Methods for biotechnological production of aldehyde mixtures

Abstract

To provide biotechnological methods for production of saturated and unsaturated aldehydes and mixtures thereof using at least one α-dioxygenase and at least one aldehyde dehydrogenase.SOLUTION: The method may be implemented either fermentatively or enzymatically. The present invention also relates to a vector system, as well as sequences and recombinant microorganisms encoding the enzymes that can be used to produce the aldehydes and mixtures according to the invention. The present invention further relates to compositions obtained by the methods according to the present invention.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a biotechnological process for the production of saturated and unsaturated aldehydes and mixtures thereof using at least one α-dioxygenase and at least one aldehyde dehydrogenase. The process may be carried out either by fermentation or by enzymatic methods. Furthermore, the present invention relates to vector systems, as well as sequences and recombinant microorganisms encoding the enzymes that can be used to produce the aldehydes and mixtures according to the invention. Furthermore, the present invention relates to compositions obtainable by the process according to the invention. [Background technology]

[0002] In the field of industrial production of flavors, there is a constant demand for efficient and cost-effective methods of synthesizing flavors. Examples of such flavors are (un)saturated aldehydes. Examples of aldehydes are 8Z-tetradecenal or 1-decenal. Substances of this type are found mainly in meat preparations and citrus oils. However, currently, no generally applicable biotechnological process for the production of these aldehydes and the corresponding acids has been described.

[0003] Processes for the preparation of unsaturated aldehydes are known from the prior art. For example, 8Z,11Z-heptadecadienal can be obtained by chemical oxidation of unsaturated alcohols (Patent Document 1). Aldehydes can also be produced from fatty acids in a known manner by alpha-oxidation using various plant enzymes, as described in Patent Document 2. The use of algal biomass to produce unsaturated aldehydes is also known (Non-Patent Document 1). Furthermore, processes for the preparation of 4,7,10,13,16-nonadecapentaenal or 3,6,9,12,15,18-heneicosahexaenal are known (Preparation of Halopolyenes and Their Intermediates, Patent Document 3).

[0004] Additionally, numerous processes are known that use aldehyde dehydrogenases to oxidize aldehydes to acids (Non-Patent Document 2; Non-Patent Document 3).

[0005] However, the above mentioned chemical methods as well as methods based only on very specific enzymes have the disadvantage that they cannot be applied to a comprehensive range of aldehydes or their mixtures. Moreover, some of these methods are based on reactions with chemical reagents as well as catalysts, so that they are not considered as natural processes in the sense of 4. No combinatorial method is known that specifically combines α-dioxygenase with aldehyde dehydrogenase to specifically expand the scope of production. In recombinant reaction systems with two or more enzymes, where the desired catalytic reaction will proceed sequentially and specifically with respect to substrate specificity and selectivity, it is necessary to precisely match the enzymes used so that they are not hindered by the endogenous enzymes of the producing organism, for example in the case of whole cell catalysis. Thus, reaction control is crucial to ensure that intermediates are also recognized by the second or each subsequent enzyme in the cascade and available to the latter. Such adjustments or combinations are always technically challenging, cannot be straightforwardly predicted and require intensive analysis of the enzymes of interest in the respective biotechnological context.

[0006] It has been reported that several unsaturated aldehydes with one or more double bonds, such as 8Z-heptadecenal, 8Z,11Z-heptadecadienal or 8Z,11Z,14Z-heptadecatrienal, occur naturally in the juice of cucumber (Non-Patent Document 5) or the green alga Ulva pertusa (Non-Patent Document 6). The contents of the aldehydes 8Z-heptadecenal, 8Z,11Z-heptadecadienal and 8Z,11Z,14Z-heptadecatrienal have been reported in the literature to be 2%, 19.9% ​​and 26.1%.

[0007] The odor of 8Z,11Z-heptadecadienal has been described as being typical of algae (Non-Patent Document 6).

[0008] In contrast, the taste of 4Z,7Z,10Z,13Z-nonadecatetraenal, 4Z,7Z,10Z,13Z,16Z-nonadecapentaenal or 3,6,9,12,15,18-heneicosahexaenoic acid has not been described.

[0009] It is known that 8Z-pentadecenal is a natural component of cucumber (Non-Patent Document 7). The flavor of 8Z-pentadecenal is primarily described as oily, and its flavor-enhancing properties for chicken and grilled beef are also described. In addition, a method for its production from 1-nonyne and 6-bromo-1-hexanol is also known (Patent Document 4), but this cannot be classified as a natural production process according to Non-Patent Document 4.

[0010] The taste of 4Z,7Z,10Z,13Z-nonadecatetraenal is unknown, and until now there has been no method for producing it.

[0011] Various aldehydes can be produced from fatty acids by α-oxidation using plant enzymes, as described in Patent Document 2. The use of green algae biomass for the production of unsaturated aldehydes is also known.

[0012] Other processes for the production of fatty acids from the corresponding oils or fats by enzymatic cleavage are also known from the prior art. For this purpose, mainly enzymes from Aspergillus niger, Rhizopus oryzae, Penicillium camembertii, Mucor juvanicus, Penicillium roquefortii, porcine pancreas, Candida rugosa, Rhizomucor miehei, Candida antarctica or Rhizops delemar are used (Non-Patent Document 8).

[0013] It was therefore an object of the present invention to provide a method for the production of an aldehyde or a mixture of aldehydes, which can be classified as a natural production process according to Friedrichsson, 1999. Moreover, it was an object to provide a biotechnological process, integrated by selective regulation of enzymatic metabolic steps, capable of providing the product of interest in high yield and purity without intermediate chemical steps. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Publication No. 63-233914 [Patent Document 2] International Publication No. 2012025629(A1) [Patent Document 3] JP 05-000974(A) [Patent Document 4] Patent Publication No. 2014-043409 [Non-patent literature]

