Method for producing vanillin

Abstract

To provide methods for producing vanillin and / or its derivatives.SOLUTION: There is provided an expression system for recombinant expression of a polypeptide having isoeugenol oxidizing activity, which expression system comprises a single nucleic acid construct carrying: a. a nucleotide sequence (A) encoding a polypeptide having enzymatic isoeugenol oxidizing activity; and b. at least one nucleotide sequence (B) encoding at least one helper polypeptide that alone or in cooperation assists a functional expression of the polypeptide encoded by the nucleotide sequence (A); where the expression system provides for a co-expression of the nucleotide sequences (A) and (B). Further provided is a method for producing vanillin comprising a step of contacting the polypeptide having isoeugenol oxidation activity to isoeugenol in the presence of oxygen.SELECTED DRAWING: None

Description

[Technical field]

[0001] Provided herein are biochemical methods for producing vanillin and related compounds and derivatives, involving the use of novel polypeptide combinations, together with novel enzyme variants applicable in the biochemical production of vanillin.

[0002] Background technology Vanillin is a globally important compound for use in food, beverages, and medicines. Only a small portion of the world's production is naturally obtained by extraction from vanilla pods, however the availability of these natural plant sources is low and the production methods are laborious and time-consuming. While complex extracts obtained from the pods of the orchids V. planifolia or V. tahitensis offer unique and complex mixtures and olfactory properties at high cost, the majority of vanillin (>99%, i.e. 16,000 tonnes per year) is produced from petrochemical feedstocks, with still a small fraction obtained from lignin waste from the paper industry. However, there is a small but growing market for natural "vanillin not from the bean" (NFB), supplying mainly North America and Europe. The regulatory requirements for claiming natural status are different in these two markets.

[0003] Various starting materials, including ferulic acid, curcumin, eugenol, or isoeugenol, can be converted to vanillin using various biochemistries. Biochemical methods for producing vanillin have been investigated [Gallage NJ, Moller BL. 2015. Mol Plant 8, 40-57; Kaur, B, Chakraborty, D. 2013. Appl Biochem Biotechnol 169,1353-1372; Walton NJ, Mayer MJ, Narbad A. 2003. Phytochem 63, 505-515].

[0004] Recently, microorganisms have been engineered that can convert simple carbon sources, e.g. sugars such as glucose, into vanillin [Hansen EH, Lindberg Moller B, Kock GR, Buenner CM, C Kristensen, Jensen OR, Okkels FT, Olsen CE, Motawia MS, Hansen J. 2009. Appl Environ Microbiol 75, 2765-2774; Ni J, Tao F, Du H, Xu P. 2015. Sci Rep 5, 1-11].

[0005] Isoeugenol is a phenylpropenoid found in many plants. However, it appears to be less abundant than the eugenol traditionally obtained by isomerization under heat in the presence of a strong base. That method no longer complies with European legislation on natural flavouring substances. However, plants rich in isoeugenol could be identified.

[0006] Isoeugenol can be converted to vanillin by various biochemical methods. For example, non-heme iron-containing enzymes, such as lipoxygenase, or iron porphyrin-containing enzymes (e.g. horseradish peroxidase) have been used to oxidatively cleave isoeugenol to vanillin. Recently, purified hemin alone was found to be as good as non-specific heme-containing enzymes for such biotransformation [Mutti FG. 2012. Bioorg Chem Appl 2012,1-13; Li YH, Sun ZH, Zhao LQ, Xu Y. 2005. Appl Biochem Biotechnol 125, 1-10; Mutti FG, Lara M, Kroutil M, Kroutil W. 2010. Chem Eur J 16, 14142-14148]. However, these methods tend to form by-products.

[0007] Interestingly, isoeugenol oxidases of microbial origin, including but not limited to isoeugenol monooxygenase, have been described to convert isoeugenol to vanillin [Ryu JY, J Seo, S Park, JH Ahn, Y Chong, MJ Sadowsky, HG Hur. 2013. Biosci Biotechnol Biochem 77, 289-294; Yamada M, Y Okada, T Yoshida, T Nagasawa. 2007. Appl Microbiol Biotechnol 73, 1025-1030; Yamada M, Y Okada, T Yoshida, T Nagasawa 2008. Biotechnol Lett 30, 665-670]. Oxidation of isoeugenol with such enzymes showed the formation of vanillin and acetaldehyde as the main products [Yamada M, Y Okada, T Yoshida, T Nagasawa. 2007. Arch Microbiol 187, 511-517 ; Ryu JY, J Seo, S Park, JH Ahn, Y Chong, MJ Sadowsky, HG Hur. 2013. Biosci Biotechnol Biochem 77, 289-294]. However, the approaches described therein are not aimed at industrial-scale production of vanillin.

[0008] For industrial processes, it is often necessary to overproduce one or more functional enzymes in recombinant bacteria, such as E. coli. Overproduction of an enzyme in a suitable bacterium provides a safe and economical source of that enzyme. Overproduction of key enzymes also serves to minimize side activities caused by endogenous enzymatic activities of the host organism (e.g., enzymes that can reduce or oxidize vanillin to vanillyl alcohol or vanillic acid, respectively).

[0009] Correct folding of enzymes in the host organism is important for catalytic activity. Incorrectly folded enzymes tend to aggregate (e.g. inclusion bodies) or degrade in the host, resulting in reduced or no catalytic activity. Different strategies can be applied to ensure correctly folded enzymes in the host organism. For example, it may be beneficial to reduce the expression level in the recombinant host organism by low gene dosage, low inducer concentration (in case of inducible systems) or low transcription level by low promoter strength. Low temperature during enzyme synthesis can also help to improve folding of recombinant proteins. Alternatively, incorrectly folded enzymes can be unfolded by strong denaturing chemicals and then refolded under physiological conditions. However, such procedures are time consuming and expensive.

[0010] In molecular biology, the broad category of molecular chaperones refers to proteins that assist in the folding or unfolding of covalent bonds and the assembly or disassembly of other macromolecular structures. The group of chaperonin proteins belongs to the broad category of chaperone molecules. The structure of these chaperonins resembles two donut-like structures stacked on top of each other to create a barrel. Each ring is composed of 7, 8, or 9 subunits, depending on the organism in which the chaperonin is found.

[0011] Group I chaperonins are found in bacteria and in organelles of endosymbiotic origin, chloroplasts and mitochondria. Group II chaperonins, found in the eukaryotic cytosol and archaea, are less well characterized. The GroEL / GroES complex is a group I chaperonin. Group II chaperonins are not thought to utilize GroES-type cofactors to fold their substrates.

[0012] As previously mentioned, the chaperonin system GroES / GroEL forms a barrel-like structure with a cavity that allows uptake of misfolded proteins for refolding upon consumption of ATP [Gragerov A, E Nudler, N Komissarova, GA Gaitanaris, ME Gottesman, V Nikiforov. 1992. Proc Nat Acad Sci 89, 10341-10344; Keskin O, Bahar I, Flatow D, Covell DG, Jernigan RL. 2002. Biochem 41, 491-501].

[0013] The applicability of any group of the large classification of chaperones for the recombinant production of monooxygenases, and in particular isoeugenol monooxygenase, for the enzymatic synthesis of vanillin has not been investigated so far.

[0014] Although there are several reports of natural vanillin production by bioengineering (e.g., WO2013 / 022881, KR101163542(B1)), there is still a need to find a simpler, more efficient and cost / or effective process for the production of natural vanillin. In particular, higher enzyme specific activity allows for lower catalyst loading and improves process performance and economics. Furthermore, there is a demand for further improved enzymes applicable especially in the biochemical production of vanillin from isoeugenol as starting material.

[0015] summary The present invention addresses the above-mentioned drawbacks associated with known systems applied so far for the biochemical production of vanillin.

[0016] The present inventors have surprisingly observed that certain helper polypeptides can be successfully used in the large-scale production of high functional activity of certain known and certain novel isoeugenol oxidases. In particular, the present inventors have surprisingly found that co-expression with the chaperonins GroES and GroEL significantly improves the amount of catalytically active isoeugenol monooxygenase enzyme produced in the bacterium Escherichia coli.

[0017] Furthermore, the inventors have surprisingly been able to genetically engineer a microbial oxidase enzyme from a microorganism of the species Pseudomonas putida, so that for the first time it can be applied for the conversion of isoeugenol, in particular (E)-isoeugenol, to vanillin in the presence of molecular oxygen.

[0018] The inventors have therefore surprisingly been able to improve the biochemical approach of vanillin production from isoeugenol by applying a generated and / or genetically engineered isoeugenol oxidase, as described in more detail below. [Brief description of the drawings]

[0019] [Figure 1] One-step conversion of isoeugenol to vanillin and acetaldehyde. [Diagram 2] Polycistronic constructs for co-expression of isoeugenol monooxygenase (IEM1) from P. nitroreducens Jin1 with chaperonins GroES and GroEL using low and high copy plasmids. Plasmid names for low copy number constructs are shown to the left of each construct. Numbers above horizontal bars indicate the following elements: 1: RBS (strong); 2: IEM1; 3: GroES; 4: native RBS; 5: GroEL; 6: spacer; 7: RBS (moderate); 8: RBS (weak). [Diagram 3]First generation catalyst based on IEM1: Product titers at different biocatalyst loadings under non-limiting substrate concentration at pH 10.5. Symbols: vanillin (black diamonds), remaining trans-isoeugenol (■), remaining (Z)-isoeugenol (▲). Average data from two experiments are shown. [Figure 4] Second generation catalysts: product titers at different catalyst loadings expressed as different optical cell densities: OD600=18 (vertical stripes), OD600=10 (horizontal stripes), OD600=5 (dotted line), OD600=2 (hatched line), OD600=1 (black). Biocatalysts were previously grown in LB medium. IEM1: isoeugenol monooxygenase of P. nitroreducens Jin1. Co-expressed chaperones are also shown. IEM1 (control) represents the activity of wild-type isoeugenol monooxygenase of P. nitroreducens Jin1 expressed in the absence of any chaperone according to Example 1. Values ​​reflect averaged endpoint measurements with a deviation of within 10%. [Diagram 5] Product titers from small scale reactions with catalyst loading OD600=5 using one of the first generation catalysts (vertical line), one of the second generation catalysts (horizontal line) and one of the third generation catalysts (dotted line). Negative controls were obtained with empty cells (filled). The averaged endpoint concentrations of duplicate experiments (maximum 10% deviation) are shown. [Figure 6] Vanillin titers obtained with single, double and triple mutants of isoeugenol monooxygenase of P. putida IE27 (IEM2). "Ref" refers to the (inactive) isoeugenol monooxygenase of P. putida IE27 used as reference. Cells producing isoeugenol monooxygenase of P. nitroreducens Jin1 (IEM1) served as positive control (C+). Cells containing the empty vector served as negative control (C-). Data points were averaged from duplicate experiments. [Figure 7]Biotransformation of isoeugenol to vanillin at different catalyst loadings shown for the triple codon mutant of isoeugenol monooxygenase (IEM2) of P. putida IE27 (horizontal lines) and for isoeugenol monooxygenase (IEM1) of P. nitroreducens Jin1 (vertical lines). E. coli cells carrying a two-plasmid system for co-expressing isoeugenol monooxygenase and the chaperonins GroES and GroEL served as catalysts. The isoeugenol monooxygenase of P. nitroreducens Jin1 was produced in a high copy construct, whereas the triple mutant of P. putida IE27 was produced in a low copy construct. The average product titers of two experiments are shown with a deviation of less than 10%. [Figure 8] Amino acid sequence alignment of IEM1 from Pseudomonas nitroreducens Jin1 and IEM2 from Pseudomonas putida IE27. [Figure 9] Annotation of plasmid pIEM2_C154_T222_A1318 for expressing the triple mutant of IEM2 of Pseudomonas putida IE27 and the corresponding plasmid map. [Figure 10] Relative activity of mutants of isoeugenol monooxygenase of P. putida IE27 obtained by individual saturation mutagenesis at amino acid positions T52, Q74 or D440. The corresponding amino acids at these positions are indicated on the horizontal axis. The relative activity of each mutant is expressed as a percentage of the average vanillin titer obtained with mutant "C154" (i.e. mutant a154c; corresponding to PQD) under standard assay conditions. For comparison, the previously identified mutants "T222" (i.e. mutant g222t; corresponding to THD) and G1318 (i.e. mutant g1318a; corresponding to TQN) are also shown. Empty cells served as negative control (C-). [Figure 11] General scheme of the engineered insertion constructs for expressing IEMs such as the triple mutant of isoeugenol monooxygenase IE27 and the chaperonins GroES and GroEL described herein. [Figure 12]Normalized product titers of vanillin obtained under standard assay conditions in strains of E. coli containing different plasmid constructs made according to the general scheme of Fig. 11 for expressing the triple mutant of isoeugenol monooxygenase IE27 together with the chaperonins GroES and GroEL. The plasmid constructs differed in the promoter sequences for expressing the chaperonins and optionally contained a transcription terminator after the ORF of isoeugenol monooxygenase, respectively indicated as +term or -term. The polycistronic construct PC1_triple is indicated as reference C+.

[0020] Abbreviations used: bp base pair kb kilobase DNA deoxyribonucleic acid cDNA Complementary DNA DTT Dithiothreitol GC Gas chromatograph IPTG Isopropyl-D-thiogalacto-pyranoside IEM Isoeugenol monooxygenase LB lysogeny medium MS mass spectrometer / mass spectrometry PCR Polymerase Chain Reaction RBS Ribosome binding site RNA Ribonucleic acid mRNA Messenger Ribonucleic Acid miRNA MicroRNA siRNA Small interfering RNA rRNA ribosomal RNA tRNA Transfer RNA P. Pseudomonas.

[0021] Specific Definitions The term "isoeugenol" refers to 2-methoxy-4-(prop-1-en-1-yl)phenol (CAS Registry Number: 97-54-1) and any isomeric mixture of trans-isoeugenol and cis-isoeugenol, or (E)-isoeugenol and (Z)-isoeugenol, respectively.

