EP4654815A1

EP4654815A1 - Unsaturated aldehydes via rice biotransformation

Info

Publication number: EP4654815A1
Application number: EP24702334.4A
Authority: EP
Inventors: Torsten Geißler; Holger Zorn; Jean-Philippe KANTER; Jakob Peter Ley; Lars MILKE; Christoph Harms; Esther-Corinna SCHWARZE; Egon Gross; Marco Alexander Fraatz
Original assignee: Symrise AG
Current assignee: Symrise AG
Priority date: 2023-01-27
Filing date: 2024-01-26
Publication date: 2025-12-03
Also published as: WO2024156360A1; WO2024156880A1

Abstract

The present invention relates to a method for producing α-dioxygenase from rice seed un- der particular conditions, to a mixture comprising a purified α-dioxygenase obtainable or obtained by such a method, to the use of such a mixture for transforming a fatty acid and/or a mixture of fatty acids to the corresponding fatty aldehyde and/or a mixture of the corresponding fatty aldehydes and to the use of rice seed for transforming a fatty acid to the corresponding fatty aldehyde.

Description

Unsaturated aldehydes via rice biotransformation

The present invention relates to a method for producing a-dioxygenase from rice seed under particular conditions, to a mixture comprising a purified a-dioxygenase obtainable or obtained by such a method, to the use of such a mixture for transforming a fatty acid and/or a mixture of fatty acids to the corresponding fatty aldehyde and/or a mixture of the corre- sponding fatty aldehydes and to the use of rice seed or mixtures thereof, for transforming a fatty acid to the corresponding fatty aldehyde.

Fatty aldehydes are a component of many natural ingredients such as the essential oils of various citrus fruits. For example, decanal, octanal and citral are a component of orange peel and other citrus fruits. Fatty aldehydes are frequently used as fragrances and flavour- ings. For example, decanal has a sweet, flowery odor, which imparts an orange peel odor, is used, among other things, as a flavoring agent in the food industry and as a perfume in the perfume industry.

Fatty aldehydes are frequently obtained by reaction of fatty acids to fatty aldehydes. One possibility for this reaction is the biotransformation via a-dioxygenase: fatty acid (n) 2-hydroperoxy fatty acid (n) aldehyde (n-1 ) In such methods, large amounts of a-dioxygenases are needed to produce fatty aldehydes on industrial scale, such that these may be further used or processed by the food or perfume industry.

Methods for heterologous expression of a-dioxygenase encoding genes are already known. In these methods, genetically modified organisms (GMOs) are used. However, for several applications, a GMO strain shall or must not be used. Frequently, many consumers have concerns or reject products obtained from or with the participation of GMOs.

Thus, it is preferred to produce fatty aldehydes without utilization of GMOs for these cases and applications.

Genes encoding an a-dioxygenase have been found in some organisms, such as rice. Rice is a plant, which is generally suitable for cultivating and producing enzymes on industrial scale. However, if at all - or under which conditions - the respective genes are expressed in rice has not been reported yet. The mere presence of a gene in the genome of an organism does not guarantee that the gene is expressed. For example, the expression of a gene may depend on the developmental stage of an organism or on the presence of particular substances in the organism’s surrounding.

Furthermore, how to achieve an expression satisfying the needs of producing a-dioxygen- ases on industrial scale has not been described. Many factors may influence the expression levels of a gene, if expressed at all.

Thus, a primary object of the present invention was to produce the endogenous a-dioxy- genase from rice, preferably such that high levels of a-dioxygenase are obtained for applying the method on industrial scale.

The primary object of the present invention is solved by a method for producing a-dioxy- genase, comprising the steps i) providing rice seed, ii) cultivating the seed provided in step i) on a medium base, preferably agar, for at least 7 days, to allow germination wherein for at least 7 days, the cultivation is substantially in the absence of light, and wherein the concentration of CuSC in the medium base is at most 25 mM, preferably at most 20 mM, preferably at most 15 mM, preferably at most 10 mM, preferably at most 7.5 mM, based on the total medium base, and/or wherein the concentration of NaCI in the medium base is at most 500 mM, preferably at most 450 mM, preferably at most 400 mM, preferably at most 350 mM, preferably at most 300 mM, preferably at most 250 mM, preferably at most 200 mM, based on the total medium base, and/or iii) optionally: purifying the produced a-dioxygenase.

It was surprisingly found that with the method according to the invention, the a-dioxygenase gene was expressed on a high level and a-dioxygenase was obtained.

Preferably, the term “a-dioxygenase”, as used herein, refers to an a-dioxygenase encoded in the rice genome. Particularly preferably, the term refers to an a-dioxygenase encoded by a gene having a nucleic acid sequence according to SEQ ID NO:1 or a nucleic acid sequence have at least 90%, preferably at least 91 %, preferably at least 92 %, preferably at least 93 %, preferably at least 94 %, preferably at least 95 %, preferably at least 96 %, preferably at least 97 %, preferably at least 98 %, preferably at least 99 % sequence identity to the sequence according to SEQ ID NO:1 . Preferably, the term refers to an a-dioxygenase having an amino acid sequence according to SEQ ID NO:2 or an amino acid sequence have at least 90%, preferably at least 91 %, preferably at least 92 %, preferably at least 93 %, preferably at least 94 %, preferably at least 95 %, preferably at least 96 %, preferably at least 97 %, preferably at least 98 %, preferably at least 99 % sequence identity to the sequence according to SEQ ID NO:2.

