Patent Application
20230407349
The present invention relates to biotechnological methods for the production of saturated and unsaturated aldehydes, as well as mixtures thereof, using at least one alpha-dioxygenase and at least one aldehyde dehydrogenase. The method may be carried out either fermentatively or enzymatically. Furthermore, the present invention 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. Further, the present invention relates to compositions obtained by the methods according to the present invention.
In the field of industrial production of flavors, there is a constant need for efficient and cost-effective ways of synthesizing flavors. An example of such flavors are (un)saturated aldehydes. An exemplary aldehyde is 8Z-tetradecenal or 1-decenal. This class of substances is mainly found in meat preparations and citrus oils. At present, however, no generally applicable biotechnological process for the production of these aldehydes and the corresponding acids has been described.
Processes for the preparation of unsaturated aldehydes are known from the prior art. For example, 8Z,11Z-heptadecadienal can be obtained by the chemical oxidation of the unsaturated alcohol (JP 63233914). Various aldehydes can also be produced in a known manner from fatty acids by alpha-oxidation using plant enzymes, as described in WO2012025629 A1. The use of algal biomass to produce unsaturated aldehydes is also known (“Concise synthesis of (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,3Z)-7,10,13-hexadecatrienal, and (8Z11Z)-8,11-heptadecadienal, components of the essential oil of marine green alga Ulva pertusa,” Biosci Biotechnol Biochem. 2005 July; 69(7):1348-52). 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, JP05000974A).
Furthermore, numerous processes using aldehyde dehydrogenases are known to oxidize aldehydes to acids (Takeru Ishige et al., Appl. Environ. Microbiol. 2000, 66, 3481-3486; Tomohisa Kato et al., Extremophiles 2010, 14, 33.).
The above-mentioned chemical methods as well as methods based exclusively on the use of a very special enzyme alone have the disadvantage, however, that no comprehensive spectrum of aldehydes or mixtures thereof is accessible. Furthermore, some of the methods are based on the reaction with chemical reagents as well as catalysts, which is why such methods are not considered natural processes in the sense of EC 1334/2008. A combined method that specifically combines an alpha-dioxygenase with an aldehyde dehydrogenase and specifically expands the production spectrum is not known. In a recombinant reaction system with two or more enzymes, where the desired catalytic reactions are to 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 interfered with by, for example in the case of whole-cell catalysis, endogenous enzymes of the production organism. The reaction control is therefore crucial to ensure that the intermediates are also recognized by the second or respective next enzyme in the cascade and are available for the latter. Such coordination or combination is always technically challenging and cannot be predicted in a straight line, but rather requires intensive analyses on the enzymes of interest in the respective biotechnological context.
The natural occurrence of some unsaturated aldehydes with one or more double bonds such as 8Z-heptadecenal, 8Z,11Z-heptadecadienal or 8Z,11Z,14Z-heptadecatrienal has been reported in pressed juices of cucumber (Cucumber Aroma formation of cucumber (Cucumis sativus L.) and bitter gourd (Momordica charantia L.) by salt-squeezing, Journal of Home Economics of Japan Vol. 60 (2009) No. 10 p. 877-885) or the alga Ulva pertusa (Production of bioflavor by regeneration from protoplasts of Ulvapertusa (Ulvales, Chlorophyta) Hydrobiologia, 1990, Vol. 204, pp 143-149). The content of the aldehydes 8Z-heptadecenal, 8Z,11Z-heptadecadienal, 8Z,11Z,14Z-heptadecatrienal is reported in the literature as 2%, 19.9% and 26.1%.
The odor of 8Z,11Z-heptadecadienal is described as typical of algae (Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales, Chlorophyta), Hydrobiologia, 1990, Vol. 204, pp 143-149).
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.
8Z-pentadecenal is known to be a naturally occurring constituent of cucumber (Kemp, A C15 aldehyde from Cucumis sativus, Phytochemistry, Volume 16, Issue 11, 1977, Pages 1831-1832). The flavor of 8Z-pentadecenal is described primarily as oily, and flavor-enhancing properties on chicken and grilled beef are also described. Furthermore, a method for the production from 1-nonine and 6-bromo-1-hexanol is known, but this cannot be classified as a natural production process according to EC 1334/2008 (JP 2014043409).