[0022] The term "isoeugenol monooxygenase" or "polypeptide having isoeugenol monooxygenase activity" or "isoeugenol monooxygenase protein" or "isoeugenol oxidase" or "polypeptide having isoeugenol oxidation activity" or "isoeugenol oxidation protein" or "IEM" relates to a polypeptide capable of catalyzing the synthesis of vanillin starting from isoeugenol in the presence of molecular oxygen under the formation of acetaldehyde, without being limited to a specific molecular mechanism of action of said enzyme. Preferably, said enzyme stereospecifically converts the (E)- or trans-isomer of isoeugenol. Vanillin is preferably obtained as main product.

[0023] "Isoeugenol oxidation activity" is determined under "standard conditions" which are described in more detail in the Examples herein below: it is determined using recombinant IEM-expressing cells, disrupted IEM-expressing cells, fragments thereof, or concentrated or purified IEM enzyme, in a reaction medium having a pH in the range of 8.5 to 11, preferably 9 to 10, preferably a buffered reaction medium, in the presence of molecular oxygen, at a temperature in the range of about 20 to 30°C, in the presence of a reference substrate, here isoeugenol, in particular trans-isoeugenol, at an initial concentration in the range of 1 to 40 mg / ml, preferably 1 to 10 mg / ml, more preferably 3 to 7 mg / ml.

[0024] The terms "biological function", "function", "biological activity" or "activity" refer to the ability of isoeugenol oxidase to catalyze the formation of vanillin from isoeugenol.

[0025] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been modified to harbor at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or nucleic acid sequence, that results in a polypeptide for use as described herein upon transcription. The host cell is in particular a bacterial cell, a fungal cell or a plant cell. The host cell may contain a recombinant gene that is integrated into the nuclear or organelle genome of the host cell. Alternatively, the host may contain the recombinant gene extrachromosomally.

[0026] "Homologous sequences" include orthologous or paralogous sequences. Methods for identifying orthologous or paralogous sequences, including phylogenetic methods, sequence similarity, and hybridization methods, are known to those of skill in the art and are described herein.

[0027] "Paralogues" or paralogous sequences result from gene duplication, resulting in two or more genes with similar sequences and similar functions. Paralogs are typically formed by gene duplication within related plant species that cluster together. Paralogs are found in groups of similar genes using pairwise Blast analysis, or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences can be identified characteristically for sequences within related genes and sequences with similar functions of genes.

[0028] "Orthologs" or orthologous sequences are sequences that are similar to each other because they are found in species that are descended from a common ancestor. For example, plant species that share a common ancestor are known to contain many enzymes with similar sequences and functions. Those skilled in the art can identify orthologous sequences and predict the function of the orthologs by constructing a polygenic tree for a gene family of a species, for example, using the CLUSTAL or BLAST programs. A method for identifying or confirming similar functions in homologous sequences is by comparing the transcription profiles in host cells or organisms, such as plants or microorganisms, that overexpress or delete (knock out / knock down) the relevant polypeptide. Those skilled in the art understand that genes that have similar transcription profiles, usually with more than 50% regulated transcription, or usually with more than 70% regulated transcription, or usually with more than 90% regulated transcription, have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences of the present invention are expected to function in a similar manner by engineering host cells, organisms, such as plants or microorganisms, that produce isoeugenol monooxygenase.

[0029] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or plant parts or plant organs, such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to practice the methods of the embodiments herein.

[0030] It is meant that a particular organism or cell is "capable of producing vanillin" if it naturally produces vanillin prior to or together with transformation with a nucleic acid described herein, or if it does not naturally produce vanillin but is transformed to produce vanillin. Also encompassed by "organisms or cells capable of producing vanillin" are organisms or cells that have been transformed to produce higher amounts of vanillin than occurs naturally.

[0031] As used herein, the terms "purified," "substantially purified," and "isolated" refer to a state in which the compound of the invention is free of other distinct compounds with which it is normally associated in its natural state, and thus "purified," "substantially purified," and "isolated" objects comprise at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% by weight of a given sample. In one embodiment, these terms refer to a compound of the invention that comprises at least 95%, 96%, 97%, 98%, 99%, or 100% by weight of a given sample. As used herein, when referring to a nucleic acid or protein, the terms "purified," "substantially purified," and "isolated" nucleic acid or protein also refer to a purified or enriched state that is different from that which occurs naturally, e.g., in a prokaryotic or eukaryotic environment, e.g., in a bacterial or fungal cell, or in a mammalian, particularly human, body. Any degree of purification or enrichment greater than that occurring in nature is within the meaning of "isolated," including (1) purification from other related structures or compounds, or (2) association with structures or compounds with which it is not normally associated in said prokaryotic or eukaryotic environment. The nucleic acids or proteins or types of nucleic acids or proteins described herein may be isolated or associated with structures or compounds with which it is not normally associated in nature according to a variety of methods and processes known to those of skill in the art.

[0032] In the context of the description provided herein and the appended claims, the use of "or" means "and / or" unless specifically stated otherwise.

[0033] Similarly, the words "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0034] Moreover, it should be further understood that where the descriptions of various embodiments use the term "comprising," those of ordinary skill in the art will understand that, in certain instances, an embodiment may instead be described using language such as "consisting essentially of" or "consisting of."

[0035] The term "about" indicates a potential variation of ±25%, particularly ±15%, ±10%, and more particularly ±5%, ±2% or ±1% of the indicated value.

[0036] The term "substantially" denotes a range of values ​​from about 80 to 100%, such as 85 to 99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9%, in particular 99 to 99.9%.

[0037] "Mainly" refers to a proportion in the range of more than 50%, for example, in the range of 51-100%, particularly in the range of 75-99.9%, more specifically in the range of 85-98.5%, for example, 95-99%.

[0038] "Major product" in the context of the present invention denotes a single compound or a group of at least two compounds, e.g. two, three, four, five or more, in particular two or three compounds, which is "predominantly" produced by the reaction described herein and which is included in said reaction in a major proportion based on the total amount of the constituents of the product formed by said reaction. Said proportion may be a molar proportion, a weight proportion or an area proportion calculated from the corresponding chromatograms of the reaction products, preferably based on chromatographic analysis.

[0039] A "by-product" in the context of the present invention refers to a single compound or a group of at least two compounds, e.g. two, three, four, five or more, in particular two or three compounds, which single compound or group of compounds is not produced "predominantly" by the reaction described herein.

[0040] Due to the reversibility of enzymatic reactions, the present invention relates to both reaction directions of the enzymatic or biocatalytic reactions described herein, unless otherwise stated.

[0041] "Functional variants" of the polypeptides described herein include "functional equivalents" of the polypeptides as defined below.

[0042] The term "stereoisomer" specifically includes conformational isomers.

[0043] In accordance with the present invention, all "stereoisomers" of the compounds described herein, such as structural isomers, and in particular stereoisomers and mixtures thereof, such as optical isomers, or geometric isomers, such as E and Z isomers, and combinations thereof, are generally included. In cases where several asymmetric centers are present in one molecule, the present invention encompasses all combinations of the different configurations of these asymmetric centers, such as enantiomeric pairs.

[0044] "Stereoselectivity" refers to the ability to produce a particular stereoisomer of a compound in stereoisomerically pure form or to specifically convert a particular stereoisomer from multiple stereoisomers in the enzyme-catalyzed methods described herein. More specifically, this means that the product of the invention can be enriched with respect to a particular stereoisomer or the educt can be depleted with respect to a particular stereoisomer. This can be achieved by reacting a compound of the formula: %ee=[X A -X B ] / [X A +X B ]*100 The purity may be quantified by the purity % ee parameter calculated according to A and X B represents the molar ratio of stereoisomers A and B (Molenbruch).

[0045] The "yield" and / or "conversion" of the reaction according to the invention is determined over a defined period of time, for example 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, 20 h, 24 h, 36 h or 48 h, during which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example "standard conditions" as defined herein.

[0046] Different yield parameters ("yield" or Y P / S "Specific Productivity Yield", or Space-Time Yield (STY), is determined as is well known in the art and described in the literature.

[0047] "Yield" and "Y P / S " (expressed as mass of product produced / mass of material consumed, respectively) are used synonymously herein.

[0048] The specific productivity yield represents the amount of product, e.g. vanillin, produced per hour, and L fermentation broth per g biomass. The amount of wet cell mass, indicated as WCW, represents the amount of bioactive microorganisms in the biochemical reaction. This value is expressed in g product per WCW per hour (i.e. g / gWCW). -1 h -1 Alternatively, the amount of biomass may be expressed as the amount of dry cell mass, denoted as DCW. Additionally, biomass concentration can be expressed as the optical density at 600 nm (OD 600 ) and using an experimentally determined correlation coefficient to estimate the corresponding wet or dry cell mass, respectively.

[0049] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzymatic activities contained or occurring in said microorganism) to produce a compound in cell culture utilizing at least one carbon source added to the incubation.

[0050] The term "fermentation broth" is understood to mean a liquid, in particular an aqueous or aqueous / organic solution, that is based on the fermentation process and that is, for example, untreated or treated as described herein.

[0051] "Enzyme-catalyzed" or "biocatalytic" methods refer to carrying out the methods under the catalytic action of an enzyme, including an enzyme variant, as defined herein. Thus, the methods can be carried out in the presence of the enzyme, isolated (purified, enriched) or crude, or in the presence of a cell system, particularly a natural or recombinant microorganism, that contains the enzyme in an active form and has the ability to catalyze the conversion reactions disclosed herein.

[0052] The term "selectively converting" or "increasing selectivity" generally means that a particular stereoisomer, such as the E-form of an unsaturated hydrocarbon, is converted in a higher proportion or amount (compared on a molar basis) than the corresponding Z-form during the entire course of the reaction (i.e., between the start and end of the reaction), at a particular time point in the reaction, or during an "interval" of the reaction. In particular, the selectivity may be observed during an "interval" corresponding to a conversion of 1-99%, 2-95%, 3-90%, 5-85%, 10-80%, 15-75%, 20-70%, 25-65%, 30-60, or 40-50% of the initial amount of substrate. The higher proportion or amount may be, for example, in view of the following: - a higher maximum yield of isomers observed during the entire course of the reaction or during intervals therebetween; - a higher relative amount of an isomer at a defined percentage of conversion of the substrate; and / or - Identical relative amounts of isomers at higher % degrees of conversion; each of which is preferably observed in comparison to a reference method, said reference method being performed under conditions otherwise identical to the known chemical or biochemical means.

[0053] "E-stereoselectivity" or "E-selectivity" refers to the ability to produce the E-isomer of a particular C=C-double bond in E-isomerically pure or essentially pure or enriched form, or to specifically or essentially specifically convert the E-isomer in an enzyme-catalyzed manner as described herein from a mixture of multiple other isomers or E- and Z-isomers at said particular position of the double bond.

[0054] In general, all "isomers" of the compounds described herein are included in accordance with the present invention, such as constitutional isomers, and in particular stereoisomers and mixtures thereof, such as optical or geometric isomers, such as E and Z isomers, and combinations thereof. In cases where several asymmetric centers are present in a molecule, the present invention includes all combinations of the different configurations of these asymmetric centers, such as pairs of enantiomers, or any mixtures of stereoisomeric forms.

[0055] Where the present disclosure sets forth features, parameters and ranges thereof of differing preference (generally including features, parameters and ranges thereof that are not expressly preferred features), unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof is included in the disclosure herein, regardless of their respective preference.

[0056] "Polycistronic" refers to a nucleic acid molecule, particularly an mRNA or the corresponding cDNA, that can separately encode more than one polypeptide within the same nucleic acid molecule.

[0057] "Derived from" a "polycistronic" construct or nucleic acid molecule means that the molecule may be modified by introducing one or more identical or different regulatory sequences at appropriate positions, such as promoters, RBSs and / or terminators, in order to influence or regulate the transcription and / or translation of at least one coding sequence contained in said nucleic acid molecule.

[0058] Detailed Description of the Invention a. Specific embodiments of the present invention The invention relates in particular to the following embodiments:

[0059] 1. An expression system for the recombinant expression of a polypeptide having isoeugenol oxidizing activity, comprising a combination of at least two different nucleic acid sequences contained in at least one recombinant nucleic acid construct; a. a nucleotide sequence (A) encoding a polypeptide having isoeugenol oxidizing activity, particularly in the presence of oxygen, particularly molecular oxygen, to catalyze the formation of vanillin and acetaldehyde from isoeugenol; and b. at least one nucleotide sequence (B) encoding at least one, e.g. one, two or three, preferably two, helper polypeptides encoded by said nucleotide sequence (A), which alone or preferably in concert assist in the functional expression of the polypeptide, in particular the correct folding of said expressed polypeptide; and providing simultaneous expression of said nucleotide sequences (A) and (B).

[0060] 2. The expression system according to embodiment 1, a. a combination of at least two, preferably acting or functioning independently of each other, nucleic acid constructs carrying said nucleic acid sequence (A) and said at least one nucleic acid (B), respectively; and b. a single nucleic acid construct carrying said nucleic acid sequence (A) and said at least one nucleic acid (B), which is ultimately transcribed into a single mRNA molecule comprising (A) a coding sequence for a polypeptide having enzymatic isoeugenol oxidizing activity and (B) a coding sequence for a helper polypeptide, which may for example be a nucleic acid construct or vector carrying multiple cloning sites or, more preferably, a polycistronic nucleic acid construct. The expression system is selected from the group consisting of: a) a nucleic acid sequence (A) and (B) comprising a nucleic acid sequence (A) and (B) comprising a nucleic acid sequence (B) that is a nucleic acid molecule that is a nucleic acid molecule of the present invention; b) a nucleic acid sequence (B) that is ...