Preferably, whenever the present disclosure refers to sequence identities of nucleic acid or amino acid sequences in terms of percentages, such references are to values as can be calculated using EMBOSS Water Pairwise Sequence Alignments (nucleotides) (http://www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html) for nucleic acid sequences and EMBOSS Water Pairwise Sequence Alignments (proteins) (http://www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences, respectively. Local sequence alignment tools provided by the European Molecular Biology Laboratory (EMBL) European Bioninformatics Institute (EBI) use a modified Smith-Waterman algorithm (see http://www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195- 197). Furthermore, here, when performing the respective pairwise alignment of two sequences using the modified Smith-Waterman algorithm, reference is made to the default parameters currently given by EMBL-EBI. These are (i) for amino acid sequences: Matrix = BLOSUM62, Gap open penalty = 10 and Gap extend penalty = 0.5 and (ii) for nucleic acid sequences: Matrix = DNAfull, Gap open penalty = 10 and Gap extend penalty = 0.5.

The term “medium base”, as used herein, describes a base, which is suitable to receive seed and which allows cultivating the received seed. Preferably, the medium base is solid, semisolid or liquid. Preferably, the medium base is solid. Preferably, the medium base is semisolid. Preferably, the medium base is liquid.

The term “cultivating”, as used herein, describes exposing of the seed to conditions under which they grow and develop more biomass. Preferably, the cultivation is performed at daily average temperatures of from 15°C to 35°C, preferably of from 20°C to 30°C, further preferably of from 25°C to 30 °C, even further preferably of from 27°C to 29°C. Preferably, the cultivation is performed at daily average relative humidity of from 70 to 95 %, preferably of from 75 to 92.5 %, preferably of from 80 to 90 %.

Typically, rice seed is cultivated with light. However, it has been surprisingly found that if against the common procedure, the rice seed cultivation is performed in substantial absence of light, the germination and growth of the seeds as well as the expression of the a- dioxygenase was advantageously increased.

It is preferred that the whole or substantially the whole step ii) of the method according to the invention is performed substantially in the absence of light.

The term “substantially in the absence of light”, as used herein, shall be understood such that short times of light are included, e.g. for allowing the general maintenance, examination and handling of the seeds I plants. However, it is preferred that the absence of light is applied for at least 65 %, preferably at least 70 %, preferably at least 75 %, preferably at least 80 %, preferably at least 85 %, preferably at least 90 %, preferably at least 95 %, preferably at least 96 %, preferably at least 97%, preferably at least 98 %, preferably at least 99% of the time.

Furthermore, the term “absence of light” as used herein describes a brightness of at most 100 lux, preferably at most 75 lux, preferably at most 50 lux, preferably at most 25 lux, preferably at most 10 lux, preferably at most 9 lux, preferably at most 8 lux, preferably at most 7 lux, preferably at most 6 lux, preferably at most 5 lux, preferably at most 4 lux, preferably at most 3 lux, preferably at most 2 lux, preferably at most 1 lux.

Typically for providing absence of light, the seeds / plants are kept in a separate room, in which the light can be controlled, e.g. a room providing no daylight but only artificial light, if necessary.

Furthermore, it was surprisingly found that CuSC and NaCI had a negative impact on the germination and growth of the seeds as well as on the expression of the a-dioxygenase gene. Thus, it is particularly advantageous if the concentrations of CuSC and NaCI do not exceed the concentrations described above.

The term “concentration in the medium base”, as used herein, preferably refers to the medium base before receiving the seed.

Typically, the expression of the a-dioxygenase gene may be measured by RT-PCR and/or by measuring the protein levels of a-dioxygenase, each according to standard methods.

It was surprisingly found that the amounts of a-dioxygenase in the seedlings was particularly high starting from day 7 of the cultivation in step ii), with continuously increasing amounts. Furthermore, it was surprisingly found that a particularly strong increase was observed after 14 days, which continued with each further day.

It is thus preferred that the cultivation in step ii) is performed for at least 8 days, preferably for at least 9 days, preferably for at least 10 days, preferably for at least 11 days, preferably for at least 15 days, preferably for at least 18 days, preferably for at least 20 days, preferably for at least 21 days, preferably for at least 22 days, preferably for at least 23 days, preferably for at least 24 days, preferably for at least 25 days, preferably for at least 26 days, preferably for at least 27days, preferably for at least 28 days, preferably for at least

29 days, preferably for at least 30 days. Particularly preferably, the cultivation in step ii) is performed for at least 25 days, particularly preferably for a time in the range of from 25 to

30 days.

Preferably, for at least 8 days, preferably for at least 9 days, preferably for at least 10 days, preferably for at least 11 days, preferably for at least 15 days, preferably for at least 18 days, preferably for at least 20 days, preferably for at least 21 days, preferably for at least 22 days, preferably for at least 23 days, preferably for at least 24 days, preferably for at least 25 days, preferably for at least 26 days, preferably for at least 27days, preferably for at least 28 days, preferably for at least 29 days, preferably for at least 30 days, particularly preferably for a time in the range of from 25 to 30 days, the cultivation is substantially in the absence of light, as described herein.

It is further preferred that the cultivation in step ii) is performed for a time in the range of from 20 to 30 days, preferably in the range of from 22 to 27 days, preferably in the range of from 24 to 26 days, particularly preferably in the range of from 25 to 30 days. It is further preferred that the cultivation is substantially in the absence of light, as described herein.

Moreover, it was surprisingly found that a medium base with only a low concentration of supplements and/or nutrients or with no supplements particularly increased the germination and growth of the seeds as well as the expression of the a-dioxygenase. However, the presence of ZnSC slightly increased the expression of the a-dioxygenase.

It is thus preferred that the medium base is or comprises a medium comprising calcium chloride, preferably is or comprises agar medium comprising calcium chloride (calcium chloride agar) or Murashige and Skoog medium, preferably a mixture of Murashige and Skoog medium and agar.

Murashige and Skoog medium is well known to a skilled person and is described in Murashige, T; Skoog, F (1962). "Xt Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures". Physiologia Plantarum. 15 (3): 473-497.

Preferably, the medium base, particularly the Murashige and Skoog medium, comprises one, two, three, four or all of the substances selected from the group consisting of ammonium nitrate, calcium chloride, magnesium sulfate, monopotassium phosphate and potassium nitrate.

Preferably, the concentration of ammonium nitrate (NH4NO3) is in a range of from 1 ,600 to 1 ,700 mg/l, based on the volume of the medium base.

