Lipoxygenase-catalyzed production of unsaturated C
Described herein are methods for the lipoxygenase (LOX)-catalyzed production of aliphatic unsaturated C 10 -aldehyde compounds from polyunsaturated fatty acid (PUFA) sources, and the isolation and characterization of novel, preferably bifunctional LOXs from different algae sources and the identification of structurally and/or functionally related LOXs from different bacterial sources. Also described herein are the provision of enzyme mutants derived from the newly identified enzymes, and corresponding coding sequences of the enzymes, recombinant vectors, and recombinant host cells suitable for the production of such LOXs and for performing the novel production methods of aliphatic unsaturated C 10 -aldehyde compounds. Further describes herein is the use of particular aldehydes or aldehyde mixtures as a flavor ingredient or ingredient for food or feed compositions.
1 . A method for preparing at least one mono- or polyunsaturated aliphatic aldehyde, which method comprises
(1) contacting at least one polyunsaturated fatty acid (PUFA) substrate with a polypeptide
which comprises the enzymatic activity of a lipoxygenase, thereby converting said at least one PUFA substrate to a reaction product comprising at least one mono- or polyunsaturated aliphatic aldehyde; and
(2) isolating at least one mono- or polyunsaturated aliphatic aldehyde as obtained in step (1);
wherein said PUFA is selected from C 16 -C 22 ,
wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 3, 6, 9, 12 or 15,
b) SEQ ID NO: 18,
and
c) amino acid sequences having at least 90% sequence identity to at least one of the sequences of a) or b) and retaining said enzymatic activity of a lipoxygenase.
2 . The method of claim 1 , wherein the polypeptide comprises the enzymatic activity of a bifunctional lipoxygenase.
3 . The method of claim 1 , wherein said at least one mono- or polyunsaturated aliphatic aldehyde is a decadienal or a decatrienal, wherein said decadienal is selected from 2E,4E-decadienal and 2E,4Z-decadienal and mixtures thereof; and wherein said decatrienal is selected from 2E,4E, 7Z-decatrienal and 2E,4Z,7Z-decatrienal and mixtures thereof.
4 . The method of claim 1 , wherein step (1) is performed in vivo in cell culture in the presence of oxygen, or in vitro in a liquid reaction medium in the presence of oxygen.
5 . The method of claim 1 , wherein step (1) is carried out by cultivating a non-human host organism or cell expressing at least one of said polypeptides having the enzymatic activity of a lipoxygenase in the presence of a PUFA substrate under conditions conducive to the peroxidation and subsequent cleavage of at least one PUFA.
6 . The method of claim 1 , wherein said PUFA substrate is an isolated PUFA compound or a natural or synthetic composition comprising at least one PUFA convertible by said lipoxygenase.
7 . The method of claim 1 , which further comprises a chemical or enzymatic isomerization of an obtained mono- or polyunsaturated aliphatic aldehyde; or a chemical or enzymatic conversion of an obtained mono- or polyunsaturated aliphatic aldehyde to the corresponding alcohol or hydrocarbyl ester.
8 . The method of claim 1 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 3, 6, 9, 12 or 15;
b) SEQ ID NO: 18;
and
c) amino acid sequences having at least 95% sequence identity to at least one of the sequences of a) or b) and retaining said enzymatic activity of a lipoxygenase.