Bioflavours Natural Vanillin - Bioprocesses Prof. Dr. Jrgen - - PowerPoint PPT Presentation

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

O H H3CO CHO

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]

O H H3CO CHO

2

From Curcumin to Vanillin

• Dolfini et al. (1990):

Chemical Hydrolysis of Curcumin to Vanillin

• Bharti & Gupta (2011): 3,56 mg L‐1 Vanillin

Conversion of Curcumin with Rhodococcus rhodochrous

[J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A] [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

• Bharti & Gupta (2011):

Conversion of Curcumin with Rhodococcus rhodochrous

• Group of Berger (2015):

isolated enzymes

[J. E. Dolfini, J. Glinka, A. C. Bosch; US4927805A] [C. Bharti, A. Nagpure, R. K. Gupta; Journal of Chemistry (2011); 50B, p. 1119-1122] [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

Eugenol hydroxylase Coniferyl alcohol dehydrogenase OH OH COOH OH COOH CH CH O OH S-CoA OH CH CH O Feruloyl-CoA- synthetase Ferulic acid Feruloyl-CoA OH Acetyl-CoA CHO Enoyl-CoA- hydratase/lyase Vanillin Coniferyl aldehyde dehydrogenase Eugenol Coniferyl alcohol Coniferyl aldehyde Vanillin dehydrogenase Vanillic acid Protocatechuic acid Vanillate - O - demethylase

[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)

C C

ech

OCH3 OH CH CH O OH S-CoA OCH3 OH CH CH O

Feruloyl-CoA- synthetase fcs

Ferulic acid Feruloyl-CoA

OCH3 OH Acetyl-CoA CHO

Enoyl-CoA- hydratase/lyase

Vanillin

• Steinbüchel

– deletion the Vanillin dehydrogenase gene – constitutive expression of ech and fcs – concentration: 19.3 g/liter; molar yield: 94.9%

Vanillin from Ferulic acid

Feruloyl-CoA- synthetase Enoyl-CoA- hydratase/lyase Vanillin- dehydrogenase

fcs

Ferulic acid Feruloyl-CoA Vanillin

ech

Acetyl-CoA Vanillic 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

Vanillin from Ferulic acid

A collaborative MOOC-Project by: part of the