[0015] [Non-Patent Document 1] "Concise synthesis of (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,3Z)-7,10,13-hexadecatrienal and (8Z,11Z)-8,11-heptadecadienal, components of the essential oil of the marine green alga Ulva pertusa," Biosci Biotechnol Biochem, vol. 69, no. 7: pp. 1348-52 (July 2005) [Non-Patent Document 2] Takeru Ishige et al., Appl. Environ. Microbiol., vol. 66, pp. 3481-3486 (2000) [Non-Patent Document 3] Tomohisa Kato et al., Extremophiles, Vol. 14, p. 33 (2010) [Non-Patent Document 4] Regulation (EC) No. 1334 / 2008 of the European Parliament and of the Council [Non-Patent Document 5] Aroma production from cucumber (Cucumis sativus L.) and bitter melon (Momordica charantia L.) by rubbing with salt, Journal of the Japan Society of Home Economics, Vol. 60 (2009) No. 10, pp. 877-885 [Non-Patent Document 6] Production of bioflavors by regeneration from protoplasts of Ulva anabae (Ulvales, Chlorophyta), Hydrobiologia, vol. 204, pp. 143-149 (1990) [Non-Patent Document 7] Kemp, A., C15 aldehydes from cucumber (Cucumis sativus), Phytochemistry, Vol. 16, No. 11, pp. 1831-1832 (1977) [Non-Patent Document 8] Industrial applications of microbial lipases, Enzyme and Microbial Technology, Vol. 39, No. 2, pp. 235-251 (June 26, 2006) Summary of the Invention [Means for solving the problem]

[0016] Thus, according to a first aspect, the present invention relates to providing a method for producing saturated and unsaturated aldehydes using at least one α-dioxygenase and at least one aldehyde dehydrogenase.

[0017] Furthermore, according to one embodiment, the present invention relates to a fermentative process for the production of saturated and unsaturated aldehydes and mixtures thereof. In another embodiment of the process according to the invention, an enzymatic process is provided.

[0018] In a further embodiment, the method according to the invention concerns the use of reactants (educts) which are either of biotechnological, natural or chemical origin.

[0019] In yet another embodiment, the process according to the invention relates to preferred reactants selected from the group of carboxylic acids and carboxylic acids esterified with alcohols.

[0020] Furthermore, the method according to the invention relates to preferred product mixtures, the microorganisms used in the method as well as the amino acid sequences of the enzymes preferably used and the nucleotide sequences or nucleic acid segments which specify the sequences of the enzymes used.

[0021] Another aspect of the invention relates to the provision of vector systems encoding the enzymes used in accordance with the invention.

[0022] Another aspect of the invention relates to a composition obtainable by the process according to the invention. [Brief description of the drawings]