The taste of 4Z,7Z,10Z,13Z-nonadecatetraenal is not known and no manufacturing methods existed until now.
Various aldehydes can be produced from fatty acids by alpha-oxidation using plant enzymes, as described in WO2012025629 A1. The use of algal biomass for the production of unsaturated aldehydes is also known.
Other processes for the production of fatty acids from the corresponding oils or fats by enzymatic cleavage are also known from the prior art. Bacterial lipases such as enzymes from Aspergillus niger, Rhizopus oryzae, Penicillium camembertii, Mucor juvanicus, Penicillium roquefortii, porcine pancreas, Candida rugosa, Rhizomucor miehei, Candida antarctica or Rhizops delemar are mainly used for this purpose (Industrial applications of microbial lipases, Enzyme and Microbial Technology Volume 39, Issue 2, 26 Jun. 2006, Pages 235-251).
It was therefore an object of the present invention to provide a method for the production of aldehydes or aldehyde mixtures which can be classified as a natural production process according to EC 1334/2008. Furthermore, it was an object to provide an integrated biotechnological process by selective tuning of enzymatic metabolic steps, which can provide the products of interest in high yield and purity without intermediate chemical steps.
Accordingly, in accordance with a primary aspect, the present invention relates to providing a method for producing saturated and unsaturated aldehydes using at least one alpha-dioxygenase and at least one aldehyde dehydrogenase.
Furthermore, according to one embodiment, the present invention relates to a fermentative method for the production of saturated and unsaturated aldehydes and mixtures thereof. In another embodiment of the method according to the present invention, an enzymatic method is provided.
In a further embodiment, the method according to the present invention relates to the use of reactants (educts) which are either of biotechnological, natural or chemical origin.
In yet another embodiment, the method according to the invention relates to preferred reactants selected from the group of carboxylic acids and carboxylic acids esterified with alcohols.
Furthermore, the method according to the invention relates to preferred product mixtures, microorganisms used in the method as well as amino acid sequences of the enzymes to be preferably used and nucleotide sequences or nucleic acid segments coding for the enzymes to be used.
Another aspect of the present invention relates to the provision of a vector system encoding the enzymes used according to the invention.
Another aspect of the present invention relates to a composition obtained by a method according to the invention.
SEQ ID NO 1: Nucleic acid sequence encoding the enzyme Os-alpha-DOX from 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: 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: Amno acid sequence encoding the enzyme Os-alpha-DOX from Oryza sativa.
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.
SEQ ID NO 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 a forward primer.
SEQ ID NO 16: Nucleic acid sequence encoding a reverse primer.
SEQ ID NO 17: Nucleic acid sequence encoding the enzyme At-alpha-DOX from Arabidopsis thaliana.
SEQ ID NO 18: Nucleic acid sequence encoding the enzyme At-alpha-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 Cucumis sativus.
SEQ ID NO 22: Nucleic acid sequence encoding the enzyme CsaDOX from Cucumis sativus.
SEQ ID NO 23: Nucleic acid sequence encoding the enzyme CsaDOxlike1 from Cucumis sativus.
SEQ ID NO 24: Nucleic acid sequence encoding the enzyme Le-alpha-DOX1 from Solanum lycopersicum.
SEQ ID NO 25: Nucleic acid sequence encoding the enzyme Le-alpha-DOX2 from Solanum lycopersicum.
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-alpha-DOX from Nicotiana attenuata.
SEQ ID NO 28: Nucleic acid sequence encoding the enzyme Nt-alpha-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-alpha-DOX from Pisum sativum.
SEQ ID NO 31: Nucleic acid sequence encoding the enzyme StaDOX2 from Solanum lycopersicum.
SEQ ID NO 32: Nucleic acid sequence encoding the enzyme StaDOX1.1 from Solanum lycopersicum.