Preferably the concentration of calcium chloride (CaCh) is in a range of from 400 to 500 mg/l, based on the volume of the medium base.

Preferably the concentration of magnesium sulfate (MgSC ) is in a range of from 300 to 400 mg/l, based on the volume of the medium base. Preferably the concentration of monopotassium phosphate (KH2PO4) is in a range of from 100 to 200 mg/l, based on the volume of the medium base.

Preferably the concentration of potassium nitrate (KNO3) is in a range of from 1 ,600 to 1 ,700 mg/l, based on the volume of the medium base.

Furthermore, it is preferred that the medium base comprises ZnSCM, preferably wherein the medium base comprises at least 0.5 mM ZnSCM, preferably at least 1 mM ZnSCM, preferably at least 2 mM ZnSC>4, preferably at least 3 mM ZnSC>4, preferably at least 4 mM ZnSCM, preferably at least 5 mM ZnSCM, preferably at least 6 mM ZnSCM, preferably at least 7 mM ZnSCM, preferably at least 8 mM ZnSCM, preferably at least 9 mM ZnSCM, preferably at least 10 mM ZnSCM.

Furthermore, it is preferred that the medium base comprises CaCh, preferably wherein the medium base comprises CaCh in a range of from 0.2 to 12.5 mM, preferably in a range of from 0.5 to 10 mM, preferably in a range of from 0.75 to 7.5 mM, preferably in a range of from 1 to 5 mM, preferably in a range of from 1.25 to 4 mM, preferably in a range of from 1 .5 to 3 mM, particularly preferably wherein the medium base is agar comprising CaCh in a range of from 0.2 to 12.5 mM, preferably in a range of from 0.5 to 10 mM, preferably in a range of from 0.75 to 7.5 mM, preferably in a range of from 1 to 5 mM, preferably in a range of from 1 .25 to 4 mM, preferably in a range of from 1 .5 to 3 mM.

Preferably, the term “agar comprising CaCh” describes a mixture of agar (a galactose polymer), which was dissolved in water, and CaCh.

It was surprisingly found that particularly high amounts of a-dioxygenase were obtained in the shoots or roots of the seedlings.

It is thus preferred that the method according to the invention further comprises the steps ii.1) separating the shoots from the seedling, preferably separating the shoots and the roots from the seedling, and/or ii.2) homogenizing the shoots separated in step ii.b) or, respectively, the shoots and roots separated in step ii.b).

The numbering ii.1) and ii.2) indicates that these steps, if present, are performed in addition to step ii) as described herein. Preferably, step ii.1) is present. Preferably, step ii.2) is present. Preferably, steps ii.1) and ii.2) are present.

Furthermore, it is preferred that step iii) is present in the method according to the invention and wherein the purification is or includes an extraction.

The term “purification”, as used herein, includes any method for physical separation of a chemical substance of interest from foreign or contaminating substances. The term includes but is not restricted to an isolation of said chemical substance of interest. Methods for purification include centrifugation, chromatography such as column chromatography, extraction with a particular solvent or mixture of solvents, filtration, evaporation, distillation or crystallization as well as any other methods known to a skilled person.

The term “extraction”, as used herein, describes a purification using a solvent (or a mixture of solvents) for separating a chemical substance of interest from foreign or contaminating substances.

Furthermore, it is preferred that step iii) is present in the method according to the invention and wherein the purification includes a centrifugation step, preferably a centrifugation step in the range of from 2.500 x g to 25.000 x g, preferably in the range of from 2.750 x g to 22.500 x g, preferably in the range of from 3.000 x g to 20.000 x g, preferably in the range of from 3.250 x g to 17.500 x g, preferably in the range of from 3.500 x g to 15.000 x g, preferably in the range of from 3.750 x g to 12.500 x g, preferably in the range of from 4.000 x g to 10.000 x g, preferably in the range of from 4.250 x g to 7.500 x g, preferably in the range of from 4.500 x g to 5.000 x g.

Moreover, it is preferred in the method according to the invention, that step iii) is present, and the method further comprises the step iv) contacting the purified a-dioxygenase with a fatty acid to transform the fatty acid to the corresponding fatty aldehyde. Typically, the transformation of the fatty acid to the corresponding fatty aldehyde is achieved by the following reaction: fatty acid (n) 2-hydroperoxy fatty acid (n) aldehyde (n-1 )

It is thus preferred that the term “corresponding fatty aldehyde” describes the fatty aldehyde, which has a chain length shorter by 1 carbon atom (i.e. n-1) than the fatty acid, which was transformed (i.e. n).

It is preferred that the, one, two, three or more or all fatty acids have a carbon chain length (n) in the range of from 3 to 30 carbon atoms, preferably 11 to 23 carbon atoms.

It is preferred that the, one, two, three or more or all fatty acids are selected from the group consisting of saturated fatty acids with a carbon chain length (n) in the range of from 10 to 22, preferably 12 to 20, preferably wherein n is an even number, and unsaturated fatty acids with a carbon chain length in the range of from 10 to 22, preferably 12 to 20, preferably wherein n is an even number.

It is preferred that the, one, two, three or more or all fatty acids are selected from the group consisting of branched fatty acids, preferably with a carbon chain length (n) in the range of from 10 to 22, preferably 12 to 20, preferably wherein the term “carbon chain length” includes all branches of the branched carbon chain.

