Vanillin manufacturing method

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, comprising the use of novel combinations of polypeptides. There are further provided novel enzyme variants applicable in the biochemical production of vanillin.

[0002] Background Art Vanillin is a globally important compound for use in foods, beverages and pharmaceuticals. Only a small fraction of global production is obtained naturally from extraction from vanilla pods; however, the availability of these natural plant sources is low, and the production method is laborious and time-consuming. While complex extracts obtained from pods of orchid species *V. planifolia* or *V. tahitensis* provide unique, complex mixtures and olfactory properties at high cost, the vast majority of vanillin (>99%, i.e., 16,000 tons per year) is produced from petrochemical feedstocks, with still a small fraction obtained from lignin waste from the paper manufacturing industry. However, there is a small but growing market for natural "vanillin not from the bean" (NFB) supplied mainly to North America and Europe. Regulatory requirements for claiming natural status differ between these two markets.

[0003] Various starting materials including ferulic acid, curcumin, eugenol or isoeugenol can be converted to vanillin using different 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, such as sugars like 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 eugenol, which is traditionally obtained by isomerization under heat in the presence of strong bases. This method is no longer compliant with European legislation regarding natural flavoring substances. Nevertheless, plants that are rich in isoeugenol have been identified.

[0006] Isoeugenol can be converted into 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 for the oxidative cleavage of isoeugenol to vanillin. In recent years, it has been found that purified hemin alone is as effective as non-specific heme-containing enzymes for such biotransformation[Mutti FG. 2012. Bioorg Chem Appl 2012,1-13; Li Y-H, Sun Z-H, Zhao L-Q, 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 for converting 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 resulted in 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 intended for the industrial-scale production of vanillin.

[0008] Industrial processes often require the overproduction of one or more functional enzymes in recombinant bacteria, such as Escherichia coli (E. coli). Overproduction of enzymes in suitable bacteria provides a safe and economical source of those enzymes. Overproduction of key enzymes also helps minimize side activities resulting from the endogenous enzyme activity of the host organism (e.g., enzymes that can reduce or oxidize vanillin to vanillyl alcohol or vanillic acid, respectively).

[0009] The correct folding of enzymes in host organisms is crucial for catalytic activity. Improperly folded enzymes tend to aggregate (e.g., form inclusion bodies) or degrade in the host, resulting in reduced or no catalytic activity. Various strategies can be applied to ensure that enzymes are correctly folded in host organisms. For example, reducing the expression level in recombinant host organisms by using low gene doses, lowering the inducible substance concentration (in the case of an induceable system), or lowering the transcription level by using low promoter strength may be beneficial. Low temperatures during enzyme synthesis can also help improve the folding of recombinant proteins. Alternatively, improperly folded enzymes may not fold using strong denaturing chemicals and then be refolded under physiological conditions. However, such procedures are time-consuming and costly.

[0010] In molecular biology, the broad classification of molecular chaperones refers to proteins that assist in the folding or unfolding of covalent bonds and the construction or degradation of other macromolecular structures. The group of chaperonin proteins belongs to this broad classification of chaperone molecules. The structure of these chaperonins resembles two donut-shaped structures stacked on top of each other to form a barrel. Each ring consists of seven, eight, or nine subunits, depending on the organism in which the chaperonin was found.

[0011] Group I chaperonins are found in bacteria and in endosymbiotic organelles such as chloroplasts and mitochondria. Group II chaperonins, found in eukaryotic cytosols and archaea, are not 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 mentioned above, the chaperonin system GroES / GroEL forms a barrel-like structure with a cavity that allows for the uptake of misfolded proteins for refolding when ATP is consumed [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 chaperones from a broad classification for the recombination production of monooxygenases, particularly isoeugenol monooxygenase, for the enzymatic synthesis of vanillin has not been investigated to date.