SEQ ID NO 33: Nucleic acid sequence encoding the enzyme StaDOX1.2 from Solanum lycopersicum.
SEQ ID NO 34: Nucleic acid sequence encoding the enzyme StaDOX1.3 from Solanum lycopersicum.
SEQ ID NO 35: Nucleic acid sequence encoding the enzyme StaDOX1 from Solanum tuberosum.
SEQ ID NO 36: Nucleic acid sequence encoding the enzyme StaDOX2 from Solanum tuberosum.
SEQ ID NO 37: Amino acid sequence encoding the enzyme At-alpha-DOX2 from Arabidopsis thaliana.
SEQ ID NO 38: Amino acid sequence encoding the enzyme At-alpha-DOX from Arabidopsis thaliana.
SEQ ID NO 39: Amino acid sequence encoding the enzyme CaaDOX from Capsicum annuum.
SEQ ID NO 40: Amino acid sequence encoding the enzyme CbaDOX from Cercospora beticola.
SEQ ID NO 41: Amino acid sequence encoding the enzyme CsaDOX2 from Cucumis sativus.
SEQ ID NO 42: Amino acid sequence encoding the enzyme CsaDOX from Cucumis sativus.
SEQ ID NO 43: Amino acid sequence encoding the enzyme CsaDOxlike1 from Cucumis sativus.
SEQ ID NO 44: Amino acid sequence encoding the enzyme Le-alpha-DOX1 from Solanum lycopersicum.
SEQ ID NO 45: Amino acid sequence encoding the enzyme Le-alpha-DOX2 from Solanum lycopersicum.
SEQ ID NO 46: Amino acid sequence encoding the enzyme NaaDOX2 from Nicotiana attenuata.
SEQ ID NO 47: Amino acid sequence encoding the enzyme Na-alpha-DOX from Nicotiana attenuata.
SEQ ID NO 48: Amino acid sequence encoding the enzyme Nt-alpha-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-alpha-DOX from Pisum sativum.
SEQ ID NO 51: Amino acid sequence encoding the enzyme StaDOX2 from Solanum lycopersicum.
SEQ ID NO 52: Amino acid sequence encoding the enzyme StaDOX1.1 from Solanum lycopersicum.
SEQ ID NO 53: Amino acid sequence encoding the enzyme StaDOX1.2 from Solanum lycopersicum.
SEQ ID NO 54: Amino acid sequence encoding the enzyme StaDOX1.3 from Solanum lycopersicum.
SEQ ID NO 55: Amino acid sequence encoding the enzyme StaDOX1 from Solanum tuberosum.
SEQ ID NO 56: Amino acid sequence encoding the enzyme StaDOX2 from Solanum tuberosum.
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 an NAD(P)H oxidase from Lactobacillus sanfranciscensis.
SEQ ID NO 62: Amino acid sequence encoding an NAD(P)H oxidase from Lactobacillus sanfranciscensis.
SEQ ID NO 63: Nucleic acid sequence encoding an NADH oxidase from Lactobacillus brevis.
SEQ ID NO 64: Amino acid sequence encoding an NADH oxidase from Lactobacillus brevis.
The term vector system as used herein means a system consisting of or containing at least one or more vectors or plasmid vectors. Thus, a vector system may include a (plasmid) vector encoding multiple different target genes. A vector system may further comprise multiple (plasmid) vectors, which in turn comprise at least one target gene according to the present disclosure. Thus, a vector system may comprise only one (plasmid) vector construct or multiple (plasmid) vector constructs, the latter of which may be stably or transiently transformed simultaneously or sequentially into the corresponding recombinant host organism such that the target genes encoded by the individual constructs may be transcribed and translated by the host organism.
The terms amino acid, protein and polypeptide are used interchangeably herein. The amino acids disclosed herein, or functional portions thereof, have enzymatic function after correct folding. Accordingly, the term enzyme is also used herein interchangeably for the term amino acid.