[0061] 3. The expression system according to embodiment 1 or 2, wherein the nucleotide sequence (A) is a. a polypeptide having isoeugenol oxidizing activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2 (i.e., IEM1 isolated from native Pseudomonas nitroreducens Jin1); or b. A polypeptide having isoeugenol oxidizing activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4 (i.e., IEM2 isolated from original Pseudomonas putida IE27), wherein at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4. the polypeptides, wherein the amino acid sequence having 100% or 100% identity to the polypeptides of the present invention comprises at least one mutation at an amino acid sequence position selected from T52, Q74, D440, and optionally at least one, such as 1, 2, 3, 4 or 5, further mutations at an amino acid sequence position selected from N120, T121, F281, M298 and L470, in particular N120I, T121P, F281Q, M298K and L470S. The expression system encoding the

[0062] 4. An expression system as described in embodiment 3, wherein the polypeptide having isoeugenol oxidizing activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 is a mutant polypeptide comprising at least one mutation at an amino acid sequence position selected from T52, Q74 and D440.

[0063] 5. The expression system according to embodiment 4, wherein the mutant is the IEM2 mutant: a. Single mutants (T52X1), (Q74X2) and (D440X3) b. Double mutants (T52X1, Q74X2), (T52X1, D440X3) and (Q74X2, D440X3), and c. Triple mutant (T52X1, Q74X2, D440X3) is selected from During the ceremony, X1 is P, K or M or another natural or non-natural, in particular natural, amino acid substitution resulting in a functional vanillin producing enzyme variant, preferably X1 is P or M, most preferably P; X2 is H or A or another naturally occurring or non-natural, particularly naturally occurring, amino acid substitution resulting in a functional vanillin producing enzyme variant, most preferably X2 is H; X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y or other natural or non-natural, in particular natural, amino acid substitution resulting in a functional vanillin producing enzyme variant, preferably X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V or Y and most preferably N, The expression system.

[0064] Non-limiting examples of such double mutants expressed by such expression systems may include: (T52X1, Q74X2): wherein X1 is P and X2 is H or A; or X1 is K and X2 is H or A; or X1 is M and X2 is H or A. (T52X1, D440X3) wherein X1 is P and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; or X1 is K and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; or X1 is M and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y. (Q74X2, D440X3) wherein X2 is H and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; or X2 is A and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y.

[0065] A preferred double mutant is: (T52P, Q74H); (T52P, D440X3), where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y. (Q74H, D440X3), wherein X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0066] Non-limiting examples of such triple mutants expressed by such expression systems may include: (T52X1, Q74X2, D440X3) wherein X1 is P, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; X1 is K, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; X1 is M, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; X1 is P, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; X1 is K, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; X1 is M, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y.

[0067] A preferred triple mutant is: (T52P, Q74H, D440X3), where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0068] Said single, double or triple mutants may optionally be further modified by at least one, such as one, two, three, four or five, preferably one, two or three, most preferably one further mutation in the amino acid sequence positions of SEQ ID NO: 4 selected from N120, T121, F281, M298 and L470, in particular selected from the mutants N120I, T121P, F281Q, M298K and L470S.

[0069] 6. The expression system according to any one of the preceding embodiments, wherein the at least nucleotide sequence (B) comprises nucleotide sequences (B1) and (B2); a. (B1) encodes a polypeptide having chaperonin activity comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8 (GroEL); and b. (B2) encodes a polypeptide having chaperonin activity comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 (GroES); The expression system.

[0070] A non-limiting example of a preferred expression system of the present invention is the IEM1 expression system, which comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO:11 (PC1), SEQ ID NO:12 (PC2), SEQ ID NO:13 (PC3), SEQ ID NO:14 (PC4), SEQ ID NO:15 (PC5); and which is applicable according to the present invention to express a functional polypeptide having isoeugenol oxidizing activity and comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2 (IEM1).

[0071] Further non-limiting examples of preferred expression systems may be derived from such IEM-1 expression systems of SEQ ID NO: 11, 12, 13, 14 or 15 by replacing the coding sequence of IEM1 with a nucleotide sequence encoding a functional IEM2 mutant derived from SEQ ID NO: 4, as described in embodiment 5 above. Such IEM2 expression systems are also preferred.

[0072] 7. A polypeptide having isoeugenol oxidizing activity and obtained by recombinant expression using an expression system as defined in any one of embodiments 1 to 6.

[0073] 8. A polypeptide having isoeugenol oxidizing activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 (i.e. IEM2), wherein the amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 comprises at least one mutation at an amino acid sequence position selected from T52, Q74, D440, and optionally further mutations at amino acid sequence positions selected from N120, T121, F281, M298 and L470, in particular selected from N120I, T121P, F281Q, M298K and L470S.

[0074] 9. A polypeptide according to embodiment 8, which is a mutant polypeptide comprising at least one mutation at an amino acid sequence position selected from T52, Q74 and D440 of SEQ ID NO:4.

[0075] 10. The polypeptide of embodiment 9, wherein the variant is: a. Single mutants (T52X1), (Q74X2) and (D440X3) b. Double mutants (T52X1, Q74X2), (T52X1, D440X3) and (Q74X2, D440X3), and c. Triple mutant (T52X1, Q74X2, D440X3) is selected from During the ceremony, X1 is P, K or M or another natural or non-natural, in particular natural, amino acid substitution resulting in a functional vanillin producing enzyme variant, preferably X1 is P or M, most preferably P; X2 is H or A or another naturally occurring or non-natural, particularly naturally occurring, amino acid substitution resulting in a functional vanillin producing enzyme variant, most preferably X2 is H; X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y or other natural or non-natural, in particular natural, amino acid substitution resulting in a functional vanillin producing enzyme variant, preferably X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V or Y and most preferably N, Polypeptides.

[0076] For non-limiting examples of such double and triple mutants of the present invention, see embodiment 5 above.

[0077] Said single, double or triple mutant may optionally be further modified by at least one, such as one, two, three, four or five, preferably one, two or three, most preferably one further mutation in the amino acid sequence positions of SEQ ID NO: 4 selected from N120, T121, F281, M298 and L470, in particular selected from N120I, T121P, F281Q, M298K and L470S.

[0078] 11. A recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of embodiments 8 to 10.

[0079] 12. A recombinant nucleic acid comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% or less than 100%, e.g., 99.9%, sequence identity to SEQ ID NO:1, 3 or 5, or its reverse complement.

[0080] Related embodiments provide nucleic acid sequences that are complementary to a nucleic acid sequence according to SEQ ID NO: 1, 3 or 5, or to a nucleic acid sequence that hybridizes under stringent conditions to at least a portion of a nucleotide sequence according to SEQ ID NO: 1, 3 or 5.

[0081] Particular examples of recombinant nucleic acids are those that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% or less than 100%, e.g., 99.9%, sequence identity to SEQ ID NO: 3 or 5 and comprise a nucleotide sequence encoding an IEM2 variant as described above for embodiment 5, or a reverse complement thereof.

[0082] Such recombinant nucleic acids may be contained in monocistronic or polycistronic expression vectors, as further described herein.

[0083] 13. A recombinant nucleic acid construct comprising a recombinant nucleic acid of embodiment 11 or embodiment 12, or a reverse complement thereof.

[0084] 14. The following: a. The recombinant expression system according to any one of embodiments 1 to 6, or one of said partial nucleic acid constructs, or a reverse complement thereof; or b. a nucleic acid as defined in embodiment 11 or 12, or c. The construct of embodiment 13. 1. An expression vector comprising:

[0085] 15. An expression vector according to embodiment 14, wherein the vector is a prokaryotic vector, a viral vector, a eukaryotic vector, or one or more plasmids.

[0086] The nucleic acid molecule or vector may be such that upon expression a monocistronic or, in particular, polycistronic mRNA is transcribed, encoding the isoeugenol oxidase of the invention and one or more of the helper polypeptides described herein.

[0087] 16. Optionally stably integrated into the genome; a. a recombinant expression system according to any one of embodiments 1 to 6 or one of said partial nucleic acid constructs; or b. a nucleic acid as defined in embodiment 11 or 12 or its reverse complement; or c. A construct according to embodiment 13, or d. The vector of embodiment 14 or 15. A non-human host organism or host cell comprising:

[0088] 17. A non-human host organism or host cell according to embodiment 16, selected from a prokaryotic or eukaryotic microorganism, a plant, or a cell derived therefrom.

[0089] 18. A non-human host organism or host cell according to embodiment 17, wherein the microorganism is a bacterium or a fungus, in particular a yeast.

[0090] 19. A non-human host organism or host cell according to embodiment 18, wherein the bacterium is selected from the genus Escherichia, in particular from the species Escherichia coli, and the yeast is selected from the genus Saccharomyces or Pichia, in particular from the species Saccharomyces cerevisiae or Pichia pastoris.

[0091] 20. A method for producing an isolated catalytically active polypeptide having isoeugenol oxidation activity, comprising co-expression, e.g. substantially simultaneous, and in particular simultaneous expression, of said polypeptide having isoeugenol oxidation activity and at least one helper polypeptide, each encoded by an expression system as defined in any of embodiments 1 to 6, in a host cell system, and optionally isolating said polypeptide having isoeugenol oxidation activity.

[0092] "Co-expression" or "co-expressing" should be understood broadly as long as it is performed in a manner that results in the functional expression of a polypeptide having isoeugenol oxidizing activity (IEM), in particular the cooperative action of a helper polypeptide that helps the correct folding of said expressed IEM polypeptide. The simultaneous or substantially simultaneous co-expression of both polypeptides is one of the non-limiting alternatives in particular. Another non-limiting alternative may be found in the sequential expression of both polypeptides in a timely manner, starting with the expression of a helper polypeptide, followed by the expression of an IEM polypeptide. Another non-limiting alternative may be found in the overlapping co-expression of both polypeptides in a timely manner, where in an initial stage only the helper polypeptide is expressed and in an overlapping stage both polypeptides are expressed. Other alternatives may be developed by the skilled reader without inventive efforts.

[0093] 21. The method according to embodiment 20, producing an isoeugenol oxidase as defined in any one of embodiments 7 to 10.

[0094] 22. The method according to embodiment 20 or 21, wherein a non-human host organism or host cell as defined in any one of embodiments 16 to 19 is applied for expression.

[0095] 23. The following: a. contacting isoeugenol with a polypeptide having isoeugenol-oxidizing activity as defined in any one of embodiments 7 to 10 or a polypeptide having isoeugenol-oxidizing activity produced by a method according to any one of embodiments 20 to 22 in the presence of oxygen, in particular molecular oxygen, to produce vanillin and acetaldehyde; b. Optionally, isolating the vanillin produced in step a. A method for producing vanillin, comprising:

[0096] In a preferred embodiment, the vanillin is isolated.

[0097] In another preferred embodiment, vanillin (and acetaldehyde) is obtained as the main product of the isoeugenol oxygenation process.

[0098] The vanillin produced in any of the processes described herein may be converted to a derivative, such as, but not limited to, a hydrocarbon, ester, amide, glycoside, ether, epoxide, aldehyde, ketone, alcohol, diol, acetal or ketal.

[0099] Vanillin derivatives may be obtained by chemical methods such as, but not limited to, oxidation, reduction, alkylation, acylation, and / or rearrangement.

[0100] Alternatively, vanillin derivatives may be obtained using biochemical methods by contacting vanillin with enzymes such as, but not limited to, oxidoreductases, monooxygenases, dioxygenases, transferases, etc. The biochemical conversion may be carried out in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0101] 24. The method according to embodiment 23, comprising transforming a non-human host organism or host cell with an expression system as defined in any one of embodiments 1 to 6 to express the coding nucleotide sequences (A) and (B) thereof.

[0102] 25. The method according to embodiment 23 or 24, wherein isoeugenol is contacted with a host cell, a cell lysate of a host cell, or a culture medium containing said host cell and / or a polypeptide having isoeugenol oxidizing activity as defined in any one of embodiments 7 to 10 isolated from the host cell, the cell lysate or the culture medium.

[0103] The isoeugenol substrate may be added to the applied reaction medium or, in an alternative embodiment, a non-human host organism or host cell already capable of producing isoeugenol is transformed with an expression system as defined in any of embodiments 1 to 6 to express at least one polypeptide having isoeugenol monooxygenase activity and cultured under conditions leading to the production of vanillin.

[0104] 26. The method of embodiment 25, wherein vanillin is produced by the non-human host organism or host cell by fermentation.

[0105] 27. The method according to embodiment 25, wherein vanillin is enzymatically produced by conversion of isoeugenol with an isolated polypeptide having isoeugenol oxidizing activity as defined in any one of embodiments 7 to 10 in the presence of oxygen and optionally further adjuvants.

[0106] 28. The method according to any one of embodiments 23 to 27, further comprising, prior to step a, a step of chemically or biochemically isomerizing eugenol to isoeugenol, and optionally isolating isoeugenol from the reaction medium.

[0107] 29. A method for producing a mutant polypeptide having isoeugenol oxidizing activity, comprising the steps of: a. selecting a nucleic acid according to embodiment 11 or 12; b. modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c. providing a host cell or a unicellular organism with the variant nucleic acid sequence to express the polypeptide encoded by the variant nucleic acid sequence; d. screening at least one mutant polypeptide for activity in the oxidation of isoeugenol; e. optionally, if the mutated polypeptide does not have the desired activity, repeating process steps a. to d. until a polypeptide having the desired activity is obtained; and f. Optionally, if a mutant polypeptide having a desired activity is identified in step d. or e., isolating the corresponding mutant nucleic acid. The method comprising:

[0108] Further aspects and embodiments of the invention described above with reference to certain preferred embodiments are described in the following paragraphs.

[0109] b. Polypeptides applicable according to the present invention In this description, the following definitions apply:

[0110] The generic terms "polypeptide" or "peptide", which may be used interchangeably, refer to natural or synthetic linear chains or series of consecutive peptide-linked amino acid residues containing from about 10 to over 1000 residues. Shorter polypeptides of up to 30 residues are also called "oligopeptides".