[0023] [Figure 1] Plasmid pET28a_OSaDOX carrying the gene encoding α-dioxygenase. [Diagram 2] The plasmid pET-Duet_VhFALDH harboring the gene encoding aldehyde dehydrogenase. [Diagram 3] The plasmid pET-Duet_LsNOX_VhFALDH carries genes encoding aldehyde dehydrogenase and NADH-oxidase. [Figure 4]Biocatalytic conversion of a mixture of oleic and linoleic acids to an aldehyde mixture containing 8,11-heptadecadienal and 7,10-hexadecadienal. [Diagram 5] Time course up to 1 h for the biocatalytic conversion of a mixture of oleic and linoleic acids to the corresponding aldehyde mixture by α-dioxygenase, aldehyde dehydrogenase and NADH oxidase. [Figure 6] Time course of biocatalytic conversion of a mixture of oleic and linoleic acids to the corresponding aldehyde mixture by α-dioxygenase and aldehyde dehydrogenase in the absence of NADH oxidase up to 24 h. [Figure 7] Enhanced time course of biocatalytic conversion of a mixture of oleic and linoleic acids to the corresponding aldehyde mixture by α-dioxygenase and aldehyde dehydrogenase with NADH oxidase up to 24 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] A brief description of the sequence SEQ ID NO: 1: Nucleic acid sequence encoding the enzyme Os-α-DOX from rice (Oryza sativa). SEQ ID NO: 2: Nucleic acid sequence encoding the enzyme VhFALDH from Vibrio harveyi. SEQ ID NO: 3: Nucleic acid sequence encoding the 175Q mutant of the enzyme VhFALDH from Vibrio harveyi. SEQ ID NO: 4: Nucleic acid sequence encoding the enzyme LsNOX from Lactobacillus sanfranciscenis. SEQ ID NO: 5: Nucleic acid sequence encoding the enzyme ReALDH from Rhodococcus erythropolis UPV-1. SEQ ID NO: 6: The nucleic acid sequence encoding the enzyme GtALDH from Geobacillus thermodenitrificans. SEQ ID NO: 7: Nucleic acid sequence encoding the enzyme Maqu3410, an aldehyde dehydrogenase from Marinobacter aquaeolei VT8. SEQ ID NO: 8: Ammonoic acid sequence encoding the enzyme Os-α-DOX from rice. SEQ ID NO: 9: Amino acid sequence encoding the enzyme VhFALDH from Vibrio harveyi. SEQ ID NO: 10: Amino acid sequence encoding the 175Q mutant of the enzyme VhFALDH from Vibrio harveyi. Sequence number 11: Amino acid sequence encoding the enzyme LsNOX from Lactobacillus sanfranciscenis. SEQ ID NO: 12: Amino acid sequence encoding the enzyme ReALDH from Rhodococcus erythropolis UPV-1. SEQ ID NO: 13: Amino acid sequence encoding the enzyme GtALDH from Geobacillus thermodenitrificans. SEQ ID NO: 14: Amino acid sequence encoding the enzyme Maqu3410, an aldehyde dehydrogenase from Marinobacter aquaeolei VT8. SEQ ID NO: 15: Nucleic acid sequence encoding the forward primer. SEQ ID NO: 16: Nucleic acid sequence encoding the reverse primer. SEQ ID NO: 17: Nucleic acid sequence encoding the enzyme At-α-DOX2 from Arabidopsis thaliana. SEQ ID NO: 18: Nucleic acid sequence encoding the enzyme At-α-DOX from Arabidopsis thaliana. SEQ ID NO: 19: Nucleic acid sequence encoding the enzyme CaaDOX from Capsicum annuum. SEQ ID NO: 20: Nucleic acid sequence encoding the enzyme CbaDOX from Cercospora beticola. SEQ ID NO: 21: Nucleic acid sequence encoding the enzyme CsaDOX2 from cucumber (Cucumis sativus). SEQ ID NO: 22: The nucleic acid sequence encoding the enzyme CsaDOX from cucumber. SEQ ID NO: 23: Nucleic acid sequence encoding the enzyme CsaDOxlike1 from cucumber. SEQ ID NO: 24: Nucleic acid sequence encoding the enzyme Le-α-DOX1 from tomato (Solanum lycopersicum). SEQ ID NO: 25: Nucleic acid sequence encoding the enzyme Le-α-DOX2 from tomato. SEQ ID NO: 26: Nucleic acid sequence encoding the enzyme NaaDOX2 from Nicotiana attenuata. SEQ ID NO: 27: Nucleic acid sequence encoding the enzyme Na-α-DOX from Nicotiana tabacum. SEQ ID NO: 28: Nucleic acid sequence encoding the enzyme Nt-α-DOX from Nicotiana tabacum. SEQ ID NO: 29: Nucleic acid sequence encoding the enzyme PpaDOX from Physcomitrella patens. SEQ ID NO: 30: Nucleic acid sequence encoding the enzyme Ps-α-DOX from pea (Pisum sativum). SEQ ID NO: 31: Nucleic acid sequence encoding the enzyme SlaDOX2 from tomato. SEQ ID NO: 32: Nucleic acid sequence encoding the enzyme StaDOX1.1 from tomato. SEQ ID NO: 33: Nucleic acid sequence encoding the enzyme StaDOX1.2 from tomato. SEQ ID NO: 34: Nucleic acid sequence encoding the enzyme StaDOX1.3 from tomato. SEQ ID NO: 35: Nucleic acid sequence encoding the enzyme StaDOX1 from potato (Solanum tuberosum). SEQ ID NO: 36: Nucleic acid sequence encoding the enzyme StaDOX2 from potato. SEQ ID NO: 37: Amino acid sequence encoding the enzyme At-α-DOX2 from Arabidopsis thaliana. SEQ ID NO: 38: Amino acid sequence encoding the enzyme At-α-DOX from Arabidopsis thaliana. SEQ ID NO: 39: Amino acid sequence encoding the enzyme CaaDOX from Capsicum. SEQ ID NO: 40: Amino acid sequence encoding the enzyme CbaDOX from CbaDOX. SEQ ID NO: 41: Amino acid sequence encoding the enzyme CsaDOX2 from cucumber. SEQ ID NO: 42: Amino acid sequence encoding the enzyme CsaDOX from cucumber. SEQ ID NO: 43: Amino acid sequence encoding the enzyme CsaDOxlike1 from cucumber. SEQ ID NO: 44: Amino acid sequence encoding the enzyme Le-α-DOX1 from tomato. SEQ ID NO: 45: Amino acid sequence encoding the enzyme Le-α-DOX2 from tomato. SEQ ID NO: 46: Amino acid sequence encoding the enzyme NaaDOX2 from Nicotiana tabacum. SEQ ID NO: 47: Amino acid sequence encoding the enzyme Na-α-DOX from Nicotiana tabacum. SEQ ID NO: 48: Amino acid sequence encoding the enzyme Nt-α-DOX from Nicotiana tabacum. SEQ ID NO: 49: Amino acid sequence encoding the enzyme PpaDOX from Physcomitrella patens. SEQ ID NO: 50: Amino acid sequence encoding the enzyme Ps-α-DOX from pea. SEQ ID NO: 51: Amino acid sequence encoding the enzyme SlaDOX2 from tomato. SEQ ID NO: 52: Amino acid sequence encoding the enzyme SlaDOX1.1 from tomato. SEQ ID NO: 53: Amino acid sequence encoding the enzyme SlaDOX1.2 from tomato. SEQ ID NO: 54: Amino acid sequence encoding the enzyme SlaDOX1.3 from tomato. SEQ ID NO: 55: Amino acid sequence encoding the enzyme SlaDOX1 from tomato. SEQ ID NO: 56: Amino acid sequence encoding the enzyme SlaDOX2 from tomato. SEQ ID NO: 57: Nucleic acid sequence encoding the enzyme Ald1 from Acinetobacter sp. strain M1. SEQ ID NO: 58: Nucleic acid sequence encoding the enzyme HFD4, an aldehyde dehydrogenase from Yarrowia lipolytica. SEQ ID NO: 59: Amino acid sequence encoding the enzyme Ald1 from Acinetobacter sp. strain M1. SEQ ID NO: 60: Amino acid sequence encoding the enzyme HFD4, an aldehyde dehydrogenase from Yarrowia lipolytica. SEQ ID NO: 61: Nucleic acid sequence encoding NAD(P)H oxidase from Lactobacillus sanfranciscenis. Sequence number 62: Amino acid sequence encoding NAD(P)H oxidase from Lactobacillus sanfranciscenis. SEQ ID NO: 63: Nucleic acid sequence encoding NADH oxidase from Lactobacillus brevis. SEQ ID NO: 64: Amino acid sequence encoding NADH oxidase from Lactobacillus brevis.

[0025] definition The term vector system as used herein means a system consisting of or including at least one or more types of vectors or plasmid vectors. Thus, a vector system may include (plasmid) vectors encoding multiple different target genes. A vector system may further include multiple (plasmid) vectors according to the present disclosure, which in turn include at least one target gene. Thus, a vector system may include only one (plasmid) vector construct or multiple (plasmid) vector constructs, the latter of which may be simultaneously or sequentially, stably or transiently transformed into a corresponding recombinant host organism, so that the target genes encoded by each construct are transcribed and translated by the host organism.

[0026] The terms amino acid, protein and polypeptide are used interchangeably herein. The amino acids disclosed herein, or functional parts of those amino acids, have an enzymatic function after correct folding. Thus, the term enzyme is also used interchangeably herein with the term amino acid.

[0027] Whenever this disclosure refers to sequence homology or sequence identity in percentages for nucleic or protein sequences, such references refer to values ​​that 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. The local sequence alignment tool provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) uses a modified Smith-Waterman algorithm (see http: / / www.ebi.ac.uk / Tools / psa / and Smith, TF and Waterman, MS, "Identification of common molecular subsequences", Journal of Molecular Biology, vol. 147, no. 1, pp. 195-197 (1981)). Furthermore, in this specification, when performing pairwise alignment of each of the 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; (ii) for nucleic acid sequences, matrix=DNAfull, gap open penalty=10, gap extend penalty=0.5.

[0028] The growth and cultivation, isolation and purification of recombinant microorganisms or fungi or proteins or enzymes encoded by nucleic acids according to the present disclosure are known to those skilled in the art.

[0029] The terms protein, polypeptide and enzyme are used interchangeably because of the invariant enzymatic function of the gene products disclosed herein. Similarly, for the purposes of this disclosure, the terms gene and nucleic acid (segment) are used interchangeably.