Whenever the present disclosure refers to sequence homologies or sequence identities of nucleic or protein 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 growing and cultivation, isolation and purification of a recombinant microorganism or fungus or a protein or enzyme encoded by a nucleic acid according to the disclosure of the present invention is known to those skilled in the art.
The terms protein, polypeptide and enzyme are used interchangeably due to the invariably enzymatic function of the gene products disclosed herein. Likewise, the terms gene and nucleic acid (segment) are used interchangeably for purposes of the present disclosure.
The nucleic acids, nucleotide sequences or nucleic acid segments (these terms are used interchangeably for DNA sequences encoding a functional enzyme, or a functional region thereof) used according to the present invention to express a desired target protein may optionally be codon-optimized, i.e., the codon usage of a gene is adapted to that of the recombinant microorganism or fungus selected as the expression strain. It is known to those skilled in the art that a desired target gene encoding a protein of interest can be modified without altering the translated protein sequence to accommodate specific species-dependent codon usage. Thus, the nucleic acids of the present invention to be transformed can be specifically adapted to the codon usage of E. coli or another bacterium, Saccharomyces spp. or another yeast.
The disclosed carboxylic acids can be present either in their free form or esterified to alcohols, which occur, for example, as components of lipids.
Where the configuration(s) of (Z)/(E) isomers is not specifically indicated herein for a named compound, the indication includes all possible configurational isomers of the corresponding compound, or a mixture thereof. It should be noted that certain enzymes mentioned herein are capable of recognizing and converting both isomers of a compound of interest.
The problem of the present invention is primarily solved by providing a biotechnological method 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, the method comprising providing at least one alpha-dioxygenase and at least one aldehyde dehydrogenase.
The aldehyde obtained is always shortened by one carbon atom compared to the reactant used. A saturated aldehyde in the context of the present invention refers to an aldehyde without double bonds, whereas an unsaturated aldehyde refers to aldehydes with double bonds present. The corresponding carboxylic acid in this case is a compound with the same chain length and the same degree of saturation as the aldehyde obtained, but which carries at least one carboxyl group. The at least one alpha-dioxygenase used according to the invention catalyzes the oxidation of carboxylic acids of chain length C6-C22 at the alpha carbon atom, so that the aldehyde corresponding to the carboxylic acid and shortened by one carbon atom is formed.
Due to its substrate specificity, the at least one aldehyde dehydrogenase used catalyzes the oxidation of an aldehyde to the corresponding carboxylic acid, which in turn can be further converted to an aldehyde shortened by one carbon atom with the alpha-dioxygenase. Thus, the method of the invention can be used to selectively produce aldehyde mixtures with aldehydes that have different chain lengths.
In one embodiment of the method according to the invention, the alpha-oxidation and the aldehyde oxidation can be carried out simultaneously, whereas in another embodiment, the alpha-oxidation can be carried out first and then the aldehyde oxidation, whereby the reaction cycle can be continued in both embodiments until the desired product is obtained.
In a preferred embodiment of the method according to the invention, the biotechnological production method may be a fermentative method comprising the following steps:
A fermentative method in the context of the present invention refers to a method in which, in at least one step, a recombinant microorganism is cultured that expresses the at least one alpha-dioxygenase and the at least one aldehyde dehydrogenase required to produce the product of the invention. The expression product here is the at least one alpha-dioxygenase and/or the at least one aldehyde dehydrogenase. Thereby, according to one embodiment, the reaction of the reactant can take place by secretion of the enzyme into the reaction mixture in which the reactants are present, whereas according to another embodiment, the reaction can take place in the cytosol of the at least one microorganism and the product can subsequently be secreted.
Providing in the context of the present invention always means involving an active step to make available a reactant, an enzyme or the like. According to one embodiment, providing may always be a technical step, such as cloning a reaction organism or producing a reactant.
