin • Dolfini et al. (1990): Chemical Hydrolysis of Curcumin to Vanillin [J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A ]
From Curcumin to Vanillin • Dolfini et al. (1990): Chemical Hydrolysis of Curcumin to Vanillin [J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A ] H 3 CO CHO 2 H O
From Curcumin to Vanillin • Dolfini et al. (1990): Chemical Hydrolysis of Curcumin to Vanillin [J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A ] • Bharti & Gupta (2011): 3,56 mg L ‐1 Vanillin Conversion of Curcumin with Rhodococcus rhodochrous [ C. Bharti, A. Nagpure, R. K. Gupta; Journal of Chemistry (2011); 50B, p. 1119-1122 ]
From Curcumin to Vanillin • Dolfini et al. (1990): Chemical Hydrolysis of Curcumin to Vanillin [J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A ] • Bharti & Gupta (2011): Conversion of Curcumin with Rhodococcus rhodochrous [C. Bharti, A. Nagpure, R. K. Gupta; Journal of Chemistry (2011); 50B, p. 1119-1122] • Group of Berger (2015): isolated enzymes [V. Esparan, U. Krings, M. Struch, RG. Berger; Molecules (2015); 20(4); p. 6640-6653; DOI: 10.3390/molecules20046640. ]
From Isoeugenol to Vanillin • one of the first precursors • Disclosed in 1991 • Serratia marcescens 3,8 g/L vanillin after 9 days Rabenhorst J, Hopp R (1991) EP 405197
From Eugenol to Vanillin • essential oil of the clove tree, Syzygium aromaticum • Metabolized by Pseudomonas sp. HR 199 Hopp R, Rabenhorst J (1992) EP 583687
From Eugenol to Vanillin Coniferyl aldehyde Ferulic acid Coniferyl alcohol O Eugenol CH CH OH Eugenol Coniferyl alcohol Coniferyl aldehyde hydroxylase dehydrogenase dehydrogenase Feruloyl-CoA- OH synthetase Vanillin Feruloyl-CoA Protocatechuic acid O Vanillic acid CHO CH CH COOH COOH S-CoA Enoyl-CoA- Vanillin Vanillate - O - hydratase/lyase dehydrogenase demethylase OH OH Acetyl-CoA OH OH [H. Priefert, J.Rabenhorst, A. Steinbüchel; J. Bacteriol (1997); 179, 2595 -2607]
Challenges and Difficulties • Vanillin dehydrogenase is very active – not possible to detect trace amounts of vanillin in the wild type strain.
Challenges and Difficulties • Vanillin dehydrogenase is very active – not possible to detect trace amounts of vanillin in the wild type strain. • Constructing a deletion mutant of the vdh‐gene – Vanillin production is possible
Challenges and Difficulties • Vanillin dehydrogenase is very active – not possible to detect trace amounts of vanillin in the wild type strain. • Constructing a deletion mutant of the vdh‐gene – Vanillin production is possible – In flask scale: • concentration very low due to limited concentration of eugenol which can be added
Challenges and Difficulties • Fed‐batch fermentation process: – Significant increase of concentration. – But coniferyl aldehyde dehydrogenase has also a vdh activity
Vanillin from Ferulic acid • The commercially successful process based on: – Microbial biotransformation of ferulic acid. • phenolic phytochemical • mainly found in plant cell walls • esterified with polysaccharides, flavonoids, hydroxycarboxylic acids, and long chain alcohols
Vanillin from Ferulic acid • Several groups studied a number of different organisms – Vanillin as an intermediate in the degradation of ferulic acid
Vanillin from Ferulic acid • Several groups studied a number of different organisms – Vanillin as an intermediate in the degradation of ferulic acid • Haarmann & Reimer: Amycolatopsis sp. HR 167 ( 11,5 g/L in 1,5 days) Ferulic acid Feruloyl-CoA Vanillin O O CHO CH CH C CH CH C OH S-CoA Feruloyl-CoA- Enoyl-CoA- synthetase hydratase/lyase fcs ech OCH 3 OCH 3 OCH 3 OH OH Acetyl-CoA OH
Vanillin from Ferulic acid • Steinbüchel – deletion the Vanillin dehydrogenase gene Ferulic acid Feruloyl-CoA Vanillin Vanillic acid Vanillin- Feruloyl-CoA- Enoyl-CoA- dehydrogenase synthetase hydratase/lyase fcs ech Acetyl-CoA – constitutive expression of ech and fcs – concentration: 19.3 g/liter; molar yield: 94.9%
Vanillin from Ferulic acid • Steinbüchel – deletion the Vanillin dehydrogenase gene and constitutive expression of ech and fcs • concentration: 19.3 g/liter; molar yield: 94.9% – improved feeding strategy • concentration: 22.3 g/liter; lower molar yield
Vanillin from Ferulic acid • Zheng et al (2005): fungal strains – 1. Aspergillus niger produced vanillic acid – 2. reduction to vanillin by Pycnoporus cinnabarinus. – concentration: 2,2 g/L of vanillic acid and 2,8 g/L vanillin
A collaborative MOOC-Project by: part of the
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