[0030] The nucleic acids, nucleotide sequences or nucleic acid segments (these terms are used indistinguishably with DNA sequences encoding functional enzymes or functional regions of such DNA sequences) used according to the present invention to express a desired target protein may optionally be codon-optimized, i.e. the codon usage of the gene is adapted to that of the recombinant microorganism or fungus chosen as the expression strain. It is known to those skilled in the art that the desired target gene encoding a protein of interest can be modified without changing the translated protein sequence to accommodate a specific codon usage depending on the species. Thus, the nucleic acid of the present invention to be converted can be specifically adapted to the codon usage of E. coli or another bacterial Saccharomyces species or another yeast.

[0031] The disclosed carboxylic acids may be either present in their free form or esterified to alcohols, which occur, for example, as components of lipids.

[0032] Where the (Z) / (E) isomeric configuration of a compound named herein is not specifically indicated, the representation includes all possible configurational isomers of the corresponding compound or mixtures thereof, although certain enzymes referenced herein are capable of recognizing and converting both isomers of the compound of interest.

[0033] Detailed Description The problem of the present invention is primarily solved by providing a biotechnological process for the production of at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid, which comprises providing at least one α-dioxygenase and at least one aldehyde dehydrogenase.

[0034] The resulting aldehyde is always one carbon atom shorter compared to the reactants used. Saturated aldehydes in the context of the present invention refer to aldehydes without a double bond, whereas unsaturated aldehydes refer to aldehydes in which a double bond is present. The corresponding carboxylic acids in this case are compounds with the same chain length and the same degree of saturation as the resulting aldehyde, but with at least one carboxyl group. The at least one α-dioxygenase used according to the present invention catalyzes the oxidation at the α-carbon atom of carboxylic acids with a chain length of C6 to C22, resulting in the formation of the corresponding aldehydes, which are one carbon atom shorter.

[0035] Due to its substrate specificity, the at least one aldehyde dehydrogenase used catalyzes the oxidation of an aldehyde to the corresponding carboxylic acid, which can then be converted by an α-dioxygenase to an aldehyde that is in turn shortened by one carbon atom. Thus, the method of the present invention can be used to selectively produce an aldehyde mixture having aldehydes with different chain lengths.

[0036] In one embodiment of the method according to the invention, the α-oxidation and the aldehyde oxidation can be carried out simultaneously, while in another embodiment, the α-oxidation can be carried out first and then the aldehyde oxidation, thereby continuing the reaction cycle until the desired product is obtained in both embodiments.

[0037] In a preferred embodiment of the method according to the invention, the biotechnological production method comprises: i. providing at least one recombinant microorganism or fungus containing a nucleic acid segment comprising at least one gene encoding an α-dioxygenase and / or at least one gene encoding an aldehyde dehydrogenase; ii. culturing at least one recombinant microorganism under conditions allowing expression of the corresponding expression product; iii. adding to the cultured at least one recombinant microorganism at least one carboxylic acid or at least one carboxylic acid esterified with an alcohol; iv. reacting at least one α-dioxygenase and at least one aldehyde dehydrogenase with the at least one carboxylic acid or carboxylic acid esterified to an alcohol of step iii to obtain at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid; The process may be a fermentation process comprising:

[0038] A fermentative process in the context of the present invention refers to a process in which a recombinant microorganism is cultured that expresses at least one α-dioxygenase and at least one aldehyde dehydrogenase necessary for producing the product of the present invention in at least one step. The expression products in this context are at least one α-dioxygenase and / or at least one aldehyde dehydrogenase. Thereby, according to one embodiment, the reaction of the reactants can be carried out by secretion of the enzyme into the reaction mixture in which the reactants are present, whereas according to another embodiment, the reaction can be carried out in the cytosol of at least one microorganism, followed by secretion of the product.

[0039] In the context of the present invention, providing always means including an active step that makes available a reactant, an enzyme or similar. According to one embodiment, providing can always be a technical step, for example the cloning of a reaction organism or the production of a reactant.

[0040] In another preferred embodiment of the method according to the invention, the biotechnological production method comprises the steps of: i. providing at least one α-dioxygenase and at least one aldehyde dehydrogenase, wherein the at least one α-dioxygenase and / or the at least one aldehyde dehydrogenase may be naturally, chemically or biotechnologically produced; ii. providing at least one carboxylic acid or at least one carboxylic acid esterified with an alcohol to the enzyme of step i; iii. reacting at least one α-dioxygenase and at least one aldehyde dehydrogenase with the at least one carboxylic acid or carboxylic acid esterified to an alcohol of step ii to obtain at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid; In some cases, the method is enzymatic, involving

[0041] Enzymatic method in the context of the present invention means a method in which at least one α-dioxygenase and at least one aldehyde dehydrogenase can be purified or partially purified and present outside the microorganism. In one embodiment, these two enzymes can be present as a cell lysate, which means the state of the microorganism that can no longer be cultivated and has been digested by physical, chemical or enzymatic processes. In a further embodiment, the enzymes can be present with a purity of >80% and purified by purification processes known to those skilled in the art. In yet another embodiment, the enzymes can be present with a purity of <80% and partially purified by purification processes known to those skilled in the art. In yet another embodiment, the enzymes can be obtained by protein synthesis and optionally purified.

[0042] Reactants (starting materials) of biotechnological origin are reactants produced by microbial fermentation. Reactants of natural origin may be obtained by microbial fermentation from natural sources, e.g. plants, fungi, animals, microorganisms or soil. Reactants of chemical origin may be produced by chemical synthesis from reactant precursors.

[0043] In a further preferred embodiment, at least one of the reactants of the method according to the invention may be of biotechnological, natural or chemical origin. The above applies to reactants of biotechnological, natural or chemical origin.

[0044] According to a further preferred embodiment of the process of the invention, the reactants can be selected from the group of carboxylic acids and carboxylic acids esterified with alcohols. Preferred reactants are palmitoleic acid, arachidonic acid, α-linolenic acid, linoleic acid, γ-linolenic acid, oleic acid, punicic acid, α-eleostearic acid, docosahexaenoic acid, eicosapentaenoic acid, petroselinic acid, schormugic acid, α-licaric acid, olive oil, canola oil, macadamia nut oil, sea buckthorn fruit oil, tung oil, fish oil, borage oil, tallow oil, parsley oil, oiticica oil, pomegranate seed oil, coconut oil, sunflower oil, fieldstone seed oil and grape seed oil, and also oleic acid. and fungal oils obtained from genera independently selected from the genera Conidiobolus, Flammulina, Fomes, Ganoderma, Mortierella, Panellus, Pleurotus, Psathyrella, Stereum, and Umbelopsis.