It is preferred that the, one, two, three or more or all fatty acids are selected from the group consisting of 11 -Methyltridecanoic acid, 11-Methyldodecanoic acid, 11-Dodecenoic acid, 11Z-Octadecenoic acid, 11Z-Hexadecenoic acid, 12-Methyltridecanoic acid, 12-Methyl- tetradecanoic acid, 12Z-eicosaenoic acid, 13-Methylpentadecanoic acid, 13-Methyltetra- decanoic acid, 13Z-Eicosaenoic acid, 14-Methylhexadecanoic acid, 14-Methylpentadeca- noic acid, 14Z-Eicosaenoic acid, 6Z,9Z,12Z-Octadecatrienoic acid (gamma-linolenic acid), 5Z,8Z,11Z,14Z,17Z-Eicosapentaenoic acid (EPA), 4Z,7Z,10Z,13Z,16Z,19Z-Docosahex- aenoic acid (DHA), 5Z,8Z,11Z,14Z-Eicosatetraenoic acid (arachidonic acid), 5Z-Dode- cenoic acid, 6Z,9Z-Pentadecadienoic acid, 6Z-Octadecenoic acid (petroselinic acid), 7E,10E-Hexadecadienoic acid, 7Z,10Z,13Z-Octadecatrienoic acid, 6Z,9Z,12Z,15Z-Octa- decatetraenoic acid (stearidonic acid), 7Z,10Z-Hexadecadienoic acid, 8Z,11Z,14Z-Hepta- decatrienoic acid, 8Z,11Z-Heptadecadienoic acid, 8Z,11Z-Pentadecadienoic acid, 8Z,10E,12E-Heptadecatrienoic acid, 8Z,10E,12Z-Heptadecatrienoic acid, 8Z,10E-Hepta- decadienoic acid, 8Z-Tridecenoic acid, 8Z-heptadecenoic acid, 8Z-Hexadecenoic acud, 8Z-Pentadecenoic acid, 9E,11Z-Heptadecadienoic acid, 9Z,11 E,13E-Octadecatrienoic acid, 9Z,11 E,13Z-Octadecatrienoic acid, 9Z,11 E-Octadecadienoic acid, 9Z,12Z,15Z-Octa- decatrienoic acid (alpha-linoleic), 9Z,12Z-Octadecadienoic acid (linoleic acid), 9Z,12Z-Hex- adecadienoic acid, 9Z-Octadecenoic acid (oleic acid), 9E-Octadecenoic acid (elaic acid), 9Z-Heptadecenoic acid, 9Z-Hexadecenoic acid (palmitoleic acid), 9Z-Hexadecenoic acid, 8Z-Pentadecenoic acid, 9Z-Tetradecenoic acid (myristoleic acid), 10E,12Z-Octadecadi- enoic acid, 10-Methyldodecanoic acid, 10Z-Heptadecenoic acid, 10Z-Pentadecenoic acid, Decanoic acid, Dodecanoic acid (lauric acid), Tetradecanoic acid (myristic acid), Hexadecenoic acid (palmitic acid), Octadecanoic acid (stearic acid), Eicosanoic acid, and Docosanoic acid.

It is preferred that the, one, two, three or more or all fatty aldehydes have a carbon chain length (n-1) in the range of from 9 to 21 carbon atoms.

It is preferred that the, one, two, three or more or all fatty aldehydes are selected from the group consisting of 10-Methyldodecanal, 10-Methylundecanal, 10-Undecenal, 10Z-Hepta- decenal, 10Z-Pentadecenal, 11-Methyldodecanal, 11 -Methyltridecanal, 11Z-Nonadecenal,

12-Methyltetradecanal, 12-Methyltridecanal, 12Z-Nonadecenal, 13-Methylpentadecanal,

13-Methyltetradecanal, 13Z-Nonadecenal, 5Z,8Z,12Z-heptadecatrienal, 4Z,7Z,10Z,13Z,16Z-Nonadecapentaenal, 3Z,6Z,9Z,12Z,15Z,18Z-Heneicosanhexaenal, 4Z,7Z,10Z,13Z-Nonadecatetraenal, 4Z-Undecenal, 5Z,8Z,11Z-Heptadecatrienal, 5Z,8Z,11Z,14Z-Heptadecatetraenal, 5Z,8Z-Tetradecadienal, 5Z-Heptadecenal, 6E,9E- Pentadecadienal, 6Z,9Z,12Z-Heptadecatrienal, 6Z,9Z-Pentadecadienal, 7Z,10Z,13Z-Hex- adecatrienal, 7Z,10Z-Hexadecadienal, 7Z,10Z-Tetradecadienal, 7Z,9E,11 E-Hexadeca- trienal, 7Z,9E,11Z-Hexadecatrienal, 7Z,9E-Hexadecadienal, 7Z-Dodecenal, 7Z-hexade- cenal, 7Z-Pentadecenal, 7Z-Tetradecenal, 8E,10Z-Hexadecadienal, 8Z,10E,12E-Hepta- decatrienal, 8Z,10E,12Z-Heptadecatrienal, 8Z,10E-Heptadecadienal, 8Z,11Z,14Z-Hepta- decatrienal, 8Z,11Z-Heptadecadienal, 8Z,11Z-Pentadecadienal, 8Z-heptadecenal, 8E- heptadecenal, 8Z-Hexadecenal, 8Z-Pentadecenal, 7Z-tetradecenal, 8Z-Tridecenal, 9E,11Z-Heptadecadienal, 9-Methylundecanal, 9Z-Hexadecenal, 9Z-Tetradecenal, nonanal, undecanal, tridecanal, pentadecanal, heptadecanal, nonadecanal, heneicosanal. It was surprisingly found that the a-dioxygenase obtained as described herein was active in a pH in the range of from 5 to 8.5, wherein the highest activity was observed at a pH in the range of from 6.5 to 7.25.

It is thus preferred that step iv) is performed at a pH value in the range of from 5 to 8.5, preferably in the range of from 5.5 to 8, preferably in the range of from 6 to 7.75, preferably in the range of from 6.25 to 7.5, preferably in the range of from 6.5 to 7.25.

It was surprisingly found that the a-dioxygenase obtained as described herein was active at a temperature in the range of from 27.5 to 45 °C, wherein the highest activity was observed at a temperature in the range of from 30 to 42 °C.

It is thus preferred that step iv) is performed at a temperature in the range of from 27.5 to 45 °C, preferably in the range of from 30 to 42 °C.