[0111] The term "protein" refers to a macromolecular structure consisting of one or more polypeptides. The amino acid sequence of that polypeptide indicates the "primary structure" of the protein. The amino acid sequence also predetermines the "secondary structure" of the protein by the formation of specific structural elements within the polypeptide chain, such as alpha-helix and beta-sheet structures. The arrangement of several such secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. When a protein comprises one or more polypeptide chains, said chains are spatially arranged to form the "quaternary structure" of the protein. Correct spatial arrangement or "folding" of a protein is a prerequisite for protein function. Denaturation or unfolding destroys protein function. If such destruction is reversible, the function of the protein can be restored by refolding.

[0112] A typical protein function referred to herein is an "enzymatic function", i.e. the protein acts as a biocatalyst on a substrate, e.g. a chemical compound, catalyzing the conversion of said substrate to a product. Enzymes may exhibit a high or low degree of substrate and / or product specificity.

[0113] Thus, a "polypeptide" referred to herein as having a particular "activity" implicitly refers to a correctly folded protein that exhibits the indicated activity, e.g., a specific enzymatic activity.

[0114] Thus, unless otherwise indicated, the term "polypeptide" also encompasses the terms "protein" and "enzyme."

[0115] Similarly, the term "polypeptide fragment" encompasses the terms "protein fragment" and "enzyme fragment."

[0116] The term "isolated polypeptide" refers to an amino acid sequence that has been removed from its natural environment by any method or combination of methods known to those of skill in the art, including recombinant, biochemical and synthetic methods.

[0117] "Targeting peptide" refers to an amino acid sequence that targets a protein or a polypeptide to an intracellular organelle, i.e., mitochondria, or plastids, or to the extracellular space (secretory signal peptide). A nucleic acid sequence encoding a targeting peptide may be fused to a nucleic acid sequence encoding the amino terminus, e.g., the N-terminus, of a protein or polypeptide, or may be used to replace a naturally occurring targeting polypeptide.

[0118] The present invention also relates to "functional equivalents" (also designated "analogs" or "functional variants") of the polypeptides specifically described herein.

[0119] For example, a "functional equivalent" refers to a polypeptide that exhibits at least 1-10%, or at least 20%, or at least 50%, or at least 75% or at least 90% higher or lower IEM activity in an assay used to measure enzymatic isoeugenol oxidation activity, or more specifically IEM activity, than the respective polypeptide specifically defined herein.

[0120] A "functional equivalent" may for example result from the functional expression of another, preferably enzymatically active polypeptide, in particular from a helper polypeptide as described above which aids in the correct folding of said expressed polypeptide, e.g. a polypeptide having isoeugenol oxidizing activity or more particularly IEM activity. Such a modified helper polypeptide may be considered as functional as long as it improves the correct expression or folding of an enzymatically active polypeptide compared to the expression of the same enzymatically active polypeptide under otherwise identical conditions but in the absence of such helper polypeptide.

[0121] "Functional equivalents" according to the invention also encompass specific variants which, at at least one sequence position of the amino acid sequences mentioned herein, have an amino acid different from the specifically mentioned amino acid but which exhibit one of the aforementioned biological activities, e.g. enzymatic activity. Thus, "functional equivalents" include variants obtained by addition, substitution, in particular conservative substitution, deletion and / or inversion of one or more, for example 1-20, in particular 1-15 or 5-10, amino acids, the mentioned changes may occur at any sequence position, as long as they lead to a variant having a property profile according to the invention. Functional equivalents are in particular when the activity patterns qualitatively correspond between the variant and the unchanged polypeptide, i.e. when the interaction rates with, for example, identical agonists or antagonists or substrates are different (i.e. EC 50 or IC 50 The present invention also provides suitable (conservative) amino acid substitutions (where the substitutions are expressed as values ​​of amino acid residues or other parameters appropriate to the state of the art). Examples of suitable (conservative) amino acid substitutions are shown in the following table: [Table 1]

[0122] "Functional equivalents" in the above sense also refer to the "precursors" of the polypeptides described herein, as well as to the "functional derivatives" and "salts" of the polypeptides.

[0123] A "precursor" then is a natural or synthetic precursor of a polypeptide, which may or may not have the desired biological activity.

[0124] The expression "salts" refers to salts of carboxyl groups and acid addition salts of amino groups of protein molecules according to the invention. Salts of carboxyl groups may be produced in a known manner and include inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases, such as salts with amines, such as triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, such as salts with inorganic acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are also included in the present invention.

[0125] "Functional derivatives" of the polypeptides according to the invention may be produced on the side chain groups of functional amino acids or at their N- or C-termini using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtained by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups produced by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups produced by reaction with acyl groups.

[0126] "Functional equivalents" of course also include polypeptides obtained from other organisms, and naturally occurring variants. For example, the extent of homologous sequence regions may be established by sequence comparison, and equivalent polypeptides may be determined based on the specific parameters of the invention.

[0127] "Functional equivalents" also include "fragments" of the polypeptides according to the invention, in particular individual domains or sequence motifs, or N- and C-terminal truncations, which may or may not exhibit a desired biological function. Preferably, such "fragments" retain at least a qualitatively desired biological function.

[0128] "Functional equivalents" are furthermore fusion proteins having one of the polypeptide sequences listed herein or functional equivalents derived therefrom and at least one further functionally different heterologous sequence in N- or C-terminal association (i.e. without substantial mutual dysfunction of the fusion protein parts). Non-limiting examples of these heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.

[0129] Also included according to the invention are "functional equivalents" of the specifically disclosed polypeptides, which have a homology (or identity) of at least 60%, preferably at least 75%, in particular at least 80% or 85%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to one of the specifically disclosed amino acid sequences, calculated according to the algorithm of Pearson and Lipman (Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448). A homology or identity expressed as a percentage of a homologous polypeptide according to the invention means in particular an identity expressed as a percentage of amino acid residues based on the full length of one of the specifically described amino acid sequences herein.

[0130] Identity data expressed as a percentage can also be determined by BLAST alignment, making use of the algorithm blastp (protein-protein BLAST) or by applying the Clustal settings as defined herein below.

[0131] In the case of possible protein glycosylation, "functional equivalents" according to the invention include the polypeptides described herein in deglycosylated or glycosylated form, as well as in modified forms obtained by altering the glycosylation pattern.

[0132] Functional equivalents or homologues of the polypeptides according to the invention may be generated by mutagenesis, for example by point mutation, extension or truncation of the protein, or as described in more detail below.

[0133] Functional equivalents or homologues of the polypeptides according to the invention can be identified by screening a combinatorial database of variants, for example truncation variants. For example, a diverse database of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides. There are numerous methods that can be used for the generation of a database of potential homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can be ligated into a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture, encoding the desired set of potential protein sequences. Methods for the synthesis of degenerate oligonucleotides are known to those skilled in the art.

[0134] Several techniques are known for screening gene products of combinatorial databases generated by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. These techniques can be adapted for the rapid screening of gene banks generated by combinatorial mutagenesis of homologs according to the invention. The most frequently used techniques for screening large gene banks based on high-throughput analysis include cloning the gene bank in replicable expression vectors, transforming suitable cells with the resulting vector database, and expressing the combined genes under conditions where detection of the desired activity facilitates the isolation of vectors encoding the genes whose products have been detected. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional variants in the database, can be used in combination with screening tests to identify homologs.

[0135] Embodiments provided herein provide orthologs and paralogs of the polypeptides disclosed herein, as well as methods for identifying and isolating such orthologs and paralogs.

[0136] c. Coding nucleic acid sequences applicable according to the present invention In this description, the following definitions apply:

[0137] The terms "nucleic acid sequence", "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a sequence of nucleotides. A nucleic acid sequence may be single- or double-stranded deoxyribonucleotides or ribonucleotides of any length and includes coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. Those skilled in the art are aware that the nucleic acid sequence of RNA is identical to that of DNA with various thymines (T) replaced by uracils (U). The term "nucleotide sequence" should also be understood to include polynucleotide or oligonucleotide molecules in the form of separate fragments or as components of a larger nucleic acid.

[0138] An "isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is in an environment different from that of a naturally occurring nucleic acid or nucleic acid sequence and may include those that are substantially free of contaminating endogenous material. As used herein, the term "naturally occurring" as applied to nucleic acids refers to a nucleic acid that is found in the cells of an organism in nature and that has not been intentionally modified by man in the laboratory.

[0139] A "fragment" of a polynucleotide or nucleic acid sequence refers in particular to a contiguous nucleotide of the polynucleotide of the embodiments herein that is at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp, and / or at least 60 bp in length. In particular, a fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of the embodiments herein. Without being limited thereto, a fragment of a polynucleotide herein may be used as a PCR primer and / or as a probe, or for antisense gene silencing or RNAi.

[0140] As used herein, the term "hybridization" or hybridization under certain conditions is intended to refer to hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be conditions under which sequences that are at least about 70%, such as at least about 80%, and at least about 85%, 90%, or 95% identical to each other remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein below. Appropriate hybridization conditions can also be selected by those skilled in the art with minimal experimentation, as described in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Further stringent conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0141] A "recombinant nucleic acid sequence" is a nucleic acid sequence that results from the use of experimental methods (e.g., molecular cloning) to bring together genetic material from one or more sources to create or modify a nucleic acid sequence that does not occur in nature and is not otherwise found in a biological organism.

[0142] "Recombinant DNA techniques" refers to molecular biology methods for generating recombinant nucleic acid sequences, e.g., as described in the Laboratory Manuals edited by Weigel and Glazebrook (2002 Cold Spring Harbor Lab Press), and Sambrook et al. (1989 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

[0143] The term "gene" refers to a DNA sequence comprising a region transcribed into an RNA molecule, e.g., an mRNA in a cell, operably linked to a suitable regulatory region, e.g., a promoter. A gene thus includes several operably linked sequences, e.g., a promoter, a 5' leader sequence, e.g., including sequences involved in translation initiation, a coding region of a cDNA or genomic DNA, introns, exons, and / or 3' untranslated sequences, e.g., including a transcription termination site.

[0144] "Polycistronic" refers to a nucleic acid molecule, particularly an mRNA or the corresponding cDNA, that can encode more than one polypeptide separately within the same nucleic acid molecule. Polycistronic genes allow for translation initiation at two or more sites on a single sequence.

[0145] "Chimeric gene" refers to any gene that is not normally found in nature in a species, particularly a gene in which there are one or more sites of nucleic acid sequence that are not naturally associated with each other. For example, a promoter is not naturally associated with part or all of the transcribed region or with other regulatory regions. The term "chimeric gene" is understood to include expression constructs in which a promoter or transcription control sequence is operably linked to one or more coding sequences, or to one antisense, i.e., the reverse complement of the sense strand, or to invert repeat sequences (sense and antisense, whereby the RNA transcript forms a double-stranded RNA for the transcript). The term "chimeric gene" also includes genes that result from combining parts of one or more coding sequences to produce a new gene.

[0146] "3'UTR" or "3' untranslated sequence" (also referred to as "3' untranslated region" or "3' end") refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, the transcription termination point and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or a variant thereof. After transcription is terminated, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added that is involved during transport of the mRNA to the site of translation, e.g., the cytoplasm.

[0147] The term "primer" refers to a short nucleic acid sequence that hybridizes to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence that is complementary to the template.

[0148] The term "selectable marker" refers to any gene whose expression may be used to select a cell or cells that contain the selectable marker. Examples of selectable markers are described below. Those skilled in the art are aware that a variety of antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to a variety of target species.

[0149] The present invention also relates to nucleic acid sequences encoding the polypeptides defined herein.

[0150] In particular, the present invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA) encoding said polypeptides and one of their functional equivalents, obtained, for example, using artificial nucleotide analogues.

[0151] The present invention relates both to isolated nucleic acid molecules encoding the polypeptides according to the invention or biologically active segments thereof, and to nucleic acid fragments which can be used, for example, as hybridization probes or primers to identify or amplify the encoding nucleic acid according to the invention.

[0152] The present invention also relates to nucleic acids that have a degree of "identity" with the sequences specifically disclosed herein. "Identity" between two nucleic acids means nucleotide identity, in each case over the entire length of the nucleic acid.

[0153] The "identity" between two nucleotide sequences (which also applies to peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) or residues that are identical in the two sequences when the alignment of these two sequences occurs. An identical residue is defined as a residue that is identical in the two sequences at a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by dividing the number of identical residues between the two sequences by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps can be introduced in one or both sequences at one or more positions of the alignment to obtain an optimal alignment. These gaps are taken into account for the calculation of the percentage of sequence identity as non-identical residues. Alignments for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be obtained in various ways using computer programs and publicly available computer programs available, for example, on the web.

[0154] In particular, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to default parameters, available at the National Center for Biotechnology Information (NCBI) website ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, may be used to obtain optimal alignments of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0155] In another example, identity may be calculated by the Vector NTI Suite 7.1 program from Informax Inc. (USA) using the Clustal Method (Higgins DG, Sharp PM. (1989)) with the following settings: [Table 2]

[0156] Alternatively, identity may be measured according to the web page of Chenna et al. (2003): http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and with the following settings: [Table 3]

[0157] All nucleic acid sequences mentioned herein (single- and double-stranded DNA and RNA sequences, e.g. cDNA and mRNA) can be produced in known manner by chemical synthesis from nucleotide building blocks, for example by fragment degeneracy of individual overlapping complementary nucleic acid building blocks of a double helix. Chemical synthesis of oligonucleotides can be carried out in known manner, for example by the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and the filling of gaps by the Klenow fragment of DNA polymerase and ligation reactions, as well as general cloning techniques, are described in Sambrook et al. (1989) (see below).

[0158] A nucleic acid molecule according to the present invention may further comprise untranslated sequences from the 3' and / or 5' end of the coding gene region.

[0159] The present invention further relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or segments thereof.

[0160] The nucleotide sequences according to the invention allow the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers generally comprise a nucleotide sequence region which hybridizes under "stringent" conditions (as defined elsewhere herein) with at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of the sense strand or the corresponding antisense strand of a nucleic acid sequence according to the invention.