[0045] In yet another preferred embodiment, the product of the process according to the invention may be at least a saturated aldehyde and its corresponding carboxylic acid and / or an unsaturated aldehyde and its corresponding carboxylic acid. In this specification, the product of the process according to the invention is preferably 7Z,10Z-hexadecadienal, 8Z,11Z-heptadecadienal, 6E,9E-pentadecadienal, 7Z-hexadecenal, 8Z-pentadecenal, 7Z-tetradecenal, 8Z,11Z,14Z-heptadecatrienal, 7Z,10Z,13Z-hexadecatrienal, 4Z,7Z,10Z,13Z-nonadecatrienal, 5 ... Decatetraenal, 8Z,10E,12E-heptadecatrienal, 8Z,10E,12Z-heptadecatrienal, 7Z,9E,11Z-hexadecatrienal, 7Z,9E,11E-hexadecatrienal, 9-decenal, 10-undecenal, 3Z-decenal, 4Z-undecenal, 7Z-dodecenal, 8Z-tridecenal, 7Z-tetradecenal, 8Z-pentadecenal Senal, 9Z-tetradecenal, 10Z-pentadecenal, 7Z-pentadecenal, 8Z-hexadecenal, 9Z-hexadecenal, 10Z-heptadecenal, 5Z,8Z-tetradecadienal, 6Z,9Z-pentadecadienal, 7Z,10Z-tetradecadienal, 8Z,11Z-pentadecadienal, 7Z,9E-hexadecadienal, 8Z,10E-heptadecadienal enal, 8E,10Z-hexadecadienal, 9E,11Z-heptadecadienal, 9-methylundecanal, 10-methylundecanal, 10-methyldodecanal, 11-methyldodecanal, 11-methyltridecanal, 12-methyltridecanal, 12-methyltetradecanal, 13-methyltetradecanal and 13-methylpentadecanal.

[0046] In the context of this invention, all compounds in which the position of the double bond is not specifically designated as to the (Z) / (E) configuration include all possible positions of the double bond and mixtures of related compounds.

[0047] According to a further embodiment of the method of the present invention, the at least one recombinant microorganism may be selected from the group consisting of E. coli, preferably E. coli BL21, E. coli MG1655, E. coli W3110 and derivatives thereof, Bacillus spp., preferably B. licheniformis, B. subitilis or B. amyloliquefaciens and derivatives thereof, Saccharomyces spp., preferably S. cerevisiae and derivatives thereof, Hansenulla or Komagatella spp., preferably K. phaffii and H. polymorpha and derivatives thereof, Kluyveromyces spp., preferably K. lactis.

[0048] According to a preferred embodiment, at least one NADH oxidase and / or at least one lipase may additionally be provided.

[0049] NADH oxidases are capable of catalyzing the oxidation of aldehydes due to their substrate specificity and regioselectivity. In a preferred embodiment of the method according to the invention, they can be used as additional enzymes to make tightly coordinated processes more efficient by recycling the corresponding cofactors.

[0050] In another preferred embodiment of the method of the invention, the at least one NADH oxidase may comprise an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 62 and 64, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. In one embodiment, the amino acid sequence encoding the NADH oxidase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 61 and 62, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0051] Furthermore, the enzyme that catalyzes the reaction according to the invention, according to various aspects and embodiments of the invention, may be a catalytically active domain or fragment of the particular enzyme from which it is derived.

[0052] In another preferred embodiment, the at least one lipase may be a commercially available lipase selected from the group consisting of Candida antarctica, Aspergillus niger, Rhizopus oryzae, Penicillium camembertii, Mucor juvanicus, Penicillium roqueforti, porcine pancreas, Candida rugosa, Rhizomucor miehei, or Rhizopus delemar.

[0053] In yet another preferred embodiment of the method of the invention, the α-dioxygenase may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 11, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55 or 56, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0054] In another preferred embodiment, the at least one aldehyde dehydrogenase may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, 13, 14, 59 or 60 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0055] Another embodiment of the invention may be directed to nucleic acid sequences or segments of such nucleic acid sequences that code for polypeptides with enzymatic functions for the purification, secretion, detection or localization of at least one α-dioxygenase and / or at least one aldehyde dehydrogenase and / or at least one lipase and / or at least one NADH oxidase. These nucleic acid segments are also called tag sequences and may precede (N-terminal) and / or follow (C-terminal) the nucleic acid segment coding for at least one α-dioxygenase and / or at least one aldehyde dehydrogenase. Preferred tag sequences are selected from the following list: polyhistidine (His) tag, glutathione S-transferase (GST) tag, thioredoxin tag, FLAG tag, green fluorescent protein (GFP) tag, streptavidin tag, maltose binding protein (MBP) tag, chloroplast transit peptide tag, mitochondrial transit peptide tag and / or secretion tag. Moreover, those skilled in the art are aware of several other suitable tag sequences for the microorganism or fungus of interest as a production strain, which allow for secretion of the polypeptide of interest directly into the culture supernatant, which in some embodiments may be advantageous in facilitating obtaining and optionally purifying the polypeptide of interest.

[0056] In another aspect of the invention, the invention relates to a vector system, preferably a plasmid vector system, comprising at least one vector or plasmid vector, comprising a nucleic acid segment (a) comprising at least one gene specifying a sequence for an α-dioxygenase, and a nucleic acid segment (b) comprising at least one gene specifying a sequence for an aldehyde dehydrogenase; optionally comprising as part of nucleic acid segment (a) and / or (b) a nucleic acid segment comprising at least one gene encoding an NADH oxidase and / or a nucleic acid segment comprising at least one gene encoding a lipase, wherein nucleic acid segment (a) and / or nucleic acid segment (b) are provided on the same vector or on two or more separate vectors.

