Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-05-03
2001-11-27
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
Reexamination Certificate
active
06323011
ABSTRACT:
The present invention relates principally to the production of vanillin (4-hydroxy-3-methoxybenzaldehyde), particularly to the production of vanillin other than by extraction from the Vanilla pod.
Vanillin is an important food and drink flavouring agent and a major flavour component of natural vanilla from the Vanilla pod. The use of natural vanilla is limited by its high price. Synthetic vanillin, commonly derived from sulphite liquors produced during the processing of wood pulp for paper manufacture, is frequently used as a low-cost vanilla substitute. Alternative biological processes for the production of natural vanillin and allied flavourings would have considerable industrial value and utility, most particularly if such processes could facilitate the production of vanillin and/or allied flavourings directly in a fermented food or beverage.
The mechanism of vanillin biosynthesis in Vanilla remains substantially uncharacterised. M. H. Zenk (
Anal. Z. Pflanzenphysiol
53, 404-414 (1965)) showed that vanillin was derived from trans-ferulate (4-hydroxy-3-methoxy-trans-cinnamate) and proposed a mechanism analogous to the classical &bgr;-oxidation of fatty acids, with cleavage of a &bgr;-keto thioester to produce acetyl SCoA and vanilloyl SCoA (4-hydroxy-3-methoxybenzoyl SCoA) and subsequent reduction and CoASH release to generate vanillin. C. Funk and P. E. Brodelius (
Plant Physiol.
94, 95-101; 102-108 (1990); 99, 256-262 (1992)), proposed a different route, in which the 4-hydroxy group of trans-ferulate became successively methylated and demethylated during the pathway of vanillin biosynthesis; however, the detailed enzymology was not elucidated. In potato tubers and in the fungus,
Polyporus hispidus
(C. J. French, C. P. Vance and G. H. N. Towers,
Phytochemistry
15, 564-566 (1976)), in cell cultures of
Lithospermum erythrorhizon
(K. Yazaki, L. Heide and M. Tabata,
Phytochemistry
30, 2233-2236 (1991)) and in cell cultures of carrot (J.-P. Schnitzler, J. Madlung, A. Rose and H. U. Seitz,
Planta
188, 594-600 (1992)), evidence was obtained from in vitro studies that the corresponding analogue of vanillin, 4-hydroxybenzaldehyde, was an intermediate in the formation of 4-hydroxybenzoate from 4-coumarate (4-hydroxy-trans-cinnamate). There was no requirement for ATP or CoASH, thus apparently ruling out a &bgr;-oxidation mechanism. Further studies with cell-free extracts of
Lithospermum erythrorhizon,
however, have in contrast recently established the presence of a &bgr;-oxidation route for the conversion of 4-coumarate to 4-hydroxybenzoate (R. Löscher and L. Heide,
Plant Physiol.
106, 271-279 (1994)); in this case, the conversion was dependent on ATP, Mg
2+
ions and NAD
+
and proceeded via 4-hydroxybenzoyl SCoA, without the intermediate formation of 4-hydroxybenzaldehyde.
In the Gram-negative bacterium,
Pseudomonas acidovorans,
trans-ferulate was shown to be catabolised to vanillate and acetate, apparently via vanillin (A. Toms and J. M. Wood,
Biochemistry
9, 337-343 (1970)). Although in cell-free extracts NAD
+
was necessary for the oxidation of vanillin to vanillate and for the further oxidation of vanillate to protocatechuate and formate, no mention was made of any other cofactor requirements. Further studies of ferulate utilisation in Pseudomonas species have been reported (V. Andreoni and G. Bestetti,
FEMS Microbiology Ecology
53, 129-132 (1988); T. Omori, K. Hatakeyama and T. Kodama,
Appl. Microbiol. Biotechnol.
29, 497-500 (1988); Z. Huang, L. Dostal and J. P. N. Rosazza,
Appl. Env. Microbiol.
59, 2244-2250 (1993)); however, these have not sought to elucidate further the mechanism of the two-carbon cleavage of ferulate. Zenk et al (1980)
Anal. Biochem.