Further, it was found that high amounts the corresponding fatty aldehyde were obtained starting from a reaction for 2 hours.

It is thus preferred in the method according to the invention that step iv) is performed for at least 2 h, preferably for at least 3 h, preferably for at least 4 h, preferably for at least 5 h, preferably for at least 6 h, preferably for at least 7 h, preferably for at least 8 h, preferably for at least 9 h.

Moreover, it was surprisingly found that the highest amounts of the corresponding fatty aldehyde were obtained when the transformation was performed for 10 hours, wherein the amounts started to slightly decrease in case of a longer reaction.

It is thus preferred in the method according to the invention that step iv) is performed for a time in the range of from 8 to 20 h, preferably in the range of from 9 to 15 h, preferably in the range of from 9.5 to 12.5 h.

It is further preferred in the method according to the invention that the seed provided in step i) is of the genus Oryza, preferably of a species selected from the group consisting of Oryza sativa, particularly preferably of a subspecies selected from the group consisting of Oryza sativa japonica and Oryza sativa indica, further preferably of the cultivars selected from the group consisting of JAPONICA IRGC 33138 (other names: IRGC 33138, JAPONICA, I 14,10.18730/2MZ4S), LEMONT IRGC 66756 (other names: IRGC 66756, LEMONT, PI 475833,10.18730/3KMBB), AKIHIKARI IRGC 76300 (other names: IRGC 76300, AKIHIKARI, 10.18730/3WR2P), AMAROO IRGC 82290 (other names: IRGC 82290, AMAROO.T1396/1990,10.18730/42K5F), JAPONICA IRGC 88722 (other names: IRGC 88722, JAPONICA, T1101/1994, 10.18730/484K*, 1451), RC 4862 IRGC 101303 (other names: IRGC 101303, RC 4862,10.18730/4H5W2), CYPRESS IRGC 124359 (other names: IRGC 124359, CYPRESS, T1039/2008, 10.18730/52RGR), Japonica IRGC 138421 (other names: IRGC 138421 .Japonica, T0092/2016/UNGB 3956, NAKM 5), NIPPONBARE IRGC 117274 (other names: 10.18730/4WZR*, IRGC 1 17274, IRGC 117274:2009DS, IRGC 12731 , IRGC 12731 :2007DS, NIPPONBARE), MING HUI IRGC 117814 (other names: IRGC 117814, MING HUI, PI 602626, T0225/2008.GSOR 301098,10.18730/4XAWJ), Van- dana IRGC 136064 (other names: IRGC 136064,Vandana,T0383/2014,3K- 0X236,10.18730/59CR8), Nipponbare IRGC 136196 (other names: IRGC 136196, Nippon- bare, 11 A13071 , T0322/2014.3K-CX140.10.18730/59EZ5) and combinations thereof.

The present invention further relates to a mixture comprising an a-dioxygenase obtainable or obtained by a method according to the invention, wherein the mixture comprises plantbased components in a range of from 0.05 to 50 wt.-%, preferably in a range of from 0.1 to 40 wt.-%, preferably in a range of from 0.25 to 30 wt.-%, preferably in a range of from 0.5 to 20 wt.-%, preferably in a range of from 0.75 to 15 wt.-%, preferably in a range of from 1 to 10 wt.-%, preferably in a range of from 1.5 to 7.5 wt.-%, preferably in a range of from 2 to 5 wt.-%, based on the total weight of the mixture, preferably wherein the plant-based components comprise components selected from the group consisting of chlorophyll, rubisco, proteins, triacylglycerides, waxes, cellulose, sugars, amino acids, phenolic acids, flavonoids, hydroxybenzoic acids, terpenoids, steroids, alkaloids, anthracenes, tocopherols, tocotrienols, their derivatives, and mixtures thereof.

Preferably, the term “phenolic acids” refers to p-hydroxybenzoic acid, caffeic acid, proto- catechuic acid, ferulic acid, sinapic acid, syringic acid, vanillic acid, ferulic acid, p-coumaric acid, and gallic acid.

Preferably, the term “flavonoids” refers to flavones, flavonols, flavanones, dihydroflavones, flavanonols, flavanols, anthocyanins, and their glycosides. Preferably, the term “flavonoids” refers to brassicin, isorhamnetin-4’-0-p-D-glucopyranoside, brassicin-4’-G-p-D-glucopyra- noside, isorhamnetin-7-O-p-D-cellobioside, 3’-0-methyltaxifolin, 3’-0-methyltaxifolin-7-0- p-D-glucopyranoside, 3’-0-methyltaxifolin-4’-0-p-D-glucopyranoside, 3’-0-methyltaxifolin- 5-O-p-D-glucopyranoside, 5,4’-dihydroxy-3’,5’-dimethoxy-7-0-p-glucopyranosylflavone, 7,4’-dihydroxy-3’,5’-dimethoxy-5-0-p-glucopyranosylflavone.

Preferably, the term “terpenoids” refers to monoterpenoids, sesquiterpenoids, diterpenoids, and triterpenoids.

Preferably, the term “steroids” refers to campesterol, p-sitosterol, and stigmasterol.

Preferably, the term “alkaloids” refers to phenylamides containing an indole ring. Preferably, the term “alkaloids” refers to 2-Acetyl-1 -pyrroline, N-feruloylagmatine, N-feruloylputres- cine, N-benzoylserotonin, N-benzoytryptamine, N-benzoyltyramine, N-trans-cinnamoylser- otonin, N-trans-cinnamoyltryptamine, N-trans-cinnamoyltyramine, N-p-coumaroylseroto- nin, and N-feruloylserotonin.

What was said herein with regard to the a-dioxygenase or features related thereto, applies accordingly for the a-dioxygenase in the mixture according to the invention.

Preferably, the plant-based components are those components originating from the rice seed or, respectively, the rice seed, shoot or root used in the method according to the invention, and which were not removed in the method according to the invention.