In another preferred embodiment of the method according to the invention, the biotechnological production method may be an enzymatic method comprising the following steps:
An enzymatic method in the context of the present invention denotes a method in which the at least one alpha-dioxygenase and the at least one aldehyde dehydrogenase may be present purified or partially purified outside a microorganism. In one embodiment, the two enzymes may be present as a cell lysate, which denotes the state of microorganisms that can no longer be cultured and that have been digested by physical, chemical or enzymatic processes. In a further embodiment, the enzymes may be present with a purity of >80%, having been purified by a purification process familiar to those skilled in the art. In yet another embodiment, the enzymes may be present at <80% purity, having been partially purified by a purification process known to those skilled in the art. In still another embodiment, the enzymes may be obtained by protein synthesis and optionally purified.
A reactant (starting material) of biotechnological origin is a reactant produced by fermentation of microorganisms, whereby a reactant of natural origin may be obtained from a natural source, for example, a plant, fungus, animal, microorganism, or soil. A reactant of chemical origin may be produced by a chemical synthesis from precursors of the reactant.
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 the reactants of biotechnological, natural or chemical origin.
According to a further preferred embodiment of the method of the invention, the reactants can be selected from the group of carboxylic acids and carboxylic acids esterified with alcohols. Preferred reactants are those selected from the group consisting of palmitoleic acid, arachidonic acid, alpha-linolenic acid, linoleic acid, gamma-linolenic acid, oleic acid, punicic acid, alpha-elaeostearic acid, docosahexaenoic acid, eicosapentaenoic acid, petroselinic acid, chaulmoogric acid, alpha-licaric acid, olive oil, canola oil, macadamia nut oil, sea buckthorn fruit oil, tung oil, fish oil, borage oil, chaulmoogra oil, parsley oil, oiticica oil, pomegranate seed oil, coconut oil, sunflower oil, field stone seed oil and grape seed oil, as well as fungal oils obtained from genera independently selected from Conidiobolus, Flammulina, Fomes, Ganoderma, Mortierella, Panellus, Pleurotus, Psathyrella, Stereum, Umbelopsis.
In a further preferred embodiment, the product of the method 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. Here, the products of the method according to the invention can preferably be selected from the group comprising 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-nonadecatetraenal, 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, 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, 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.
In the context of the present invention, all compounds in which the positions of the double bond are not specifically indicated with respect to the (Z)/(E) configuration include all possible positions of the double bond and mixtures of the relevant compounds.
According to a further embodiment of the method of the invention, the at least one recombinant microorganism may be selected from the group consisting of Escherichia coli, preferably E. coli BL21, E. coli MG1655, E. coli W3110, and their derivatives, Bacillus spp, preferably B. licheniformis, B. subitilis or B. amyloliquefaciens, and their derivatives, Saccharomyces spp, preferably S. cerevesiae, and their derivatives, Hansenula or Komagatella spp, preferably K. phaffii and H. polymorpha, and their derivatives, Kluyveromyces spp, preferably K. lactis.
According to a preferred embodiment, at least one NADH oxidase and/or at least one lipase may additionally be provided.
NADH oxidases are those that can catalyze the oxidation of an aldehyde due to their substrate specificity and regioselectivity. In a preferred embodiment of the method according to the invention, these can be used as additional enzymes to make the closely coordinated methods even more efficient by recycling the corresponding cofactor.
In another preferred embodiment of the method of the invention, the at least one NADH oxidase may comprise an amino acid sequence, wherein the amino acid sequence is independently selected from the group consisting of SEQ IDs NO: 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 IDs NO: 61 and 62, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.
Furthermore, an enzyme that catalyzes the reaction according to the invention according to the various aspects and embodiments of the present invention may be a catalytically active domain or fragment of the particular enzyme from which it is derived.
In another preferred embodiment, the at least one lipase may be a commercial 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.
In yet another preferred embodiment of the method of the invention, the alpha-dioxygenase may comprise 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.
In another preferred embodiment, the at least one aldehyde dehydrogenase may comprise or consist of an amino acid sequence, wherein the amino acid sequence may be 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.