101, 182-187 describe a procedure for the enzymatic synthesis and isolation of cinnamoyl-CoA thioesters using a bacterial system. In contrast, the enzymology and genetics of the utilisation of simple benzene derivatives, including benzoic acids and phenols, by Pseudomonas have been intensively studied (T. K. Kirk, T. Higuchi and H.-M. Chang (eds.), “
Lignin biodegradation
”, CRC Press, Boca Raton, Fla., USA (1980); D. T. Gibson (ed.), “
Microbial degradation of organic compounds
”, Marcel Dekker, New York (1984); J. L. Ramos, A. Wasserfallen, K. Rose and K. N. Timmis,
Science
235, 593-596 (1987); C. S. Harwood, N. N. Nichols, M. K. Kim, J. L. Ditty and R. E. Parales,
J. Bacteriol.
176, 6479-6488 (1994); S. Romerosteiner, R. E. Parales, C. S. Harwood and J. E. Houghton,
J. Bacteriol.
176, 5771-5779 (1994); J. Inoue, J. P. Shaw, M. Rekik and S. Harayama,
J. Bacteriol.
177, 1196-1201 (1995; )).
A survey of potential microbial routes to aromatic aldehydes, including routes (i) from trans-cinnamic acids, (ii) from benzoic acids by reduction and (iii) by conversion of aromatic amino acids to phenylpyruvic acids followed by treatment with base, has been presented by J. Casey and R. Dobb (
Enzyme Microb. Technol.
14, 739-747 (1992)).
U.S. Pat. No. 5,128,253 describes a method of producing vanillin from ferulic acid by various microorganisms and extracts thereof or enzymes derived therefrom in the presence of a sulphydryl compound but does not disclose what any of the enzymes involved in the conversion of ferulic acid to vanillin are. U.S. Pat. No. 5,279,950 is a continuation-in-part application of U.S. Pat. No. 5,128 253 which additionally describes that Vanilla calluses can be used in the process.
WO 94/13614 describes the production of vanillin from ferulic acid by the action of Vanilla root material and makes use of an adsorbent, such as charcoal, to extract vanillin but does not disclose the specific enzymes involved.
EP 0 453 368 describes that a culture of Pycnoporus can convert trans-ferulic acid into vanillin but does not disclose the specific enzymes involved.
WO 94/02621 describes the production of vanillin from trans-ferulic acid by the action of a lipoxygenase enzyme. EP 0 405 197 describes the production of vanillin from eugenol/isoeugenol by bacteria from the genera Serratia, Klebsiella and Enterobacter by oxidation.
Vanillin may also be produced from phenolic stilbenes as is mentioned in Hagedorn & Kaphammer (1994)
Ann. Rev. Microbiol.
48, 773-800.
Vanillic acid is also a useful compound as it can be polymerised into oligomers or used as a monomer in the synthesis of polyesters; similarly p-hydroxybenzoic acid is also useful for polymer synthesis.
A first aspect of the invention provides a method of producing vanillin comprising the steps of
(1) providing trans-ferulic acid or a salt thereof; and
(2) providing trans-ferulate:CoASH ligase activity (enzyme activity I), trans-feruloyl SCoA hydratase activity (enzyme activity II), and 4-hydroxy-3-methoxyphenyl-&bgr;-hydroxy-propionyl SCoA (HMPHP SCoA) cleavage activity (enzyme activity III).
The advantages of the present invention over chemical synthesis or extraction from the Vanilla pod include (i) economic advantage over extraction from Vanilla pod and freedom from geographical dependence on Vanilla growing areas; (ii) the ability to produce vanillin by a natural process, involving biological catalysts; (iii) the benefits of generating a natural flavour in situ in a fermented food or beverage, if the genes are expressed in appropriate food-grade hosts—eg lactic acid bacteria or yeasts; and (iv) the possibility of expanding the range of plants in which vanillin and related substances might be produced and from which they might be extracted. These and other examples of the methods of the invention are described in more detail below.
We have determined the mechanism of chain-shortening of trans-ferulate (trans-ferulic acid) by a strain of
Pseudomonas fluorescens
(named
Ps. fluorescens
biovar. V, strain AN103 and which we have abbreviated at some points to AN103) isolated from soil. Our data indicate clearly that vanillin (4-hydroxy-3-methoxy benzaldehyde) is an intermediate and that the mechanism does not involve &bgr;-oxidation. The vanillin pathway of
Ps. fluorescens
biovar. V, strain AN103 is described in FIG.
Gasson Michael John
Narbad Arjan
Rhodes Michael John Charles
Walton Nicholas John
Institute of Food Research
Lilling Herbert J.
Nixon & Peabody LLP
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