[0057] According to a preferred embodiment, the vector system may code for the expression of at least one polypeptide, said polypeptide being selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 59 or 60 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0058] In another aspect, the present invention relates to a composition comprising an aldehyde mixture obtainable by the process according to the invention, the composition comprising at least one, or preferably at least two or three, aldehydes selected from the group consisting of 7Z-tetradecenal, 8Z-pentadecenal, pentadecanal, 7Z-hexadecenal and 8Z-heptadecenal, the one or more aldehydes being present in an amount of about 0.0001 to 20% by weight, preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight, based on the total composition, and the composition optionally comprising a further aldehyde obtainable by the process according to any one of claims 1 to 12, or the composition comprises 7Z-tetradecenal in an amount of about 0.0001 to 20% by weight, or the composition comprises 8Z-pentadecenal in an amount of about 0.0001 to 20% by weight. The present invention relates to a composition comprising at least 8Z-pentadecenal and pentadecanal, and the proportion of 8Z-pentadecenal and pentadecanal in the entire composition is about 0.0001 to 20% by weight, or the composition comprises pentadecanal in an amount of about 0.0001 to 20% by weight, or the composition comprises 7Z-hexadecenal in an amount of about 0.0001 to 20% by weight, or the composition comprises 8Z-heptadecenal in an amount of about 0.0001 to 20% by weight, or the composition comprises at least 8Z-pentadecenal and 8Z-heptadecenal, and the proportion of 8Z-pentadecenal and 8Z-heptadecenal in the entire composition is about 0.0001 to 20% by weight.

[0059] A composition in the context of the present invention is a semi-finished or finished product composition, preferably selected from the group consisting of preparations used as nutritional or recreational or cosmetic or cleaning preparations.

[0060] Preparations used for nutrition or entertainment within the meaning of the present invention are, for example, bakery products (e.g. bread, dry cookies, cakes, other pastries), confectionery (e.g. chocolate, chocolate bar products, other bar products, fruit gums, hard and soft caramels, chewing gum), alcoholic or non-alcoholic drinks (e.g. coffee, tea, wine, wine-based drinks, beer, beer-based drinks, liquor, spirits, brandy, fruit-based sodas, isotonic drinks, soft drinks, nectars, fruit and vegetable juices, fruit or vegetable juice preparations), instant drinks (e.g. instant cocoa drinks, instant tea drinks, instant coffee drinks, instant fruit drinks), meat products (e.g. ham, fresh or raw sausage preparations, seasoned or marinated fresh or salted meat products), eggs or egg products (dried eggs, egg whites, egg yolks), cereal products (e.g. breakfast cereals, cereal bars, pre-cooked ready-to-eat rice products), dairy products (e.g. milk drinks, buttermilk drinks, milk ice cream, yogurt, etc.). These include but are not limited to: yogurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, partially or fully hydrolysed milk protein containing products), products made from soy protein or other soy fractions (e.g. soy milk and products derived therefrom, fruit drinks containing soy protein, preparations containing soy lecithin, fermented products such as tofu or tempeh or products made therefrom), fruit preparations (e.g. jams, fruit ice cream, fruit sauces, fruit fillings), vegetable preparations (e.g. ketchup, sauces, dried vegetables, frozen vegetables, pre-cooked vegetables, cooked vegetables), snacks (e.g. baked or fried potato chips or potato dough products, corn or peanut based extrusions), fats and oil based products or their emulsions (e.g. mayonnaise, remoulade sauce, dressings), other ready-to-eat meals and soups (e.g. dry soups, instant soups, pre-cooked soups), for example spices, seasoning mixtures and especially seasonings used in the snack sector.Preparations within the meaning of the present invention are also used as semi-finished products for the manufacture of further preparations used for nutrition or entertainment. Preparations within the meaning of the present invention may be in the form of capsules, tablets (uncoated as well as coated tablets, e.g. enteric coated), lozenges, granules, pellets, solid mixtures, dispersions in a liquid phase, as emulsions, as powders, as solutions, as pastes or as other preparations that can be swallowed or chewed as food supplements.

[0061] Cosmetic or washing preparations in the sense of the present invention are preparations that can be used to cleanse, groom or perfume the body or for washing.Preferred cosmetic preparations are floor cleaners, window cleaners, dishwashing detergents, bathroom and sanitary cleaners, scouring milks, solid and liquid toilet cleaners, powder and foam carpet cleaners, fabric fresheners, ironing aids, liquid detergents, powder detergents, laundry pretreatment agents such as bleaching powder, softeners, soaking agents and stain removers, laundry softeners, cleaning soaps, cleaning tablets in liquid, gel or solid form, disinfectants, surface disinfectants and air fresheners, aerosol sprays, waxes and polishes such as furniture polishes, floor waxes, solid and liquid soaps such as shoe polishes and personal care products, shower gels, shampoos, shaving soaps, shaving foams, bath oils, oil-in-water, water-in-oil and water-in-oil-in-water cosmetic emulsions such as skin creams and lotions, facial creams and lotions. , sunscreen creams and lotions, after-sun creams and lotions, hand creams and lotions, foot creams and lotions, depilatory creams and lotions, after-shave creams and lotions, suntan creams and lotions, hair care products such as hair sprays, hair gels, hair lotions for setting, hair conditioners, permanent and semi-permanent hair colorants, cold wave and hair straightening products, hair styling products such as hair tonics, hair creams and lotions, deodorants and antiperspirants such as underarm sprays, roll-ons, deodorant sticks, deodorant creams, decorative cosmetic products such as eye shadows, make-up, lipsticks, mascaras, nail varnishes, as well as candles, kerosene, perfume sticks, insecticides, insect repellents and propellants.

[0062] In a preferred embodiment, the composition obtained contains a further aldehyde, carboxylic acid or alcohol selected from the group consisting of dodecanol, tridecanal, tridecanal, tetradecanal, 8Z-tetradecenol, tetradecanol, pentadecanal, pentadecanol, octanoic acid, decanoic acid, tridecanal, tridecenal, pentadecenal and 8Z-tetradecenal depending on the embodiment of the process according to the invention.

[0063] In yet another embodiment, the individual components of the resulting mixture are partially or wholly purified by methods known to those skilled in the art. Thus, the main advantage of these mixtures is their universal applicability in a wide range of industries, since they can be used both as mixtures and as individual components.

[0064] The invention is further described below with reference to non-limiting examples and based on the disclosure provided in the sequence listing and the figures. EXAMPLES

[0065] Example 1: Creation of a single construct For the generation of single constructs used for protein expression, suitable vectors with coding sequences were generated. For this purpose, the coding sequences of each of the various target genes were synthesized and cloned between two restriction sites in the vector pET28a (Merck Chemicals GmbH, Schwalbach) or pETDuet-1 (Merck Chemicals GmbH, Schwalbach). An overview of the cloned genes and the respective restriction sites used for cloning is summarized in Table 1. For all SEQ ID NOs, the codon usage of the genes was adapted to E. coli as one of the intended expression hosts. Furthermore, SEQ ID NO: 3 has point mutations with respect to the original sequence obtained from the respective organism.