[0161] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid, and further may be substantially free of other cellular materials or culture media if produced by recombinant techniques, or free of chemical precursors or other chemicals if chemically synthesized.

[0162] The nucleic acid molecules according to the invention can be isolated by standard techniques of molecular biology and the sequence information provided according to the invention. For example, cDNA can be isolated from a suitable cDNA library using the entire specifically disclosed sequence or one of its segments as a hybridization probe and standard hybridization techniques (e.g. as described in Sambrook (1989)).

[0163] Furthermore, the nucleic acid molecule comprising one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using the oligonucleotide primer constructed based on this sequence.The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequencing.The oligonucleotide according to the present invention can also be produced by standard synthesis methods, for example using an automatic DNA synthesizer.

[0164] The nucleic acid sequences according to the invention or their derivatives, homologues or parts of these sequences can be isolated, for example, by conventional hybridization techniques or PCR techniques from other bacteria, for example through genomic or cDNA libraries, whose DNA sequences hybridize under standard conditions with the sequences according to the invention.

[0165] "Hybridize" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, while non-specific binding does not occur between non-complementary partners under these conditions. Thus, the sequences may be 90-100% complementary. The property of complementary sequences to be able to bind specifically to each other is exploited, for example, in Northern or Southern blotting, or in primer binding in PCR or RT-PCR.

[0166] Short oligonucleotides of the conserved regions are advantageously used for hybridization. However, it is also possible to use longer fragments of the nucleic acid according to the invention or the entire sequence for hybridization. These "standard conditions" vary depending on the nucleic acid used (oligonucleotide, longer fragment or entire sequence) or on the type of nucleic acid used for hybridization (DNA or RNA). For example, the melting temperature for DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0167] For example, depending on the particular nucleic acid, standard conditions mean a temperature of 42-58°C in an aqueous buffer solution at a concentration of 0.1-5xSSC (1xSSC=0.15M NaCl, 15 mM sodium citrate, pH 7.2) or even in the presence of 50% formamide, for example 42°C in 5xSSC, 50% formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1xSSC and the temperature is about 20°C-45°C, preferably about 30°C-45°C. Advantageously, the hybridization conditions for DNA:RNA hybrids are 0.1xSSC and the temperature is about 30°C-55°C, preferably about 45°C-55°C. These said temperatures of hybridization are examples of melting temperature values ​​calculated for a nucleic acid having a length of about 100 nucleotides and a G+C content of 50% in the absence of formamide. Experimental conditions for DNA hybridization are described in relevant genetics textbooks, such as Sambrook et al. (1989), and can be calculated using formulas known to those skilled in the art, depending for example on the length of the nucleic acid, the type of hybrid, or the G+C content. Those skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds) (1985), Brown (ed) (1991).

[0168] "Hybridization" may be carried out under particularly stringent conditions, such as those described in Sambrook (1989) or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0169] As used herein, the term hybridization or hybridization under certain conditions is intended to refer to hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be conditions under which sequences that are at least about 70%, such as at least about 80%, and at least about 85%, 90%, or 95% identical to each other remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein.

[0170] Appropriate hybridization conditions can be selected by one of skill in the art with minimal experimentation as described in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Further stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0171] As used herein, the defined conditions of low stringency are as follows: Filters containing DNA are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5xSSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x106 32P-labeled probes are used. Filters are incubated in the hybridization mixture for 18-20 hours at 40°C and washed for 1.5 hours at 55°C. in a solution containing 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and further incubated for 1.5 hours at 60°C. Filters are blotted dry and exposed for autoradiography.

[0172] As used herein, the defined conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 7 hours at 50°C in a solution containing 35% formamide, 5xSSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x106 32P-labeled probes are used. Filters are incubated in the hybridization mixture for 30 hours at 50°C and washed for 1.5 hours at 55°C. in a solution containing 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and further incubated for 1.5 hours at 60°C. Filters are blotted dry and exposed for autoradiography.

[0173] As used herein, the defined conditions of high stringency are as follows: Prehybridization of DNA-containing filters is performed for 8 hours to overnight at 65°C in a buffer consisting of 6xSSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C in a prehybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5-20x106 cpm of 32P-labeled probe. Washing of filters is performed for 1 hour at 37°C in a solution containing 2xSSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a 45 minute wash in 0.1xSSC at 50°C.

[0174] Other conditions of low, medium, and high stringency well known in the art (e.g., as used for cross-species hybridizations) may be used when the above conditions are inappropriate (e.g., as used for cross-species hybridizations).

[0175] A detection kit for a nucleic acid sequence encoding a polypeptide of the present invention may comprise primers and / or probes specific for the nucleic acid sequence encoding said polypeptide and associated protocols for using the primers and / or probes to detect the nucleic acid sequence encoding said polypeptide in a sample. Such a detection kit may be used to determine whether a plant, organism, microorganism or cell has been modified, i.e. transformed, with a sequence encoding said polypeptide.

[0176] To test the functionality of a variant DNA sequence according to embodiments herein, the sequence of interest is operably linked to a selectable or screenable marker gene and reporter gene expression is tested in transient expression assays, for example in microorganisms or in protoplasts or in stably transformed plants.

[0177] The present invention also relates to derivatives of the specifically disclosed nucleic acid sequences or derivable nucleic acid sequences.

[0178] Thus, further nucleic acid sequences according to the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by one or more, for example 1 to 20, in particular 1 to 15, or 5 to 10 additions, substitutions, insertions or deletions of one or several (for example 1 to 10) nucleotides and may further code for a polypeptide having a desired profile of properties.

[0179] The present invention also encompasses nucleic acid sequences which contain so-called silent mutations or which are altered in accordance with the codon usage of a particular origin or host organism compared to the specifically listed sequences.

[0180] According to certain embodiments of the present invention, variant nucleic acids may be produced to adapt the nucleotide sequence to a particular expression system. For example, bacterial expression systems are known to express polypeptides more efficiently when amino acids are encoded by specific codons. Due to the degeneracy of the genetic code, more than one codon may code for the same amino acid sequence, and multiple nucleic acid sequences can code for the same protein or polypeptide, and all these DNA sequences are included in the embodiments of the present invention. Optionally, the nucleic acid sequences encoding the polypeptides described herein may be optimized for increased expression in host cells. For example, the nucleic acids of the embodiments herein may be synthesized using codons specific to the host for improved expression.

[0181] The present invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0182] Allelic variants have at least 60% homology at the derived amino acid level, preferably at least 80% homology over the entire sequence range, very particularly preferably at least 90% homology (with regard to homology at the amino acid level, see the details provided above for polypeptides). Advantageously, the homology may be higher than partial regions of the sequence.

[0183] The present invention also relates to sequences resulting from conservative nucleotide substitutions, ie those which result in the replacement of a given amino acid with an amino acid of identical charge, size, polarity and / or solubility.

[0184] The present invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may include functional equivalents. These natural variations usually result in 1-5% variation in the nucleotide sequence of a gene. Said polymorphisms may lead to changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may include functional equivalents.

[0185] Furthermore, derivatives are also to be understood as homologues of the nucleic acid sequences according to the invention, such as animal, plant, fungal or bacterial homologues, truncated sequences, single-stranded DNA or single-stranded RNA of coding and non-coding DNA sequences, for example, homologues have at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% homology at the DNA level over a given DNA region in the sequences specifically disclosed herein.

[0186] Furthermore, derivatives should be understood to be, for example, fusions with promoters. The promoters added to the listed nucleotide sequences may be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, but without impairing the functionality or efficiency of the promoter. Furthermore, the efficiency of promoters may be increased by modifying their sequences, or may be completely replaced with more efficient promoters, even in organisms of different genera.

[0187] d. Generation of Functional Polypeptide Variants Furthermore, one of skill in the art is familiar with methods to generate functional variants that are encoded by a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the amino acids related to the SEQ ID NOs disclosed herein, and / or a nucleotide sequence that has at least 70% sequence identity to any of the amino acids related to the SEQ ID NOs disclosed herein, or their reverse complements.

[0188] Depending on the technique used, the skilled artisan can introduce completely random or more directed mutations into genes or non-coding nucleic acid regions (e.g. important for regulating expression) and subsequently generate gene libraries. The molecular biology methods required for this purpose are known to the skilled artisan and are described, for example, in Sambrook and Russell (Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001).

[0189] Methods for modifying genes, and thus the polypeptides encoded by them, have been known to the skilled artisan for a long time, for example: - site-directed mutagenesis, in which individual nucleotides or several nucleotides of a gene are replaced in a targeted manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey), - Saturation mutagenesis, which allows the codon for any amino acid to be exchanged or deleted anywhere in the gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1) - an error-prone DNA polymerase that mutates nucleotide sequences by error-prone polymerase chain reaction (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739); - SeSaM (sequence saturation method), in which favorable conversions are prevented by the polymerase, Schenk et al. (Biospektrum, Vol. 3, 2006, 277-279); - passage of genes in mutator strains, which increase the mutation rate of nucleotide sequences, for example due to defects in DNA repair mechanisms (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E.coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or - DNA shuffling, in which a pool of closely related genes is formed, digested, and the fragments used as templates for polymerase chain reaction that ultimately produces a full-length mosaic gene by repeated strand separation and rejoining (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0190] Using the so-called directed evolution (described inter alia in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200:31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), the skilled person can produce functional variants in a directed manner on a large scale. For this purpose, in a first step, a gene library for the respective polypeptide is first generated, for example using the methods described above. The gene library is expressed in a suitable manner, for example by bacteria or by a phage display system.

[0191] The relevant gene of the host organism expressing a functional mutant with a property corresponding sufficiently to the desired property may be subjected to another mutation cycle. The mutation and selection or screening steps may be repeated iteratively until the functional mutants present have the desired property to a sufficient extent. Using this iterative procedure, a limited number of mutations, for example 1, 2, 3, 4, or 5 mutations, may be carried out stepwise, evaluated and selected for their effect on the activity of interest. The selected mutants are sent to further mutation steps in the same manner. This can significantly reduce the number of individual mutants to be investigated.

[0192] The results according to the invention also provide important information regarding the structure and sequence of related polypeptides, which is necessary to generate further polypeptides with desired modified properties in a targeted manner. In particular, it is possible to define so-called "hot spots", i.e. sequence segments that are potentially suitable for modifying properties by introducing targeted mutations.

[0193] Information about the location of the amino acid sequence can also be deduced, where mutations likely to have little effect on activity may occur and be designed as potential "silent mutations".

[0194] e. Constructs for expressing the polypeptides of the invention In this description, the following definitions apply:

[0195] "Expression of a gene" encompasses "heterologous expression" and "overexpression" and includes transcription of a gene and translation of mRNA into a protein. Overexpression refers to the production of a gene product, as measured by levels of mRNA, polypeptide and / or enzymatic activity, in a transgenic cell or organism that exceeds the level of product in a non-transformed cell or organism of the same genetic background.

[0196] As used herein, "expression vector" refers to a nucleic acid molecule designed using molecular biology methods and recombinant DNA technology to introduce foreign or exogenous DNA into a host cell. Expression vectors typically contain sequences required for proper transcription of a nucleotide sequence. The coding region usually codes for a protein of interest, but may also code for RNA, such as antisense RNA, siRNA, etc.

[0197] As used herein, "expression vector" includes any linear or circular recombinant vector, including, but not limited to, viral vectors, bacteriophages, and plasmids. Those skilled in the art can select a suitable vector according to the expression system. In one embodiment, the expression vector comprises a nucleic acid of an embodiment herein operably linked to at least one "control sequence" that controls transcription, translation, initiation, and termination, such as a transcription promoter, operator or enhancer, or an mRNA ribosome binding site, and optionally includes at least one selectable marker. A nucleotide sequence is "operably linked" when the control sequence is functionally related to the nucleic acid of an embodiment herein.

[0198] As used herein, an "expression system" encompasses any combination of nucleic acid molecules required for the expression of one, or the simultaneous expression of two or more polypeptides in a given expression host in vivo or in vitro. Each coding sequence may be located on a single nucleic acid molecule or vector, e.g. a vector containing multiple cloning sites, or on a polycistronic nucleic acid, or distributed in two or more physically distinct vectors.

[0199] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification to produce or detect recombination of naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying (e.g., by polymerase chain reaction (PCR)) naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo, or in vitro.

[0200] "Control sequence" refers to a nucleic acid sequence that determines the expression level of a nucleic acid sequence of the embodiments herein and can control the rate of transcription of a nucleic acid sequence operably linked to the control sequence. Control sequences include promoters, enhancers, transcription factors, promoter elements, and the like.

[0201] "Promoter", "nucleic acid with promoter activity" or "promoter sequence" are understood according to the present invention to mean a nucleic acid which, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. "Promoter" refers in particular to a nucleic acid sequence which controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for proper transcription, including but not limited to transcription factor binding sites, repressor and activator protein binding sites. The meaning of the term promoter also includes the term "promoter control sequence". Promoter control sequences may include upstream and downstream elements which may affect the transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally occurring and synthetic sequences. The coding nucleic acid sequence is usually placed downstream of the promoter with respect to the direction of transcription starting at the transcription initiation site.

[0202] In the present context, a "functional" or "operable" linkage is understood to mean, for example, one contiguous arrangement of a nucleic acid with a regulatory sequence. For example, the promoter activity, the nucleic acid sequence to be transcribed, and optionally further regulatory elements, such as sequences ensuring the transcription of the nucleic acid, such as sequences with terminators, are linked in such a way that each regulatory element can perform its function during the transcription of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic regulatory sequences, such as enhancer sequences, can exert their function on the target sequence from more distant positions or even from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is placed behind the promoter sequence (i.e. at the 3' end) so that the two sequences are covalently linked together. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly can be less than 200 base pairs, or less than 100 base pairs, or less than 50 base pairs.

[0203] In addition to promoters and terminators, the following may be mentioned as examples of other regulatory elements: targeting sequences, enhancers, polyadenylation signals, selection markers, amplification signals, replication origins, etc. Suitable regulatory sequences are described, for example, in Goeddel (Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)).