[0014] While there are several reports of natural vanillin production by bioengineering (e.g., International Publication No. 2013 / 022881 (WO2013 / 022881), Korean Registered Patent No. 101163542 (KR101163542(B1))), the discovery of simpler, more efficient, and cost-effective / or efficient processes for the production of natural vanillin remains necessary. In particular, higher specific activity of the enzyme reduces the amount of catalyst required, improving process performance and economics. Furthermore, there is a need for even more improved enzymes applicable, especially in the biochemical production of vanillin from isoeugenol as a starting material.

[0015] summary This invention addresses the aforementioned drawbacks associated with known systems that have been applied to date for the biochemical production of vanillin.

[0016] The inventors have surprisingly observed that certain helper polypeptides can be successfully used in the large-scale production of certain known and novel isoeugenol oxidases with high functional activity. In particular, the inventors have surprisingly found that co-expression of 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 remarkably succeeded in genetically modifying a microbial oxidase enzyme from the Pseudomonas putida species, which has made it possible for the first time to apply this to the conversion of isoeugenol, particularly (E)-isoeugenol, to vanillin in the presence of molecular oxygen.

[0018] Therefore, surprisingly, the inventors were able to improve the biochemical approach to the production of vanillin by isoeugenol by applying the generated and / or genetically modified isoeugenol oxidase, as described in more detail below. [Brief explanation of the drawing]

[0019] [Figure 1] One-step conversion of isoeugenol to vanillin and acetaldehyde. [Figure 2] Polycistronic constructs for the co-expression of isoeugenol monooxygenase (IEM1) of P. nitroreducens Jin1 using chaperonin GroES and GroEL with low-copy and high-copy plasmids. The plasmid names of the low-copy constructs are shown to the left of each construct. The numbers above the horizontal bars indicate the following elements: 1: RBS (strong); 2: IEM1; 3: GroES; 4: Natural RBS; 5: GroEL; 6: Spacer; 7: RBS (moderate); 8: RBS (weak). [Figure 3]First-generation catalysts based on IEM1: Product titers under different biocatalytic loadings at unrestricted substrate concentrations at pH 10.5. Symbols: vanillin (black diamond), remaining trans isoeugenol (■), remaining (Z)-isoeugenol (▲). Average data from two experiments are shown. [Figure 4] Second-generation catalyst: Product titers at different catalytic loadings, expressed as different optical cell densities: OD600=18 (vertical stripes), OD600=10 (horizontal stripes), OD600=5 (dotted line), OD600=2 (diagonal line), OD600=1 (black). The biocatalyst was pre-grown in LB medium. IEM1: Isoeugenol monooxygenase of P. nitroredusens Jin1. Co-expressed chaperones are also shown. IEM1 (control) represents the activity of wild-type isoeugenol monooxygenase of P. nitroredusens Jin1 expressed according to Example 1 in the absence of any chaperone. Values ​​reflect averaged endpoint measurements within a 10% deviation. [Figure 5] Product titers from small-scale reactions with catalytic load OD600=5 using one of the following catalysts: first-generation catalyst (vertical line), second-generation catalyst (horizontal line), and third-generation catalyst (dotted line). Negative controls were obtained with empty cells (blacked out). Average endpoint concentrations (maximum 10% deviation) from the dual experiment are shown. [Figure 6] Vanillin titers obtained from single, double, and triple mutants of isoeugenol monooxygenase in P. petida IE27 (IEM2). "Ref" refers to the (inactive) isoeugenol monooxygenase of P. petida IE27 used as a reference. Cells producing isoeugenol monooxygenase in P. nitroredusens Jin1 (IEM1) served as a positive control (C+). Cells containing an empty vector served as a negative control (C-). Data points were averaged from the double experiments. [Figure 7]In vivo conversion of isoeugenol to vanillin under different catalytic loads is shown for the triple codon mutant of isoeugenol monooxygenase (IEM2) from P. petida IE27 (horizontal line) and the isoeugenol monooxygenase (IEM1) from P. nitroredusens Jin1 (vertical line). E. coli cells containing two plasmid systems for co-expression of isoeugenol monooxygenase and chaperonins GroES and GroEL functioned as catalysts. Isoeugenol monooxygenase from P. nitroredusens Jin1 was generated using a high-copy construct, while the triple mutant of P. petida IE27 was generated using a low-copy construct. The mean product titers of the two experiments are shown with a deviation of less than 10%. [Figure 8] Amino acid sequence alignment of IEM1 of Pseudomonas nitroredusens Jin1 and IEM2 of Pseudomonas petida IE27. [Figure 9] Annotation of plasmid pIEM2_C154_T222_A1318 for expressing a triple mutant of IEM2 in Pseudomonas petida IE27, and corresponding plasmid map. [Figure 10] Relative activity of isoeugenol monooxygenase mutants of P. petida IE27 obtained by individual saturation mutagenesis at amino acid positions T52, Q74, or D440. The corresponding amino acids at these positions are shown on the x-axis. 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. 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 controls (C-). [Figure 11] A general scheme of modified insertion structures for expressing the triple mutant of isoeugenol monooxygenase IE27 described herein, and IEMs such as the chaperonins GroES and GroEL. [Figure 12]Normalized product titers of vanillin obtained under standard assay conditions in strains of E. coli containing different plasmid constructs prepared according to the general scheme in Figure 11 to express a triple mutant of isoeugenol monooxygenase IE27 together with the chaperonins GroES and GroEL. The plasmid constructs differed in their promoter sequences for chaperonin expression and included a transcriptional terminator after the ORF of isoeugenol monooxygenase, optionally indicated as +term or -term, respectively. The polycistronic construct PC1_triple is shown 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 lysogenic 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 definition The term "isoeugenol" refers to 2-methoxy-4-(prope-1-en-1-yl)phenol (CAS registry number: 97-54-1), any mixture of isomers of trans-isoeugenol and cis-isoeugenol, or (E)-isoeugenol and (Z)-isoeugenol, respectively.