Further, the present invention relates to use a of a mixture according the invention for producing a composition comprising one or more fatty aldehydes. Typically, the production includes the transformation of a fatty acid or a mixture of fatty acids to the corresponding fatty aldehyde or a mixture of the corresponding fatty aldehydes.

It is thus preferred that the use according to the invention refers to the use of a mixture according to the invention for transforming a fatty acid or a mixture of fatty acids to the corresponding fatty aldehyde or a mixture of the corresponding fatty aldehydes.

Typically, the transformation of the fatty acid to the corresponding fatty aldehyde is achieved by the following reaction: H₂O + co_{2 0} - 7^ — ” OOH spontaneous fatty acid (n) 2-hydroperoxy faty acid (n) aldehyde (n-1 )

13-Methyltetradecanal, 13Z-Nonadecenal, 5Z,8Z,12Z-heptadecatrienal, 4Z,7Z,10Z,13Z,16Z-Nonadecapentaenal, 3Z,6Z,9Z,12Z,15Z,18Z-Heneicosanhexaenal, 4Z,7Z,10Z,13Z-Nonadecatetraenal, 4Z-Undecenal, 5Z,8Z,11Z-Heptadecatrienal, 5Z,8Z,11Z,14Z-Heptadecatetraenal, 5Z,8Z-Tetradecadienal, 5Z-Heptadecenal, 6E,9E- Pentadecadienal, 6Z,9Z,12Z-Heptadecatrienal, 6Z,9Z-Pentadecadienal, 7Z,10Z,13Z-Hex- adecatrienal, 7Z,10Z-Hexadecadienal, 7Z,10Z-Tetradecadienal, 7Z,9E,11 E-Hexadeca- trienal, 7Z,9E,11Z-Hexadecatrienal, 7Z,9E-Hexadecadienal, 7Z-Dodecenal, 7Z-hexade- cenal, 7Z-Pentadecenal, 7Z-Tetradecenal, 8E,10Z-Hexadecadienal, 8Z,10E,12E-Hepta- decatrienal, 8Z,10E,12Z-Heptadecatrienal, 8Z,10E-Heptadecadienal, 8Z,11Z,14Z-Hepta- decatrienal, 8Z,11Z-Heptadecadienal, 8Z,11Z-Pentadecadienal, 8Z-heptadecenal, 8E- heptadecenal, 8Z-Hexadecenal, 8Z-Pentadecenal, 7Z-tetradecenal, 8Z-Tridecenal, 9E,11Z-Heptadecadienal, 9-Methylundecanal, 9Z-Hexadecenal, 9Z-Tetradecenal, nonanal, undecanal, tridecanal, pentadecanal, heptadecanal, nonadecanal, heneicosanal.

Furthermore, the present invention relates to the use of rice seed for transforming a fatty acid to the corresponding fatty aldehyde, wherein the use includes the production of a-dioxygenase and optionally the purification of the produced a-dioxygenase, preferably wherein the transformation includes a purification as described herein, particularly preferably, wherein the transformation includes the step of contacting the purified a- dioxygenase with a fatty acid to transform the fatty acid to the corresponding fatty aldehyde.

What was said above with regard to the rice seed, particularly the preferred features thereof, applies accordingly.

What was said above with regard to the transformation of a fatty acid to the corresponding fatty aldehyde, particularly the preferred features thereof, applies accordingly.

What was said above with regard to the a-dioxygenase, its production and the purification of the produced a-dioxygenase, particularly the preferred features, applies accordingly.

Fig. 1 shows the distribution of a-dioxygenase in the plant material analysed in Example 2.

Fig. 2 shows the influence of time and temperature on the transformation of fatty acids to fatty aldehydes with a a-dioxygenase produced by a method according to the invention, with the reactions at 24 °C (x), 30 °C (circle), 36 °C (square) and 42 °C (triangle) for a duration of 3 hours, 6 hours or 9 hours, as analysed in Example 4a.

Fig. 3 shows the influence of the pH on the transformation of fatty acids to fatty aldehydes with an a-dioxygenase produced by a method according to the invention, as analysed in Example 4b.

Short description of the sequences:

SEQ ID NO 1 : Nucleic acid sequence encoding the enzyme a-dioxygenase from

Oryza sativa.

SEQ ID NO 2: Amino acid sequence encoding the enzyme a-dioxygenase from Oryza sativa.

Further aspects and advantages of the invention result from the subsequent description of preferred examples. Examples

Example 1 : Production of a-dioxygenase

Rice seeds were sterilised with 4% NaCIO. The seeds were subsequently soaked in sterile dFW for 2 hours and placed in calcium chloride agar. The germination was allowed for 7 days in the absence of light. A germination of approximately 80% was obtained. Approximately 45% of the obtained biomass was seeds, approximately 35 % was shoots and approximately 20 % was roots.

The shoots were separated from the seedlings, mixed with 10x volume extraction buffer (100 mM phosphate buffer, pH 7.0, 1 % Triton X-100, 5% Glycerol) and homogenized with a ball mill (25 Hz, 1 min).

Furthermore, the homogenized shoots were suspended in 50x volume of phosphate buffer (100 mM, pH 7.0) and extracted for 1 min by vortexing. Subsequently, the obtained mixture was centrifuged at 4,000 x g for 5 min at 4 °C and the supernatant was collected.

To confirm the presence of a-dioxygenase, 2 mL of the supernatant were mixed with 1 mg of a mixture comprising myristoleic acid or oleic acid. The mixture was incubated for 20 hours at 24 °C on a rotating wheel rotating with 40 rpm.

Subsequently, the reaction was stopped by acidification with 100 pL 4 M HCI. The reaction mixture was then diluted with water and analysed via HS-SPME-GC-MS.

The fatty aldehyde corresponding to myristoleic acid, in the sense of the present application text, is 8-tridecenal. The fatty aldehyde corresponding to oleic acid, in the sense of the present application text, is 8-heptadecenal.