[0066] [Table 1]

[0067] The obtained plasmids were introduced into competent E. coli BL21(DE3) cells using standard transformation methods (Maniatis et al., 1983), and the cells were grown on selective solid medium (LB + ampicillin (100 mg / L) + 6 g / L agar) at 37°C for 18 h.

[0068] Example 2: Generation of various VhAldh mutants by mutagenesis Starting from the plasmid pET-Duet_LsNOX_VhFALDH, the different enzyme mutants were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, Waldbronn, Germany). In the process, the targeted mutations were introduced into the coding sequence of VhFaldh using specific primers according to the manufacturer's instructions. For the mutant form of VhFALDH pET-Duet_LsNOX_VhFALDHT175Q, the primers VhFALDH-T175Q_fw (SEQ ID NO: 15) and VhFALDH-T175Q_rv (SEQ ID NO: 16) were used. The sequences of the nucleic acids or the corresponding translated proteins are shown in SEQ ID NO: 3 and 10, respectively.

[0069] Example 3: Protein Expression and Purification Osa-DOX To prepare the protein expression product in a volume of 50 mL, first a preculture of 5 mL LB medium (Carl Roth GmbH, Karlsruhe, Germany) with the appropriate antibiotic was prepared, and E. coli BL21(DE3) cells harboring pET28a_OsaDOX were removed from the agar plate using an inoculation loop and transferred to the preculture, which was then incubated for 16 h at 37 °C and 150 rpm. From the preculture, a main culture of 50 mL LB medium (Carl Roth GmbH, Karlsruhe, Germany) with the appropriate antibiotic was inoculated to obtain an optical density at 600 nm of 0.05. The main culture was then incubated at 37 °C and 200 rpm until an optical density at 600 nm of 0.6–1.0 was obtained. To induce protein expression, 0.5 mM isopropyl-β-D-thiogalactopyranoside was added at this point, and the culture was incubated for a further 16 h at 21 °C. Finally, the main culture was centrifuged at 17,105 × g for 10 min to obtain a cell pellet and subsequently perform protein extraction and purification. For this purpose, cell disruption was first performed using B-PER protein extraction reagent (Thermo Fisher Scientific, Bonn, Germany) according to the manufacturer's instructions. The obtained cell lysate was then either used as is or processed using a 1 mL HisPur Ni-NTA chromatography column (Thermo Fisher Scientific, Bonn, Germany) according to the manufacturer's instructions.

[0070] Example 4: Protein expression from VhFaldh and LsNOX To prepare the protein expression product in a volume of 50 mL, a preculture of 5 mL LB medium (Carl Roth, Karlsruhe, Germany) with the appropriate antibiotic was first prepared, and E. coli BL21(DE3) cells containing pET-Duet_LsNOX_VhFALDH were removed from the agar plate using an inoculation loop and transferred to the preculture, which was then incubated at 37 °C and 150 rpm for 16 h. From the preculture, a main culture of 50 mL LB medium (Carl Roth, Karlsruhe, Germany) with the appropriate antibiotic was inoculated to obtain an optical density at 600 nm of 0.1. The main culture was then incubated at 37 °C and 200 rpm until an optical density at 600 nm of 0.6–0.8 was obtained. To induce protein expression, 0.5 mM isopropyl-β-D-thiogalactopyranoside was added at this point, and the culture was incubated at 16 °C for a further 16 h. Finally, the main culture was centrifuged at 17,105 × g for 10 min to obtain a cell pellet and subsequently perform protein extraction and purification. For this purpose, cell disruption was first performed using B-PER Protein Extraction Reagent (Thermo Fisher Scientific, Bonn, Germany) according to the manufacturer's instructions. The resulting turbid cell lysate was then used as is.

[0071] Example 5: Hydrolysis of fatty acid triglycerides 5–10 g of CALB-imm (Novozym 435; 10,000 U / g) in 380 mL of tert-butanol (Carl Roth) was stirred for 0.5–3 h in a 1 L round-bottom flask. Then, 50 g of triglyceride and 15 mL of 200 mM Tris-HCl, pH 8 buffer were added. The reaction was carried out at 60 °C under vigorous stirring for 15–21 h. Subsequently, the enzyme was removed by filtration and the reaction mixture was concentrated to dryness using a rotary evaporator. The product was stored overnight at 4 °C and then taken up in 200 mL of 200 mM Tris-HCl, pH 2. Fatty acids were extracted by adding 500 mL of tert-butyl dimethyl ether (Honeywell) with stirring for 1 h. The mixture was transferred to a separatory funnel and the phase separation was waited until it occurred. Subsequently, the organic phase was transferred to a round-bottom flask and concentrated to dryness using a rotary evaporator. The products were stored at -20°C and analyzed for composition by GC-MS (20m ZB-1, 60-9-300, split 60:1). Retention indices of the products were determined by comparison with standards.

[0072] Example 6: Preparation of aldehyde mixture using Os-α-Dox and VhFaldh For the reaction of various fatty acids or hydrolysates according to Example 5, lysates from E. coli BL21(DE3) cells containing pET28a_OsaDOX according to Example 3 and from E. coli BL21(DE3) cells containing pET-Duet_LsNOX_VhFALDH according to Example 4 were used. For this purpose, 25 mM linoleic acid (Sigma-Aldrich), 40 g / L E. coli α-Dox according to Example 3, 10 g / L lysate from E. coli VhFALDH_NOX, 3.25 mM NAD + (Sigma-Aldrich), 0.5% glucose, 0.1% (wt / vol) gum arabic and 4.7 mL potassium phosphate buffer, pH 8, were suspended in a 50 mL reaction tube.

[0073] Here, buffer, linoleic acid, and NAD were mixed in a 100 mL shake flask with baffles. +, glucose and gum arabic were combined and mixed for 5 min at 200 rpm and 37° C. Cells or lysates as indicated were then added and reactions were incubated at 200 rpm and 37° C. for 24 h.

[0074] The mixture was subsequently adjusted to pH 2 with hydrochloric acid and extracted twice by adding an equal volume of ethyl acetate (Sigma-Aldrich) with vortex mixing (1 min). Separation of the organics from the aqueous phase was performed by centrifugation at 17.105×g for 10 min. Samples were analyzed by gas chromatography (20m ZB-1, df: 0.18 μm, id: 0.18 mm / 60-12-300°KAS). Retention indices for selected GC methods were determined by comparison with standards and GC-MS data.