[0022] The terms "isoeugenol monooxygenase," "polypeptide having isoeugenol monooxygenase activity," "isoeugenol monooxygenase protein," "isoeugenol oxidase," "polypeptide having isoeugenol oxidizing activity," "isoeugenol oxidized protein," or "IEM" relate to polypeptides 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 the specific molecular mechanism of action of the enzyme. Preferably, the enzyme stereospecifically converts the (E) isomer or trans isomer of isoeugenol. Vanillin is preferably obtained as the main product.

[0023] The "isoeugenol oxidation activity" is determined under the "standard conditions" described in more detail in the following examples herein: this is determined using recombinant IEM-expressing cells, disrupted IEM-expressing cells, fragments thereof, or concentrated or purified IEM enzymes 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, particularly 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 terms “host cell” or “transformed cell” mean a cell (or organism) modified to house at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or nucleic acid sequence, which yields a polypeptide for use as described herein for transcription. Host cells are, in particular, bacterial, fungal, or plant cells. A host cell may contain a recombinant gene integrated into the host cell’s nuclear genome or organelle genome. Alternatively, the host may contain the recombinant gene extrachromosomally.

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

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

[0028] Orthologs, or orthogus sequences, are sequences that are similar to one another because they are found in species that are descendants of a common ancestor. For example, it is well known that plant species sharing a common ancestor contain many enzymes with similar sequences and functions. Those skilled in the art can identify orthogus sequences and predict the function of orthologs by constructing a polygenic tree for a gene family of a species, for example, using the CLUSTAL or BLAST program. Methods for identifying or confirming similar functions in homologous sequences involve comparing transcription profiles in host cells or organisms, such as plants or microorganisms, that overexpress or delete (knockout / knockdown) the relevant polypeptides. Those skilled in the art understand that genes with similar transcription profiles, typically with more than 50% regulated transcription, or more than 70% regulated transcription, or more than 90% regulated transcription, have similar functions. Homologs, paralogs, orthologs, and any other variants of the sequence in this invention are expected to function in a similar manner by creating host cells, organisms, such as plants or microorganisms, that produce isoeugenol monooxygenase.