Another embodiment of the present invention may be directed to nucleic acid sequences or segments thereof encoding polypeptides having enzymatic function for the purpose of purification, secretion, detection or localization of the at least one alpha-dioxygenase and/or the at least one aldehyde dehydrogenase and/or the at least one lipase and/or the at least one NADH oxidase. These nucleic acid segments are also referred to as tag sequences and may precede (N-terminal) and/or follow (C-terminal) the nucleic acid segments encoding the at least one alpha-dioxygenase and/or the 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, mitochondrial transit peptide and/or secretion tag. Moreover, the skilled person is aware of a number of other suitable tag sequences for microorganisms or fungi of interest as a production strain, wherein these tag sequences allow secretion of a polypeptide of interest directly into the culture supernatant. This may be advantageous in some embodiments to facilitate obtaining and optionally purifying a polypeptide of interest.
In another aspect of the present invention, the invention relates to a vector system, preferably a plasmid vector system, which comprises at least one vector or plasmid vector comprising a nucleic acid segment (a) comprising at least one gene coding for an alpha-dioxygenase, and a nucleic acid segment (b) comprising at least one gene coding for an aldehyde dehydrogenase, and optionally comprising, as part of the 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 the nucleic acid segment (a) and/or the nucleic acid segment (b) is provided on the same vector, or on two or more separate vectors.
According to a preferred embodiment, the vector system may encode expression of at least one polypeptide, wherein said polypeptide is selected from the group consisting of SEQ ID NOs: 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.
In another aspect, the present invention relates to a composition comprising an aldehyde mixture obtained by a method according to the invention, characterized in that the composition comprises 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, wherein the one or more aldehydes are each present in a proportion of about 0.0001-20 wt. %, preferably 0.001-10 wt. %, more preferably 0.01-5 wt. %, each with respect to the total composition, and wherein the composition optionally contains further aldehydes obtained by a method according to any of claims 1 to 12, or wherein the composition comprises 7Z-tetradecenal in a proportion of about 0.0001-20 wt. %, or wherein the composition comprises 8Z-pentadecenal in a proportion of about 0.0001-20 wt %, relative to the total composition, or wherein the composition comprises pentadecanal in an amount of about 0.0001-20 wt. %, relative to the total composition, or wherein the composition comprises 7Z-hexadecenal in an amount of about 0.0001-20 wt. %, relative to the total composition, or wherein the composition comprises 8Z-heptadecenal in an amount of about 0.0001-20 wt. %, relative to the total composition, or wherein the composition comprises at least 8Z-pentadecenal and pentadecanal, wherein the proportion of 8Z-pentadecenal and pentadecanal in the total composition comprises about 0.0001-20 wt. %, or wherein the composition comprises at least 8Z-pentadecenal and 8Z-heptadecenal, wherein the proportion of 8Z-pentadecenal and 8Z-heptadecenal in the total composition comprises about 0.0001-20 wt. %.
A composition in the context of the present invention is a semi-finished or finished product composition and is preferably selected from the group consisting of a preparation serving nourishment or pleasure or a cosmetic or cleansing preparation.
Preparations serving nourishment or pleasure in the sense of the present invention are e.g. bakery products (e.g. bread, dry cookies, cakes, other pastries), confectionery (e.g. chocolates, chocolate bar products, other bar products, fruit gums, hard and soft caramels, chewing gum), alcoholic or non-alcoholic beverages (e.g., coffee, tea, wine, wine-based beverages, beer, beer-based beverages, liquors, spirits, brandies, fruit-based sodas, isotonic beverages, soft drinks, nectars, fruit and vegetable juices, fruit or vegetable juice preparations), instant beverages (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 cured meat products), eggs or egg products (dried egg, egg white, egg yolk), 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, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, partially or wholly hydrolyzed milk protein-containing products), products made from soy protein or other soybean fractions (e.g. soy milk and products made 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 deep-fried potato chips or potato dough products, corn- or peanut-based extrudates), fat- and oil-based products or emulsions thereof (e.g. mayonnaise, remoulade, dressings), other ready meals and soups (e.g. dry soups, instant soups, pre-cooked soups), spices, seasoning mixtures and in particular seasonings, which are used for example in the snack sector. The preparations in the sense of the invention can also serve as semi-finished goods for the production of further preparations serving nourishment or pleasure. The preparations within the meaning of the invention may also be in the form of capsules, tablets (uncoated as well as coated tablets, e.g. enteric coatings), lozenges, granules, pellets, solid mixtures, dispersions in liquid phases, as emulsions, as powders, as solutions, as pastes or as other preparations which can be swallowed or chewed as food supplements.