[0204] The term "constitutive promoter" refers to an unregulated promoter that allows for the continuous transcription of an operably linked nucleic acid sequence.

[0205] The term "operably linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, rather a transcriptional control sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous. The nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence may be wholly or partially synthetic. Regardless of origin, the nucleic acid sequence associated with the promoter sequence is expressed or repressed according to the promoter properties to which it is linked after binding to the polypeptide of the embodiments herein. The associated nucleic acid may code for a protein that is desired to be expressed or repressed at all times or alternatively at specific times throughout the organism or in specific tissues, cells or cell compartments. Such nucleotide sequences code for proteins that, in particular, confer desired phenotypic characteristics to the host cell or organism modified or transformed therewith. More particularly, the associated nucleotide sequence directs the production of vanillin in the cell or organism. In particular, the nucleotide sequence encodes an IEM.

[0206] The nucleotide sequences described herein above may be part of an expression cassette. The terms "expression cassette" and "expression construct" are used synonymously. A (preferably recombinant) expression construct comprises a nucleotide sequence that encodes a polypeptide according to the invention and that is under the genetic control of a regulatory nucleic acid sequence.

[0207] In the process applied according to the present invention, the expression cassette may be part of an "expression vector", in particular a recombinant expression vector.

[0208] An "expression unit" is understood to mean a nucleic acid which, according to the invention, comprises a promoter as defined herein and has an expression activity which regulates the expression, i.e. the transcription and translation of a nucleic acid or gene to be expressed, after it is functionally linked to said nucleic acid or gene. It is therefore also referred to in this context as a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements may also be present, such as enhancers.

[0209] "Expression cassette" or "expression construct" is understood according to the present invention to mean an expression unit operatively linked to a nucleic acid to be expressed or to a gene to be expressed. Thus, in contrast to an expression unit, an expression cassette contains not only nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are to be expressed as a protein as a result of transcription and translation.

[0210] The term "expression" or "overexpression" in the context of the present invention refers to the production or increase of the intracellular activity of one or more polypeptides in a microorganism that are encoded by the corresponding DNA. For this purpose, it is possible, for example, to introduce genes into the organism, to replace existing genes with other genes, to increase the copy number of the genes, to use strong promoters, or to use genes that code for the corresponding polypeptides with higher activity, optionally combining these measures.

[0211] Preferably, such constructs according to the invention comprise a promoter sequence 5'-upstream and a terminator sequence 3'-downstream of the respective coding sequence, and optionally other conventional regulatory elements, in each case operably linked to the coding sequence.

[0212] Nucleic acid constructs according to the present invention include, inter alia, sequences encoding polypeptides derived, for example, from the amino acids related to the SEQ ID NOs described herein or their reverse complements, or derivatives and homologues thereof, and nucleic acid sequences operatively or functionally linked to one or more regulatory signals to advantageously control, e.g. increase, gene expression.

[0213] In addition to these regulatory sequences, the natural regulation of these sequences may still be present in front of the actual structural gene, and may be optionally genetically modified to turn off the natural regulation and enhance gene expression.However, the nucleic acid construct may also be of a simpler structure, i.e., no additional regulatory signals are inserted in front of the coding sequence, and the natural promoter with its regulation is not removed.Instead, the natural regulatory sequence is mutated, so that regulation does not occur, and gene expression is increased.

[0214] A preferred nucleic acid construct advantageously also comprises one or more of the already mentioned "enhancer" sequences functionally linked to the promoter, which allow for enhanced expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, may be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acid according to the invention may be present in the construct. In the construct, other markers, such as genes that complement auxotrophy or antibiotic resistance, may optionally be present to select the construct.

[0215] Examples of suitable regulatory sequences are promoters, such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI. q , T7, T5, T3, H9, H10, G6, C4, gal, trc, ara, rhaP(rhaP BAD )SP6, Lambda P R Or Lambda P L These are preferably used in gram-negative bacteria. Further advantageous regulatory sequences are found, for example, in the gram-positive promoters amy and SPO2, and in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation. See, for example, Jones et al., ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci. Rep. 2015, 5, 11301.

[0216] For expression in the host organism, the nucleic acid construct is advantageously inserted into a vector, such as a plasmid or phage, which allows optimal expression of the gene in the host. Vector is also understood to mean, in addition to plasmids and phages, all other vectors known to those skilled in the art, such as viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA or artificial chromosomes. These vectors can replicate automatically in the host organism or on the chromosome. These vectors are a further development of the present invention. Binary or cpo integrating vectors can also be applied.

[0217] Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III in E. coli. 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 of Streptomyces, pUB110, pC194 or pBD214 of Bacillus, pSA77 or pAJ667 of Corynebacterium, pALS1, pIL2 or pBB116 of fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 of yeast, or pLGV23, pGHlac of plants. + , pBIN19, pAK2004 or pDH51. The above plasmids are a small selection of possible plasmids. Further plasmids are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0218] In the further development of the vector, the nucleic acid construct according to the invention or the vector comprising the nucleic acid according to the invention may advantageously be introduced into the microorganism in the form of linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA may consist of a linearized vector, for example a plasmid, or solely of the nucleic acid construct or nucleic acid according to the invention.

[0219] For optimal expression of a heterologous gene in an organism, it is advantageous to modify the nucleic acid sequence to conform to the particular "codon usage" used by the organism, which can be readily determined by computer evaluation of other known genes of the organism in question.

[0220] The expression cassette according to the invention is generated by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Conventional recombination and cloning techniques are used for this purpose and are described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0221] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector that allows optimal expression of the gene in the host. Vectors are well known to those skilled in the art and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0222] Alternative embodiments of the embodiments herein provide methods of "altering gene expression" in a host cell. For example, the polynucleotides of the embodiments herein may be enhanced or overexpressed or induced in a host cell or host organism under certain circumstances (e.g., upon exposure to certain temperatures or culture conditions).

[0223] The altered expression of the polynucleotides provided herein may also result in ectopic expression, which is a different expression pattern in the altered organism and in the control or wild-type organism. The altered expression may result from the interaction of the polypeptides of the embodiments herein with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide. This term refers to the altered expression pattern of the polynucleotides of the embodiments herein, which is altered below the detection level or completely suppressed activity.

[0224] Also provided herein, in one embodiment, is an isolated recombinant or synthetic polynucleotide that encodes a polypeptide or a variant polypeptide provided herein.

[0225] In one embodiment, the nucleic acid sequences encoding several polypeptides are co-expressed in a single host, particularly under the control of different promoters.In another embodiment, the nucleic acid sequences encoding several polypeptides may be co-transformed simultaneously, either in a single transformation vector or using separate vectors, and selecting transformants containing both chimeric genes.Similarly, one or more genes encoding polypeptides may be expressed together with other chimeric genes in a single plant, cell, microorganism, or organism.

[0226] f. Microorganisms applicable to the present invention Depending on the context, the term "microorganism" can refer to wild-type microorganisms or genetically modified recombinant microorganisms or both.

[0227] In one embodiment, the vectors according to the invention can be used to generate recombinant microorganisms, which can be transformed with at least one vector according to the invention and used to generate the polypeptides according to the invention. Advantageously, the recombinant constructs according to the invention as described above are introduced into a suitable host system and expressed. Preferably, conventional cloning and transfection methods known to those skilled in the art, such as co-precipitation, protoplast fusion, electroporation, retroviral transfection, etc., are used to express the nucleic acid of interest in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or in Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0228] In principle, all prokaryotic or eukaryotic organisms may be considered as recombinant host organisms for the nucleic acids or nucleic acid constructs according to the invention. Advantageously, microorganisms, such as bacteria, fungi or yeasts, are used as host organisms. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria from the family Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria from the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus Escherichia coli is very particularly preferred. Furthermore, other advantageous bacteria are found in the groups of the Alphaproteobacteria, Betaproteobacteria or Gammaproteobacteria.

[0229] Depending on the host organism, the organisms used in the process according to the invention are grown or cultured in a manner known to those skilled in the art. The culture can be batch, semi-batch or continuous. Nutrients can be present at the beginning of the fermentation or fed later, semi-continuously or continuously. This is also described in more detail below.

[0230] g. Recombinant production of the polypeptides according to the invention The present invention further relates to a method for the recombinant production of a polypeptide according to the invention or a functional biologically active fragment thereof, in which a polypeptide-producing microorganism is cultured, optionally expression of the polypeptide is induced by applying at least one inducer inducing gene expression, and the expressed polypeptide is isolated from the culture. The polypeptide may be produced on an industrial scale in this manner, if desired.

[0231] The microorganisms produced according to the invention may be cultivated continuously or discontinuously in the batch or fed-batch or repeated flow fed-batch process. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0232] The culture medium used should be appropriately adapted to the requirements of each strain. Descriptions of culture media for various microorganisms are provided in the American Society for Bacteriology manual "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981).

[0233] These media that can be used according to the invention usually contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0234] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars may also be added to the medium via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add a mixture of different carbon sources. Other possible carbon sources are oils and fats, such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as palmitic acid, stearic acid or linoleic acid, alcohols, such as glycerol, methanol or ethanol, and organic acids, such as acetic acid or lactic acid.

[0235] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing these compounds. Examples of nitrogen sources include ammonia gas, or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources may be used alone or as a mixture.

[0236] Inorganic salt compounds which may be present in the medium include chlorides, phosphorus or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0237] Inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds such as mercaptans and thiols may be used as sulfur sources.

[0238] Phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as the phosphorus source.

[0239] Chelating agents may be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0240] The fermentation medium used according to the invention usually also contains other growth factors, e.g. vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts often come from components of complex media, e.g. yeast extract, molasses, corn steep liquor, etc. Furthermore, suitable precursors may be added to the medium. The exact composition of compounds in the medium is highly dependent on each experiment and is determined individually for each specific case. Information on medium optimization is given in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growth media may be obtained from commercial suppliers, e.g. Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0241] All components of the medium are sterilized either by heating (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components may be sterilized together or individually as required. All components of the medium may be present at the start of the culture or may be added continuously or batchwise.

[0242] The culture temperature is usually between 15°C and 45°C, preferably between 25°C and 40°C, and can be varied or kept constant during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably about 7.0. The pH for growth may be controlled during growth by adding acidic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or basic compounds, such as phosphoric acid or sulfuric acid. Antifoaming agents, such as fatty acid polyglycol esters, may be used to control foaming. To maintain the stability of the plasmids, suitable selective substances, such as antibiotics, may be added to the medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the culture. The culture temperature is usually in the range of 20°C to 45°C. The culture is continued until a maximum of the desired product is formed. This objective is usually reached within 10 hours to 160 hours.

[0243] The fermentation broth is then further processed. Depending on the requirements, the biomass may be completely or partially removed from the fermentation broth by separation techniques such as centrifugation, filtration, decantation or a combination of these methods, or may be left completely intact.

[0244] If the polypeptide is not secreted into the medium, the cells may be lysed and the product may be obtained from the lysate by known methods for protein isolation. Optionally, the cells may be disrupted by high frequency ultrasound, high pressure, e.g. in a French press, by osmotic lysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers, or by some combination of the above methods.

[0245] The polypeptides may be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), e.g. Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional techniques, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper (TG, Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York) or in Scopes (R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin).

[0246] To isolate recombinant proteins, it may be advantageous to use vector systems or oligonucleotides that extend the cDNA with a defined nucleotide sequence and thus code for modified polypeptides or fusion proteins that serve, for example, for easier purification. Suitable modifications of this type are, for example, so-called "tags" that function as anchors, such as the modifications known as hexahistidine anchors, or epitopes that can be recognized as antigens for antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors may, for example, be provided to attach the protein to a solid carrier, such as a polymer matrix, which may be used as a packing in a chromatography column or on a microtiter plate or some other carrier.

[0247] At the same time, these anchors may also be used for the recognition of proteins, for which it is also possible to use conventional markers, such as fluorescent dyes, enzymatic markers or radioactive markers, which form detectable reaction products after reaction with a substrate, either alone or in combination with the anchors for the derivation of proteins.

[0248] For the expression of the mutants according to the invention, reference may be made to the description of the expression of the wild-type enzyme EbN1 and the expression systems available therefor in WO 2005 / 108590 (WO2005 / 108590) and WO 2006 / 094945 (WO2006 / 094945), which are expressly referenced.

[0249] h. Immobilization of Polypeptides The enzymes or polypeptides according to the invention may be used free or immobilized in the methods described herein. Immobilized enzymes are enzymes immobilized on an inert carrier. Suitable carrier materials and enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and the references cited therein. In this regard, the entire disclosure is incorporated by reference. Suitable carrier materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes, and polyolefins such as polyethylene and polypropylene. To prepare supported enzymes, the carrier material is usually used in the form of finely divided particles, preferably in porous form. The particle size of the carrier material is usually less than 5 mm, in particular less than 2 mm (particle size distribution curve). Similarly, when dehydrogenases are used as whole cell catalysts, free or immobilized forms can be selected. Carrier materials are, for example, calcium alginate, carrageenan. Enzymes and cells may be directly cross-linked (cross-linked to CLEA) with glutaraldehyde. Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes", K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out the method according to the invention can also be found in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim.

[0250] i. Reaction conditions for the biocatalyst production method of the present invention The at least one polypeptide / enzyme present in the method of the invention or in the individual steps of the multi-step method defined herein above may be present in living cells which naturally or recombinantly produce the enzyme(s), in harvested cells, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts or in essentially pure or completely pure form. The at least one enzyme may be present in solution or as an immobilized enzyme on a carrier. One or several enzymes may be simultaneously present in soluble and / or immobilized form.