[0075] Example 7: Aldehyde mixture according to the invention Tables 2 and 3 show examples of mixtures obtained by the process according to the invention. In addition to the mixtures obtained, the individual components can be purified in subsequent steps.

[0076] [Table 2]

[0077] [Table 3]

Claims

1. A biotechnological process for the production of at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid, comprising: Providing at least one α-dioxygenase and at least one aldehyde dehydrogenase; reacting the at least one α-dioxygenase and the at least one aldehyde dehydrogenase simultaneously with at least one carboxylic acid or carboxylic acid esterified with an alcohol as a starting material; Obtaining at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid by said reaction. Including, the at least one α-dioxygenase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 11, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, or 56, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto; the at least one aldehyde dehydrogenase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 12, 13, 14, 59, or 60, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto; method.

2. The biotechnological method comprises: i. providing at least one recombinant microorganism or fungus harboring a nucleic acid segment comprising at least one gene encoding an α-dioxygenase and / or at least one gene encoding an aldehyde dehydrogenase; ii. culturing said at least one recombinant microorganism under conditions that allow expression of said corresponding expression product; iii. adding to the at least one cultured recombinant microorganism at least one carboxylic acid or at least one carboxylic acid esterified with an alcohol; iv. reacting the at least one α-dioxygenase and the at least one aldehyde dehydrogenase simultaneously with the at least one carboxylic acid or carboxylic acid esterified with an alcohol of step iii to obtain at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid; 2. The method of claim 1, which is a fermentative process comprising:

3. The biotechnological production method comprises: i. providing at least one α-dioxygenase and at least one aldehyde dehydrogenase, wherein said at least one α-dioxygenase and / or said at least one aldehyde dehydrogenase can be naturally, chemically or biotechnologically produced; ii. adding to the provided enzyme of step i at least one carboxylic acid or at least one carboxylic acid esterified with an alcohol; iii. reacting the at least one α-dioxygenase and the at least one aldehyde dehydrogenase simultaneously with the at least one carboxylic acid or the carboxylic acid esterified with an alcohol of step ii to obtain at least one unsaturated aldehyde and its corresponding carboxylic acid and / or at least one saturated aldehyde and its corresponding carboxylic acid; 2. The method of claim 1, which is an enzymatic method comprising:

4. The method according to any one of claims 1 to 3, wherein at least one of said starting materials is of biotechnological, natural or chemical origin, or a combination thereof.

5. The starting materials include palmitoleic acid, arachidonic acid, α-linolenic acid, oleic acid, punicic acid, α-eleostearic acid, docosahexaenoic acid, eicosapentaenoic acid, petroselinic acid, schormugic acid, α-licaric acid, olive oil, rapeseed oil, macadamia nut oil, sea buckthorn fruit oil, tung oil, fish oil, borage oil, tallow oil, parsley oil, oiticica oil, pomegranate seed oil, coconut oil, sunflower oil, grape seed oil, and Conidiobolus spp.

5. The method according to claim 1, wherein the oil is selected from the group consisting of fungal oils obtained from any of the genera selected from the genera Flammulina, Formes, Ganoderma, Mortierella, Panellus, Pleurotus, Psathyrella, Stereum, and Umbelopsis.

6. The products of the process are 7Z,10Z-hexadecadienal, 8Z,11Z-heptadecadienal, 6E,9E-pentadecadienal, 7Z-hexadecenal, 8Z-pentadecenal, 8Z,11Z,14Z-heptadecatrienal, 7Z,10Z,13Z-hexadecatrienal, 4Z,7Z,10Z,13Z-nonadecatetraenal, 8Z,10E,12E-heptadecatrienal, 8Z,10E,12Z ... Putadecatrienal, 7Z,9E,11Z-hexadecatrienal, 7Z,9E,11E-hexadecatrienal, 9-decenal, 10-undecenal, 3Z-decenal, 4Z-undecenal, 7Z-dodecenal, 8Z-tridecenal, 7Z-tetradecenal, 8Z-pentadecenal, 9Z-tetradecenal, 10Z-pentadecenal, 7Z-pentadecenal, 8Z-hexadecenal, 9Z-hexadecenal Undecane, 10Z-heptadecenal, 5Z,8Z-tetradecadienal, 6Z,9Z-pentadecadienal, 7Z,10Z-tetradecadienal, 8Z,11Z-pentadecadienal, 7Z,9E-hexadecadienal, 8Z,10E-heptadecadienal, 8E,10Z-hexadecadienal, 9E,11Z-heptadecadienal, 9-methylundecanal, 10-methylundecanal, 10-methyldodecanal The method according to any one of claims 1 to 5, wherein the carboxylic acid is at least one saturated aldehyde and its corresponding carboxylic acid selected from the group consisting of 11-methyldodecanal, 11-methyltridecanal, 12-methyltridecanal, 12-methyltetradecanal, 13-methyltetradecanal and 13-methylpentadecanal, and / or at least one unsaturated aldehyde and its corresponding carboxylic acid.

7. 7. The method according to any one of claims 1 to 6, wherein the at least one recombinant microorganism is selected from the group consisting of Escherichia coli, Bacillus spp., Saccharomyces spp., Hansenula or Komagatella spp., Kluyveromyces spp.

8. 8. The method according to claim 1 , wherein additionally at least one NADH oxidase and / or at least one lipase is provided.

9. 9. The method of claim 8, wherein the at least one NADH oxidase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 and 64, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

10. The at least one lipase may be selected from the group consisting of Candida antarctica, Aspergillus niger, Rhizopus oryzae, Penicillium camembertii, Mucor juvanicus, Penicillium roqueforti, porcine pancreas, Candida rugosa, Rhizomucor miehei, Candida antarctica, and the like.

9. The method of claim 8, wherein the lipase is a commercially available lipase selected from the group consisting of Rhizopus antarctica or Rhizopus delemar.

11. A vector system consisting of at least one vector or plasmid vector, comprising a nucleic acid segment (a) comprising at least one gene encoding an α-dioxygenase and a nucleic acid segment (b) comprising at least one gene encoding an aldehyde dehydrogenase; with or without a nucleic acid segment comprising at least one gene encoding an NADH oxidase and / or a nucleic acid segment comprising at least one gene encoding a lipase as part of said nucleic acid segment (a) and / or (b), said nucleic acid segment (a) and / or said nucleic acid segment (b) being provided on the same vector or on two or more separate vectors, The vector system encodes for expression of at least one polypeptide, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 59 or 60, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

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