A cosmetic or cleansing preparation in the sense of the present invention is a preparation which can be used for cleaning, caring for or perfuming the body or for cleansing. Preferred cosmetic preparations are selected from the group consisting of floor cleaners, window glass cleaners, dishwashing detergents, bathroom and sanitary cleaners, scouring milks, solid and liquid toilet cleaners, powder and foam carpet cleaners, textile fresheners, ironing aids, liquid detergents, powder detergents, laundry pretreatment agents such as bleaches, softeners, soaking agents and stain removers, laundry softeners, washing soaps, washing tablets, disinfectants, surface disinfectants and air fresheners in liquid, gel or solid form, aerosol sprays, waxes and polishes such as furniture polishes, floor waxes, shoe polishes and personal care products such as solid and liquid soaps, shower gels, shampoos, shaving soaps, shaving foams, bath oils, cosmetic emulsions of the oil-in-water type, water-in-oil type and water-in-oil-in-water type, 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, tanning creams and lotions, hair care products, such as hair sprays, hair gels, setting hair lotions, hair conditioners, permanent and semi-permanent hair colorants, hair shaping products such as cold waves and hair straightening products, 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, nail polishes, make-ups, lipsticks, mascara, as well as candles, lamp oils, incense sticks, insecticides, repellents and propellants.
In a preferred embodiment, the obtained composition contains further aldehydes, carboxylic acids or alcohols selected from the group consisting of dodecanol, tridecanal, tridecanol, tetradecanal, 8Z-tetradecenol, tetradecanol, pentadecanal, pentadecanol, octanoic acid, decanoic acid, tridecanal, tridecenal, pentadecenal and 8Z-tetradecenal, depending on the embodiment of the method according to the invention.
In a further embodiment, the individual components of the mixtures obtained can be partially or completely purified by a method known to the skilled person. Accordingly, a major 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.
The present invention is further explained below with reference to non-limiting examples and based on the disclosure provided in the sequence listing and drawings.
For the generation of the single constructs used for protein expression, a suitable vector with a coding sequence was generated. For this purpose, the respective coding sequence of different target genes was synthesized and cloned between two restriction sites into the vector pET28a (Merck Chemicals GmbH, Schwalbach) or pETDuet-1 (Merck Chemicals GmbH, Schwalbach). An overview of the cloned genes with the respective restriction sites used for cloning is summarized in Table 1. For all SEQ ID Nos, the codon usage of the gene was adapted to E. coli as one of the intended expression hosts. In addition, SEQ ID NO: 3 carries a point mutation relative to the sequence of origin obtained from the respective organism.
Using standard transformation methods (Maniatis et al. 1983), obtained plasmids were introduced into competent E. coli BL21 (DE3) cells, and the cells were grown for 18 h at 37° C. on selective solid medium (LB+ampicillin (100 mg/L)+6 g/L agar).
Starting from the plasmid pET-Duet_LsNOX_VhFALDH, different enzyme variants were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, Waldbronn, Germany). In this process, targeted mutations were introduced in the coding sequence of VhFaldh using specific primers according to the manufacturer's instructions. For the variant pET-Duet_LsNOX_VhFALDHT175Q of VhFALDH, primers VhFALDH-T175Q_fw (SEQ ID NO:15) and VhFALDH-T175Q_ry (SEQ ID NO:16) were used. The sequences of the nucleic acid or the corresponding translated protein are shown in SEQ ID NO: 3 and 10, respectively.