[0251] The method according to the invention may be carried out in conventional reactors known to those skilled in the art and on different scales, for example laboratory scale (reaction volumes from a few milliliters to several tens of liters) to industrial scale (reaction volumes from a few liters to several thousand cubic meters). Chemical reactors may be used when using polypeptides in a form encapsulated by non-living, optionally permeabilized cells, in the form of more or less purified cell extracts or in a purified form. Chemical reactors usually make it possible to control the amount of at least one enzyme, the amount of at least one substrate, the pH, the temperature and the circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the method will be a fermentation. In this case, the biocatalytic production is carried out in a bioreactor (fermenter), in which the parameters necessary for suitable growth conditions for living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, etc.) may be controlled. The skilled person is familiar with chemical reactors or bioreactors, e.g. procedures for scaling up chemical or biotechnological processes from laboratory scale to industrial scale or for optimizing process parameters, and these have also been extensively described in the literature (for biotechnological processes see, e.g., Krueger und Crueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Muenchen, Wien, 1984).

[0252] Cells containing at least one enzyme may be permeabilized by physical or mechanical means, such as ultrasound or radio frequency pulses, French press, or chemical means, such as hypotonic medium, lytic enzymes and detergents present in the medium, or a combination of such methods. Examples for detergents are digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Nonidet® P40 (ethylphenol poly(ethylene glycol ether)), etc.

[0253] Instead of living cells, biomass of dead cells containing the required biocatalyst may also be applied in the biotransformation reactions of the present invention.

[0254] When at least one enzyme is immobilized, it is attached to an inert carrier as described above.

[0255] The conversion reaction may be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) may be fed at the beginning of the reaction or fed later in a semi-continuous or continuous manner.

[0256] The reactions of the present invention may be carried out in aqueous, aqueous-organic, or non-aqueous reaction media, depending on the particular reaction type.

[0257] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to a value in the range of 5 to 11, for example 6 to 10.

[0258] In the aqueous organic medium, organic solvents that are miscible, partially miscible or immiscible with water may be applied. Non-limiting examples of suitable organic solvents are given below. Further examples are mono- or polyhydric aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols, such as glycerol.

[0259] The non-aqueous medium is substantially free of water, ie, contains less than about 1% or 0.5% water by weight.

[0260] The biocatalytic process may be carried out in an organic non-aqueous medium. Suitable organic solvents include, for example, aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene, aliphatic acyclic and ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or mixtures thereof.

[0261] The reactant / substrate concentrations may be adapted to the optimal reaction conditions, which may depend on the particular enzyme applied, for example the initial substrate concentration may be, for example, 0.1-0.5 M, such as 10-100 mM.

[0262] The reaction temperature may be adapted to the optimal reaction conditions, which may depend on the particular enzyme applied. For example, the reaction may be carried out at a temperature ranging from 0 to 70° C., such as from 20 to 50° C. or from 25 to 40° C. Examples of reaction temperatures are about 30° C., about 35° C., about 37° C., about 40° C., about 45° C., about 50° C., about 55° C. and about 60° C.

[0263] The process may be continued until the equilibrium between the substrate and the product is reached, but may be stopped earlier. Typical treatment times are in the range of 1 minute to 25 hours, particularly 10 minutes to 6 hours, for example 1 hour to 4 hours, particularly 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0264] Where the host is a transgenic plant, optimal growth conditions may be provided, such as optimal light, water and nutrient conditions.

[0265] k. Product isolation The methodology of the present invention may further comprise a step of recovering the final product or intermediate product, optionally in a substantially stereoisomerically or enantiomerically pure form. The term "recovering" includes extracting, harvesting, isolating or purifying the compound from the culture or reaction medium. Recovery of the compound may be carried out according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0266] The identity and purity of the isolated products may be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g. IR, UV, NMR), colorimetry, TLC, NIRS, enzymatic assays or microbial assays (see, for example, Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 und S. 581-587; See Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0267] l. Fermentative production of vanillin The present invention also relates to a method for the fermentative production of vanillin.

[0268] The fermentation used according to the invention may be carried out, for example, in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of possible method types, including the type and geometric design of the stirrers, can be found in "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". Typical variants that can be used in the process of the invention are known to the skilled artisan or are described, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or continuous fermentation with or without biomass recycling. Depending on the production strain, air, oxygen, carbon dioxide, hydrogen, nitrogen or suitable gas mixtures can be sparged to achieve a good yield (YP / S).

[0269] The culture medium used should meet the requirements of the particular strain in an appropriate manner. Descriptions of culture media for various microorganisms are provided in the American Society for Bacteriology handbook "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981).

[0270] These media used in accordance with the present invention contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0271] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars may also be added to the medium via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add a mixture of different carbon sources. Other possible carbon sources are oils and fats, such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as palmitic acid, stearic acid or linoleic acid, alcohols, such as glycerol, methanol or ethanol, and organic acids, such as acetic acid or lactic acid.

[0272] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing these compounds. Examples of nitrogen sources include ammonia gas, or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources may be used separately or as a mixture.

[0273] Inorganic salt compounds which may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0274] Inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds such as mercaptans and thiols may be used as sulfur sources.

[0275] Phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as the phosphorus source.

[0276] Chelating agents may be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0277] The fermentation medium used according to the invention may also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts are often derived from the complex components of the medium, such as yeast extract, molasses, corn steep liquor, etc. Furthermore, suitable precursors may be added to the medium. The exact composition of compounds in the medium is highly dependent on the specific experiment and needs to be determined individually for each specific case. Information on medium optimization is given in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997). Growth media are also available from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0278] All components of the medium are sterilized either by heating (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components may be sterilized together or individually as required. All components of the medium may be present at the start of growth or optionally added continuously or batchwise.

[0279] The culture temperature is usually between 15°C and 45°C, preferably between 25°C and 40°C, and may be kept constant or varied during the experiment. The pH value of the medium should be in the range of 5 to 8.5, preferably about 7.0. The pH value for growth may be controlled during growth by adding acidic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or basic compounds, such as phosphoric acid or sulfuric acid. Antifoaming agents, such as fatty acid polyglycol esters, may be used to control foaming. To maintain the stability of the plasmid, suitable substances with a selection action, such as antibiotics, may be added to the medium. Oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the culture to maintain aerobic conditions. The culture temperature is usually between 20°C and 45°C. The culture is continued until a maximum of the desired product is formed. This is usually reached within 1 hour to 160 hours.

[0280] The process of the present invention may further comprise the step of recovering the vanillin.

[0281] The term "recovering" includes extracting, harvesting, isolating or purifying a compound from a culture or medium. Recovery of a compound may be performed according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0282] Prior to the intended isolation, the biomass of the culture broth may be removed. Methods for removing the biomass, such as filtration, sedimentation and flotation, are known to those skilled in the art. Thus, the biomass may be removed, for example, in a centrifuge, separator, decanter, filter or in a flotation separator. For maximum recovery of the valuable product, washing of the biomass is often recommended, for example in the form of diafiltration. The choice of said method depends on the biomass content and the characteristics of the biomass in the fermentation broth, as well as the interaction of the biomass with the valuable product.

[0283] In one embodiment, the fermentation broth may be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. This concentration may be carried out batchwise or continuously, as required. Pressure and temperature ranges should be selected so that firstly, no damage to the product occurs, and secondly, a minimum use of equipment and energy is required. A judicious selection of pressure and temperature levels, especially for multi-stage evaporation, allows for energy savings.

[0284] The following examples are illustrative only and are not intended to limit the scope of the invention set forth in the abstract, specification, or claims.

[0285] Numerous possible variations that will be readily apparent to those of ordinary skill in the art after considering the disclosure provided herein also fall within the scope of the present invention.

[0286] Working Example material: Unless otherwise stated, all chemical and biochemical materials and microorganisms or cells used herein are commercially available products.

[0287] Unless otherwise stated, recombinant proteins were prepared by standard methods, e.g., as described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2 ndEdition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0288] method: In a typical isoeugenol monooxygenase assay, reactions were centrifuged and extracted with MTBE prior to analysis by gas chromatography, as described in the following example.

[0289] Gas chromatography was performed with the following program: 100°C (1 min), 20°C min -1 The analysis was carried out on an Agilent Technologies'6850 equipped with an FID detector and a DB-WAX column (30 m, 250 microM, 0.25 microM) using a 500 rpm run at 245°C (10 min). Carrier gas: hydrogen, 75 cm sec -1 , spirit = 50, injection volume = 1 μL.

[0290] Example 1: Generation and testing of first generation isoeugenol monooxygenase (IEM1) The following first generation biocatalysts were used: [Table 4]

[0291] A freshly grown colony of the BL21(DE3):pIEM1 strain was picked from the LB agar plate and transferred to 20 mL of LB medium containing 100 mg / L ampicillin. It was cultured for 20 hours at 37°C with shaking at 225 rpm. 1 mL of the culture was taken and diluted with 100 mg / L ampicillin using a 1 L Erlenmeyer flask. -1 200 mL of sterile LB medium was inoculated with OD 600 The cultures were grown with shaking at 220 rpm and 37°C until the OD reached 0.4. 600The temperature was reduced to 20°C while growing until the OD reached 0.6. The culture was induced with 1 mM IPTG and grown for an additional 18 hours. Typically, after overnight incubation, the OD 600 reached 5.5 to 7.

[0292] The cells were centrifuged at 9000 g and 4° C. for 20 min. The supernatant was discarded and the cells were resuspended in 100 mM glycine-NaOH buffer (pH 10.5) containing 10% v / v DMSO (the optimum pH was later found to be about pH 9 for the isoeugenol monooxygenase of P. nitroreducens Jin1). 600 Suspensions with different cell densities corresponding to 10-90 were prepared. In a 20 mL vial, 2 mL of cell suspension with defined optical density and 75 mg of isoeugenol (Sigma Aldrich #I17206) were added. The reaction was stirred at 500 rpm for 20 h at room temperature (24 °C) using vial stoppers made of aluminum membranes with holes for oxygenation. The reaction was acidified with 6 drops of 15% HCl and diluted with 1 g L of tridecane as an internal standard before analysis by gas chromatography. -1 The extract was made with 10 mL of MTBE containing 0.01% vanillin. A negative control was performed in the same manner using empty cells. The time curve for the formation of vanillin is shown in Figure 3.

[0293] Example 2: Vanillin production by first generation isoeugenol monooxygenase (IEM1) Vanillin was produced as follows: E. coli cells expressing the isoeugenol monooxygenase of P. nitroreducens Jin1 (see Example 1) were cultured on LB agar plates containing ampicillin with 15 g L of glucose as the carbon source. -1 The cultures were transferred to 50 mL of ampicillin-containing mineral salts medium containing 15 g glucose L−1 and grown with shaking at 225 rpm at 37°C for 20 h. -1 , (NH4)2HPO45g L -1 , K2HPO416g L -1 , Citric acid 2g L -1 , MgSO4 1g L-1 , CaCl2 30 mg L -1 , trace elements, vitamins, and ampicillin 100 mg L -1 0.4 mL of this culture was then taken and used to inoculate 200 mL of mineral salts medium of the same composition in a 1 L flask. Several flasks were run in parallel. The cultures were incubated at 37°C with shaking at 180 rpm until OD 600 The cultures were grown until the OD reached 2.5. The temperature was reduced to 20°C. The cultures were incubated at 37°C for 1 h at 37°C for 2 h. 600 The culture was induced with 1 mM IPTG at 3 and grown at 20°C with shaking at 180 rpm for an additional 17 hours. 600 The final OD was 24. The culture was centrifuged at 4000 g for 50 min at 4° C. The cells were resuspended in 100 mM glycine-NaOH buffer (pH 9) and incubated at 4° C. for 1 h at 37° C. for 2 h. 600 was increased to 45.

[0294] In a 2 L reaction flask, the OD 600 1.3 L of cell suspension with a pH of 45 and 19.5 g of isoeugenol were added. The reaction was allowed to proceed with stirring at room temperature for 20 hours. Analysis of the reaction by gas chromatography revealed the presence of 7.3 g vanillin L -1 , (Z)-Isoeugenol 1.2g L -1 , and the remainder is (E)-isoeugenol 4.1 g L -1The temperature of the reaction broth was lowered to 12° C., the pH was adjusted to pH 12.5, and extracted with distilled ethyl acetate (1 L). After phase separation and removal of the organic phase, the aqueous phase was again adjusted to 12° C. and pH 12.5. A second extraction with 700 mL of ethyl acetate was performed and the organic phase was discarded. The pH of the aqueous phase was then adjusted to pH 7 with 15% HCl. The aqueous phase was extracted twice with 1 L of distilled ethyl acetate. These combined extracts contained 7.7 g of vanillin based on GC analysis. The combined organic extracts were then washed with 600 mL of water and evaporated under vacuum. 7.7 g of yellow residue was obtained. Efforts to remove the yellow color by another extraction with ethyl acetate at 10° C. and pH 12.5 failed. About 0.8 g of vanillin was lost in this operation, leaving 6.9 g of a slightly yellow solid residue. The residue was dissolved in 153 mL of hot water at 55° C. Once all the crude vanillin was dissolved, the solution was left at room temperature for 1 hour. The temperature was reduced to 4°C and maintained at this temperature for 3 hours. Finally the solution was cooled in an ice-water bath for 18 hours. The solution with the precipitated vanillin was passed through a pre-cooled paper filter. The crystals were washed with ice-cold water (30 mL). The crystals were then dried under vacuum in a desiccator at room temperature for 6 hours. 5.4 g of a slightly yellowish solid residue was recovered containing 99.7% vanillin by GC based on an internal standard. The chemical identity was verified by GC-MS, 1H-NMR and 13C-NMR. Some losses occurred during purification. Ethyl acetate is not very suitable for alkaline extraction and may be replaced by a more stable solvent, e.g. MTBE.