High levels of (Z)-8-tridecenal and high levels of (Z)-8-heptadecenal were detected in the analysed sample of the reaction mixture. It was thus concluded that high levels of a-dioxy- genase are present in the reaction mixture. Furthermore, higher levels of (Z)-8-tridecenal or (Z)-8-heptadecenal represented higher levels of a-dioxygenase in the reaction mixture. Example 2: Distribution of a-dioxygenase in the plant material

Rice seedlings were grown for 7 days and the presence of a-dioxygenase was compared between shoot, root and seed. Shoot, root and seed were separated from each other and the presence and amount of a-dioxygenase was compared as described in Example 1 .

The amounts of detected (Z)-8-heptadecenal were compared between shoot, root and seed. It was found that the highest amounts were observed in the shoot, with lower amounts in the root and only very low levels in the seed. The results of this analysis are shown in Fig. 1.

Example 3: Optimising the expression levels of a-dioxyqenase

Rice seeds were placed onto agar medium for allowing germination. Different parameters were modified for evaluating their contribution to a-dioxygenase expression. The expression of a-dioxygenase was assessed as described in Example 1.

Example 3a: Growth status

Rice seedlings were grown for 7, 14, 21 or 25 days. It was found that the amount of a- dioxygenase increased with time and was the highest after 25 days.

Example 3b: light / darkness

Rice seedlings were grown in light or in darkness. It was found that the amount of a-dioxy- genase was strongly increased in case the rice seedlings were grown in darkness.

Example 3c: Supplements

Rice seedlings were grown in the presence or absence of CuSC or NaCI. It was found that both, CuSC and NaCI slowed down I impaired the growth of the seedlings and strongly reduced the amount of a-dioxygenase, wherein this effect was proportional to the amount of CuSCM or NaCI applied.

Rice seedlings were grown in the presence or absence of ZnSC . It was found that ZnSC slightly increased the expression of the a-dioxygenase. Rice seedlings were grown in the presence or absence of 2 mM CaCh. It was found that CaCh slightly increased the expression of the a-dioxygenase.

Example 3d: Temperature

Rice seedlings were grown at different temperatures. It was found that the amount of a- dioxygenase was highest when the rice seedlings were grown at 28 °C.

Example 4: Transforming fatty acids to fatty aldehydes

Example 4a: Time and temperature

For transforming fatty acids to fatty aldehydes, a-dioxygenase was produced as described in Example 1 .

After the centrifugation step, 2 mL of the supernatant were mixed with 1 mg of oleic acid. The levels of (Z)-8-heptadecenal were determined as described in Example 1 .

The chemical reaction was stopped after 3 hours, 6 hours or 9 hours, in each case, the chemical reaction was performed at a temperature of 24 °C, 30 °C, 36 °C or 42 °C. The influence of time and temperature was assessed by comparing the levels of obtained (Z)- 8-heptadecenal.

It was found that with an increased time, the amounts of (Z)-8-heptadecenal increased as well until 9 hours. Further, it was found that increasing the temperature also increased the amounts of (Z)-8-heptadecenal. However, starting from 9 hours, the amounts of (Z)-8-hep- tadecenal obtained with 36°C exceeded the amounts obtained with 42 °C. The results of this analysis are shown in Fig. 2.

To further allow a conclusion of the influence of the time of reaction, the experiment was repeated and the chemical reaction was stopped after 6 hours, 8 hours, 10 hours or 20 hours. The reaction was performed at a temperature of 24 °C.

It was surprisingly found that the maximum amount of (Z)-8-heptadecenal was obtained after 10 hours, whereas the amount was still high after a reaction of 20 hours, but lower than after 10 hours. Example 4b: pH

The chemical reaction was performed at 24 °C for a duration of 20 hours. Furthermore, the chemical reaction was performed at a pH of 5, 6, 6.5, 7, 7.5, 8, 8.5 or 9 and the levels of obtained (Z)-8-heptadecenal were compared.

It was found that the highest amounts of (Z)-8-heptadecenal were obtained at a pH of 6.5 to 7, with a maximum value obtained for a pH of 7. The results of this analysis are shown in Fig. 3.

Example 5: Effect of light / darkness

Rice seedlings were grown as described in Example 3b.

The seedlings were grown in agar containing 2 mM CaCh, i.e. in the absence of CuSC and NaCI. The seedlings were grown for 8, 14 or 21 days with light or substantially in the absence of light (“no light”).

The presence and amount of a-dioxygenase was compared as described in Example 1 , using oleic acid and measuring the amount of 8-heptadecenal.

For each condition, triplicate samples were grown and measured.

It was found that in all samples, the growth substantially in the absence of light lead to higher amount of a-dioxygenase, since higher amounts of 8-heptadecenal were measured:

Claims

1 . Method for producing a-dioxygenase, comprising the steps i) providing rice seed, ii) cultivating the seed provided in step i) on a medium base, preferably agar, for at least 7 days, to allow germination wherein for at least 7 days, the cultivation is substantially in the absence of light, and wherein the concentration of CuSC in the medium base is at most 25 mM, preferably at most 20 mM, preferably at most 15 mM, preferably at most 10 mM, preferably at most 7.5 mM, based on the total medium base, and/or wherein the concentration of NaCI in the medium base is at most 500 mM, preferably at most 450 mM, preferably at most 400 mM, preferably at most 350 mM, preferably at most 300 mM, preferably at most 250 mM, preferably at most 200 mM, based on the total medium base, and/or iii) optionally: purifying the produced a-dioxygenase.