To prepare protein expression in a volume of 50 mL, a preculture of 5 mL LB medium (Carl Roth GmbH, Karlsruhe, Germany) was first prepared with the appropriate antibiotic, and using an inoculating loop, E. coli BL21 (DE3) cells containing pET28a_OsaDOX were removed from the agar plate and transferred to the preculture. This was then incubated for 16 h at 37° C. and 150 rpm. From the preculture, the main culture was inoculated with 50 mL of LB medium (Carl Roth GmbH, Karlsruhe, Germany) and the appropriate antibiotic so that an optical density at 600 nm of 0.05 was present. 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 time point and the culture was incubated for an additional 16 h at 21° C. Finally, the main culture was centrifuged at 17,105×g for 10 min to obtain the 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 cell lysate obtained was then either used directly or processed using a 1 mL HisPur Ni-NTA chromatography column (Thermo Fisher Scientific, Bonn, Germany) according to the manufacturer's instructions.
To prepare protein expression in a volume of 50 mL, a preculture of 5 mL LB medium (Carl Roth GmbH, Karlsruhe, Germany) was first prepared with the appropriate antibiotic, and using an inoculating loop, E. coli BL21 (DE3) cells containing pET-Duet_LsNOX_VhFALDH were removed from the agar plate and transferred to the preculture. This was then incubated for 16 h at 37° C. and 150 rpm. From the preculture, the main culture was inoculated with 50 mL of LB medium (Carl Roth GmbH, Karlsruhe, Germany) and the appropriate antibiotic to give 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 time point and the culture was incubated for an additional 16 h at 16° C. Finally, the main culture was centrifuged at 17,105×g for 10 min to obtain the 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 unclarified cell lysate obtained was then used directly.
In a 1 L round bottom flask, 5-10 g of CALB-imm (Novozym 435; 10,000 U/g) was stirred for 0.5-3 h in 380 mL of tert-butanol (Carl Roth). Then 50 g of triglycerides, and 15 mL of 200 mM Tris-HCl pH 8 buffer were added. The reaction was carried out under vigorous stirring for 15-21 h at 60° C. 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 200 mM Tris-HCl pH 2. The fatty acids were extracted by adding 500 mL of tert-butyl methyl ether (Honeywell) with stirring for 1 h. The mixture was transferred to a separatory funnel until phase separation occurred. The organic phase was subsequently transferred to a round bottom flask and concentrated to dryness using a rotary evaporator. The product was stored at −20° C. and the composition was analyzed by GC-MS (20m ZB-1, 60-9-300, split 60:1). Retention indices of the products were determined by comparison with standards.
For the reaction of different fatty acids or hydrolysates according to example E. coli BL21 (DE3) cells containing pET28a_ OsaDOX according to Example 3 and lysate 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 alpha-Dox according to Example 3, lysate from 10 g/L E. coli VhFALDH_NOX, 3.25 mM NAD+ (Sigma Aldrich), 0.5% glucose, 0.1% (w/v) gum arabic, and 4.7 mL potassium phosphate buffer pH 8 were suspended in a 50 mL reaction tube.
Here, buffer, linoleic acid, NAD+, glucose, and gum arabic were combined in a 100 mL shake flask with a baffle and mixed at 200 rpm and 37° C. for 5 min. Then indicated cells or lysates were added and the reaction was incubated at 200 rpm and 37° C. for 24 h.
Subsequently, the mixture was adjusted to pH 2 using hydrochloric acid and extracted twice by adding the same volume of ethyl acetate (Sigma-Aldrich) under vortexing (1 min). Separation of the organic from the aqueous phase was performed by centrifugation for 10 min at 17.105×g. Samples were analyzed by gas chromatography (20 m ZB-1 df:0.18 μm i.D.:0.18 mm/60-12-300° KAS). Retention indices for the selected GC method were determined by comparison with standards and GC-MS data.
Tables 2 and 3 show exemplary mixtures obtained by the process according to the invention. In addition to the mixtures obtained, the individual components can be purified in a subsequent step.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/056678 | 3/12/2020 | WO |