[0295] Example 3: Generation of second generation isoeugenol monooxygenase The following second generation biocatalysts (IEM and helper polypeptides located on two different plasmids) were tested: [Table 5]

[0296] Freshly grown cells were isolated from the agar plates and the optical density OD 600The culture was grown in 2 mL of LB medium containing ampicillin and chloramphenicol with shaking at 37 °C until the β-amyloid ratio reached 1. 1 mL of the culture was taken and diluted with 100 mg L of ampicillin. -1 and chloramphenicol 30 mg L -1 Inoculate 200 mL of LB medium containing 35 mg L FeCl in a 1000 mL flask. -1 Chaperone induction was achieved by supplementing with arabinose at 2 mg mL -1 and tetracycline 5ng mL as appropriate -1 The OD was measured at the beginning of the culture according to the manufacturer's protocol (Takara Bio Inc.). 600 The culture was grown at 37°C with shaking at 180 rpm until the OD reached 0.4. The temperature was lowered to 20°C and the 600 = 0.6 and induced with 1 mM IPTG. After overnight culture, cells were harvested by centrifugation at 3600 g and resuspended in cold 0.1 M glycine NaOH (pH 9.5) buffer to the desired optical density. Catalytic activity was tested as described in Example 1. The catalytic activity observed with the different constructs is shown in Figure 4. Most of the second generation catalysts gave higher specific activity than the first generation catalysts (control). Cells co-expressing isoeugenol monooxygenase from P. nitroreducens Jin1 with the chaperonins GroES and GroEL were the most active. Cells carrying the plasmid pKJE7 showed very poor growth and were not tested for activity.

[0297] Example 4: Fed-batch cultivation of second generation isoeugenol monooxygenase Strain BL21(DE3):pIEM1_pGro7 was tested under fed-batch cultivation conditions. Fed-batch cultivation was carried out in a reactor (Bioengineering, Switzerland) with a working volume of 3.7 L using a glucose feed based on dissolved oxygen. Yeast extract 5 g L -1 Mineral salts medium containing 0.1% glycerol and 0.1% chloramphenicol and supplemented with carbenicillin and chloramphenicol at pH 7 served as the growth medium using glycerol instead of glucose as the carbon source. Cultures were at 37°C. OD 600At 48 °C, reduce the temperature to 25 °C and grow the culture to an OD 600 51 Arabinose 1.5g L -1 The OD 600 At 59, the culture was induced with 1 mM IPTG. The feed solution contained 70% sterile aqueous glycerol. The pH was adjusted with aqueous NH3. The fermentation was continued for 43 h, with a final OD 600 The plasmid p53 activity reached 163. Plasmid stability was measured using agar plates with or without the appropriate antibiotic for selection. Bacterial cells were harvested by centrifugation as described above, resuspended in 100 mM glycine-NaOH buffer (pH 9) and tested for activity. Vanillin 3.7 g L -1 Up to OD 600 In a reaction carried out as described in Example 1, with a catalyst loading equivalent to 5, the reaction was monitored by gas chromatography.

[0298] Example 5: Generation of third generation isoeugenol monooxygenase The following third generation biocatalysts (IEM and helper polypeptides located on one single polycistronic construct) were tested: [Table 6]

[0299] A schematic diagram of the different polycistronic constructs is also shown (Figure 2). Cultivation, induction and activity tests were carried out as described in Example 1. OD 600 Comparative activity studies at a catalyst loading of 5 showed that E. coli cells expressing one of the polycistronic constructs (third generation catalyst) exhibited higher specific catalytic activity compared to the first and second generation catalysts (Figure 5).

[0300] Example 6: Generation of mutants of isoeugenol monooxygenase from P. putida IE27 Artificial mutants of isoeugenol monooxygenase from P. putida IE27 (IEM2) were induced in plasmid pJ431 (T7, R, low copy; supplier Atum, Newark, CA) and expressed in E. coli BL21(DE3)T1. Alternatively, E. coli cells producing one of the mutants of P. putida IE27 were cotransformed with the plasmid pGro7 to coproduce the chaperonins GroES and GroEL.

[0301] The following mutants were tested: [Table 7]

[0302] Freshly grown E. coli colonies carrying the plasmid-encoded mutations were picked from LB agar plates and incubated with 30 mg kanamycin L -1 The bacteria were grown overnight (16 h) at 37 °C with 220 rpm in 2 mL of LB liquid culture containing the required antibiotics. 150 μL was then transferred to a flask containing 50 mL of LB medium containing the required antibiotics and the OD 600 The culture was incubated at 37°C with shaking at 200 rpm for approximately 2 hours until the OD reached 0.45. 600 The temperature was reduced to 20°C while growing until the OD reached 0.6. After induction with 0.5 mM IPTG, the culture was grown under the same conditions for an additional 16 h. Cells of E. coli containing the mutant isoeugenol monooxygenase of P. putida IE27 were harvested by centrifugation and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5) containing 10 v / v % DMSO and finally cultured at OD 600 The OD was set to 45. For E. coli cells containing the isoeugenol monooxygenase of P. nitroreducens Jin1 (used as a positive control), an identical procedure was applied using 0.1 M glycine buffer (pH 9.0). A 20 mL vial was filled with 80 mg of isoeugenol, OD 600 2 mL of the cell suspension at 0.5% CO was added. The vial was closed with a perforated aluminum membrane for air circulation. The reaction was allowed to proceed for 17 h at room temperature (23 °C) with magnetic stirring at 450 rpm. The reaction was acidified with 3 drops of 15% HCl and 1 g L of tridecane was added as an internal standard for analysis by GC. -1The vanillin concentrations obtained with the individual mutants and the control are shown in FIG. 6. Further mutants of the isoeugenol monooxygenase of P. putida IE27 (IEM) were obtained by individual saturation mutagenesis experiments at amino acid positions T52, or Q74 or D440, carried out by a commercial manufacturer. These additional mutants were tested similarly to the mutants identified above. As shown in FIG. 10, the relative activity of each mutant is expressed as a percentage of the averaged vanillin titer obtained with mutant "C154" (i.e., mutant a154c; corresponding to PQD) under standard assay conditions.

[0303] Example 7: Triple mutant of isoeugenol monooxygenase of P. putida IE27 co-expressed with GroES and GroEL Colonies of E. coli BL21(DE3)T1 harboring plasmids pIEM2_c154_t222_a1318 (see plasmid map and annotation in FIG. 8 and SEQ ID NO: 16) and pGro7 were isolated from agar plates and added to 2 mL of LB medium containing 30 mg / L kanamycin and 30 mg / L chloramphenicol. Cultures were grown at 37° C. with shaking at 220 rpm for 16 hours. 150 μL was transferred to 50 mL of fresh LB medium containing the same antibiotics. Cells were incubated at OD 600 The cultures were grown at 37°C with shaking at 220 rpm until the OD reached 0.45. The cultures were induced with 1.5 g / L arabinose and 600 The temperature was reduced to 20°C with shaking until the OD reached 0.6. The culture was induced with 0.5 mM IPTG and grown at 20°C with shaking at 220 rpm for an additional 16 h. Cells were harvested by centrifugation and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5). An OD of 2-45 was obtained. 600Different cell dilutions corresponding to different cell densities were prepared. To an open 20 mL reaction vial, 80 mg of isoeugenol and 2 mL of cell suspensions of different optical densities were added. The reaction was allowed to proceed at room temperature for 17 h with stirring with a magnetic bar at 500 rpm. The reaction was acidified with 3 drops of 15% HCl and extracted with 10 mL of MTBE containing 1 g / L tridecane as an internal standard for analysis by GC. The vanillin concentrations observed in reactions containing different cell densities are shown in Figure 7.

[0304] Example 8: Triple mutant of isoeugenol monooxygenase of P. putida IE27 co-expressed with GroES and GroEL from different expression constructs derived from the polycistronic construct PC1_triple (SEQ ID NO: 25; containing the triple mutant of IEM IE27) Colonies of E. coli cells BL21(DE3)T1 carrying different plasmid constructs for expressing the triple mutant of isoeugenol monooxygenase IE27 together with the chaperonins GroES and GroEL (constructed according to the general scheme in FIG. 11, with nucleotide sequences selected from SEQ ID NOs: 17-24, and the corresponding constructs in which the transcription terminator of the IEM coding sequence is absent) were picked from kanamycin-containing agar plates and inoculated into 2 mL of mineral salts medium, which contains 30 g / L glycerol, 45 g / L (NH4)2HPO4, 5 g / L glycerol, and 100 g / L (NH4)2HPO4. -1 , K2HPO416g L -1 , Citric acid 2g L -1 , MgSO4 1g L -1 , CaCl2 30 mg L -1 , yeast extract 5g / L, trace elements and kanamycin 50mg / L.

[0305] The culture was shaken at 230 rpm for 16 h at 37° C. 0.25 mL of the culture was transferred to a flask containing 50 mL of the same medium and the optical density OD 600 The culture was grown at 37°C and 230 rpm until the OD reached 2.5. The temperature was lowered to 23°C and the 600 The culture was continued at 220 rpm until the rO2 reached 3. The culture was induced with 0.5 mM IPTG and maintained under the same conditions overnight.

[0306] At the end of the incubation, samples were taken to determine the optical density at 600 nm, to estimate the dry mass of the cells using standard gravimetric methods, and for activity testing. For activity testing, 15 mL of culture was centrifuged at 3500 g for 35 min at 4° C. and the supernatant was discarded. The cell pellet was frozen at −80° C. for several hours. The frozen pellet was thawed on ice and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5) to obtain a final OD 600 was set to 20.

[0307] To a 20 mL flask, add 80 mg of isoeugenol and 1800 μL of ice-cold 0.1 M glycine buffer (pH 9.5). Under magnetic stirring, the cell resuspension (OD 600 = 20) Add 200 μL to obtain a final OD 600 The reaction was stirred magnetically at 23° C. and 450 rpm for 16 h.

[0308] Tridecane 1 g L as internal standard -1 The reactions were acidified with 8 drops (80 μL) of 85% phosphoric acid before extraction with 10 mL of MTBE containing 10% vanillin. The vanillin concentration was measured by gas chromatography as described above. The vanillin concentrations obtained with the different plasmid constructs were normalized with respect to the cell dry mass of the cell suspension used in the reaction. The results are shown in FIG. 12.

[0309] The contents of the cross-referenced documents are incorporated by reference.

[0310] The sequences referred to herein are: [Table 8] [Table 9] [Table 10] [Table 11]

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

Table 38

Claims

1. An expression system for recombinant expression of a polypeptide having isoeugenol oxidizing activity, comprising: a. a nucleotide sequence (A) encoding a polypeptide having enzymatic isoeugenol oxidizing activity, and b. at least one nucleotide sequence (B) encoding at least one helper polypeptide that alone or in cooperation with the nucleotide sequence (A) aids in the functional expression of the polypeptide encoded by the nucleotide sequence (A); and providing simultaneous expression of said nucleotide sequences (A) and (B).

2. 2. The expression system of claim 1 which is a single polycistronic nucleic acid construct.

3. The nucleotide sequence (A) is a. a polypeptide having isoeugenol oxidizing activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:2; or b. A polypeptide having isoeugenol oxidizing activity comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4 and comprising at least one mutation at an amino acid sequence position selected from T52, Q74, D440, and optionally at least one further mutation at an amino acid sequence position selected from N120, T121, F281, M298, and L470. The expression system according to claim 1 or 2, which encodes

4. 4. The expression system of claim 3, wherein the polypeptide having isoeugenol oxidizing activity is a mutant polypeptide comprising at least one mutation at an amino acid sequence position selected from T52, Q74 and D440 of SEQ ID NO:

4.

5. The at least one nucleotide sequence (B) comprises nucleotide sequences (B1) and (B2), a. (B1) encodes a polypeptide having chaperonin activity comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8; and b. (B2) encodes a polypeptide having chaperonin activity comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10; An expression system according to any one of claims 1 to 4.

6. A polypeptide having isoeugenol oxidizing activity comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO:4, and an amino acid sequence comprising at least one mutation at an amino acid sequence position selected from T52, Q74, D440, and optionally at least one further mutation at an amino acid sequence position selected from N120, T121, F281, M298 and L470.

7. 7. The polypeptide of claim 6, which is a mutant polypeptide comprising at least one mutation at an amino acid sequence position selected from T52, Q74 and D440 of SEQ ID NO:

4.

8. The mutant is a. Single mutant (T52X 1 ), (Q74X 2 ) and (D440X 3 ) b. Double mutant (T52X 1 , Q74X 2 ), (T52X 1 , D440X 3 ) and (Q74X 2 , D440X 3 ),and c. Triple mutant (T52X 1 , Q74X 2 , D440X 3 ) is selected from Where: X 1 is P, M or K; X 2 is H or A, and X 3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; The polypeptide of claim 7.

9. A recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 6 to 8, or comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 3 or 5 or their reverse complements.

10. A recombinant nucleic acid construct comprising the recombinant nucleic acid of claim 9 or a reverse complement thereof.

11. below, a. a single nucleic acid construct contained by a recombinant expression system according to any one of claims 1 to 5 or its reverse complement; b. A recombinant nucleic acid according to claim 9 or its reverse complement; c. The recombinant nucleic acid construct of claim 10 or its reverse complement. An expression vector comprising:

12. A non-human host organism or host cell, optionally comprising in its genome a. a single nucleic acid construct comprised by a recombinant expression system according to any one of claims 1 to 5, or b. A recombinant nucleic acid according to claim 9, or c. The recombinant nucleic acid construct of claim 10, or d. The expression vector of claim 11. A non-human host organism or host cell, including one into which has been stably integrated.

13. A method for producing an isolated catalytically active polypeptide having isoeugenol oxidation activity, the method comprising co-expressing said polypeptide having isoeugenol oxidation activity with at least one helper polypeptide, each encoded by an expression system defined in any one of claims 1 to 5, in a host cell system, and optionally isolating said polypeptide having isoeugenol oxidation activity.

14. below, a) contacting isoeugenol with a polypeptide having isoeugenol-oxidizing activity as defined in any one of claims 6 to 8 or a polypeptide having isoeugenol-oxidizing activity produced by the method of claim 13 in the presence of oxygen to produce vanillin; b. Optionally, isolating the vanillin produced in step a. A method for producing vanillin, comprising:

15. 15. The method according to claim 14, further comprising the step of chemically or biochemically isomerizing eugenol to isoeugenol and, optionally, isolating isoeugenol from the reaction medium.

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