2. Method according to claim 1 , wherein the cultivation in step ii) is performed for at least 8 days, preferably for at least 9 days, preferably for at least 10 days, preferably for at least 11 days, preferably for at least 15 days, preferably for at least 18 days, preferably for at least 20 days, preferably for at least 21 days, preferably for at least 22 days, preferably for at least 23 days, preferably for at least 24 days, preferably for at least 25 days, preferably for at least 26 days, preferably for at least 27days, preferably for at least 28 days, preferably for at least 29 days, preferably for at least 30 days, particularly preferably for a time in the range of from 25 to 30 days, preferably wherein for at least 8 days, preferably for at least 9 days, preferably for at least 10 days, preferably for at least 11 days, preferably for at least 15 days, preferably for at least 18 days, preferably for at least 20 days, preferably for at least 21 days, preferably for at least 22 days, preferably for at least 23 days, preferably for at least 24 days, preferably for at least 25 days, preferably for at least 26 days, preferably for at least 27days, preferably for at least 28 days, preferably for at least 29 days, preferably for at least 30 days, particularly preferably for a time in the range of from 25 to 30 days, the cultivation is substantially in the absence of light.

3. Method according to claims 1 or 2, wherein the medium base is or comprises Mu- rashige and Skoog medium.

4. Method according to any one of the preceding claims, wherein the medium base comprises ZnSCM, preferably wherein the medium base comprises at least 0.5 mM ZnSCM, preferably at least 1 mM ZnSCM, preferably at least 2 mM ZnSCM, preferably at least 3 mM ZnSCM, preferably at least 4 mM ZnSCM, preferably at least 5 mM ZnSCM, preferably at least 6 mM ZnSCM, preferably at least 7 mM ZnSCM, preferably at least 8 mM ZnSCM, preferably at least 9 mM ZnSCM, preferably at least 10 mM ZnSCM.

5. Method according to any one of the preceding claims, wherein the medium base comprises CaCh, preferably wherein the medium base comprises CaCh in a range of from 0.2 to 12.5 mM, preferably in a range of from 0.5 to 10 mM, preferably in a range of from 0.75 to 7.5 mM, preferably in a range of from 1 to 5 mM, preferably in a range of from 1 .25 to 4 mM, preferably in a range of from 1 .5 to 3 mM, particularly preferably wherein the medium base is agar comprising CaCh in a range of from 0.2 to 12.5 mM, preferably in a range of from 0.5 to 10 mM, preferably in a range of from 0.75 to 7.5 mM, preferably in a range of from 1 to 5 mM, preferably in a range of from 1 .25 to 4 mM, preferably in a range of from 1 .5 to 3 mM.

6. Method according to any one of the preceding claims, further comprising the steps ii.1) separating the shoots from the seedling, preferably separating the shoots and the roots from the seedling, and/or ii.2) homogenizing the shoots separated in step ii.b) or, respectively, the shoots and roots separated in step ii.b).

7. Method according to any one of the preceding claims, wherein step iii) is present and wherein the purification includes a centrifugation step, preferably a centrifugation step in the range of from 2.500 x g to 25.000 x g, preferably in the range of from 2.750 x g to 22.500 x g, preferably in the range of from 3.000 x g to 20.000 x g, preferably in the range of from 3.250 x g to 17.500 x g, preferably in the range of from 3.500 x g to 15.000 x g, preferably in the range of from 3.750 x g to 12.500 x g, preferably in the range of from 4.000 x g to 10.000 x g, preferably in the range of from 4.250 x g to 7.500 x g, preferably in the range of from 4.500 x g to 5.000 x g.

8. Method according to any one of the preceding claims, wherein step iii) is present, and the method further comprising the step iv) contacting the purified a-dioxygenase with a fatty acid to transform the fatty acid to the corresponding fatty aldehyde.

9. Method according to claim 8, wherein step iv) is performed at a pH value in the range of from 5 to 8.5, preferably in the range of from 5.5 to 8, preferably in the range of from 6 to 7.75, preferably in the range of from 6.25 to 7.5, preferably in the range of from 6.5 to 7.25.

10. Method according to claim 8 or 9, wherein step iv) is performed at a temperature in the range of from 27.5 to 45 °C, preferably in the range of from 30 to 42 °C.

11 . Method according to any one of claims 8 to 10, wherein step iv) is performed for at least 2 h, preferably for at least 3 h, preferably for at least 4 h, preferably for at least 5 h, preferably for at least 6 h, preferably for at least 7 h, preferably for at least 8 h, preferably for at least 9 h, preferably wherein step iv) is performed for a time in the range of from 8 to 20 h, preferably in the range of from 9 to 15 h, preferably in the range of from 9.5 to 12.5 h.

12. Method according to any one of the preceding claims, wherein the seed provided in step i) is of the genus Oryza, preferably of a species selected from the group consisting of Oryza sativa, particularly preferably of a subspecies selected from the group consisting of Oryza sativa japonica and Oryza sativa indica.

13. Mixture comprising an a-dioxygenase obtainable or obtained by a method according to any one of the previous claims, wherein the mixture comprises plant-based components in a range of from 0.05 to 50 wt.-%, preferably in a range of from 0.1 to 40 wt.-%, preferably in a range of from 0.25 to 30 wt.-%, preferably in a range of from 0.5 to 20 wt.-%, preferably in a range of from 0.75 to 15 wt.-%, preferably in a range of from 1 to 10 wt.-%, preferably in a range of from 1 .5 to 7.5 wt.-%, preferably in a range of from 2 to 5 wt.-%, based on the total weight of the mixture, preferably wherein the plant-based components comprise components selected from the group consisting of chlorophyll, rubisco, proteins, triacylglycerides, waxes, cellulose, sugars, amino acids, phenolic acids, flavonoids, hydroxybenzoic acids, terpenoids, steroids, alkaloids, anthracenes, tocopherols, tocotrienols, their derivatives, and mixtures thereof.

14. Use of a mixture according to claim 13 for producing a composition comprising one or more fatty aldehydes, preferably for transforming a fatty acid or a mixture of fatty acids to the corresponding fatty aldehyde or a mixture of the corresponding fatty aldehydes.

15. Use of rice seed for transforming a fatty acid to the corresponding fatty aldehyde, wherein the use includes the production of a-dioxygenase and optionally the purification of the produced a-dioxygenase, preferably wherein the transformation includes a method according to claim 8.