[0029] The term "plant" is used without distinction to include plant protoplasts, plant tissues, plant cell tissue cultures that produce regenerated plants, or parts of plants, or plant organs, such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. The methods of the embodiments described herein can be carried out using any plant.

[0030] Certain organisms or cells are "capable of producing vanillin" if they naturally produce vanillin before or in combination with the nucleic acids described herein, or if they do not naturally produce vanillin but are transformed to produce vanillin. Organisms or cells transformed to produce higher amounts of vanillin than naturally occurring are also included in "organisms or cells capable of producing vanillin."

[0031] As used herein, the terms “purified,” “substantially purified,” and “isolated” refer to a state in which the compound of the present invention does not have other different compounds that would normally associate with it in its natural state. Therefore, “purified,” “substantially purified,” and “isolated” objects contain at least 0.5% by mass, 1% by mass, 5% by mass, 10% by mass, or 20% by mass, or at least 50% by mass or 75% by mass, of the mass of a given sample. In one embodiment, these terms refer to a compound of the present invention containing at least 95% by mass, 96% by mass, 97% by mass, 98% by mass, 99% by mass, or 100% by mass, of the mass of a given sample. As used herein, when relating to nucleic acids or proteins, the terms “purified,” “substantially purified,” and “isolated” nucleic acids or proteins also refer to a purified or concentrated state that differs from those that occur naturally, for example, in prokaryotic or eukaryotic environments, for example, in bacterial or fungal cells, or in the bodies of mammals, particularly humans. Any degree of purification or concentration greater than that which occurs naturally, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that do not normally associate in the environment of the prokaryotes or eukaryotes, falls within the meaning of “isolated.” The nucleic acids or proteins or types of nucleic acids or proteins described herein may be isolated or associated with structures or compounds that do not otherwise normally associate in nature, according to various methods and processes known to those skilled in the art.

[0032] In the descriptions and accompanying claims provided herein, the use of “or” means “and / or” unless otherwise specified.

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

[0034] Furthermore, when describing various embodiments using the term “comprising,” those skilled in the art will understand that in certain examples, one embodiment may instead be described using the language “consisting essentially of” or “consisting of.”

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

[0036] The term "effectively" refers to a range of values ​​approximately 80-100%, for example 85-99.9%, especially 90-99.9%, more specifically 95-99.9%, or 98-99.9%, especially 99-99.9%.

[0037] "Mainly" refers to percentages in the range of 51-100%, especially 75-99.9%, more specifically 85-98.5%, or even 95-99%, that is, percentages in the range of over 50%.

[0038] In the description of the present invention, “main product” refers to a single compound or at least two compounds, for example two, three, four, five or more, particularly two or three compounds, where a single compound or group of compounds is “mainly” produced by the reaction described herein and is included in the reaction in a main proportion based on the total amount of components of the product formed by the reaction. The proportion may be a molar ratio, a weight ratio, or, preferably, an area ratio calculated from the corresponding chromatogram of the reaction product by chromatographic analysis.

[0039] In the context of this invention, “by-product” refers to a single compound or at least two compounds, for example, two, three, four, five or more, particularly two or three compounds, and a single compound or group of compounds refers to those not “primarily” produced by the reactions described herein.

[0040] Due to the reversibility of enzymatic reactions, the present invention relates to the reaction direction of both enzymatic and biocatalytic reactions described herein, unless otherwise specified.

[0041] The “functional variants” of polypeptides described herein include “functional equivalents” of polypeptides as defined below.

[0042] The term "stereoisomer" includes conformational isomers in particular.

[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. When several asymmetric centers are present in a single molecule, the present invention encompasses all combinations of different stereostructures of these asymmetric centers, such as enantiomer pairs.

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

[0045] The "yield" and / or "conversion rate" of the reaction according to the present invention is determined, for example, over a defined period of 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours or 48 hours after the reaction proceeds. In particular, the reaction is carried out under precisely defined conditions, for example, the "standard conditions" defined herein.

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

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

[0048] Specific productivity yield represents the amount of product produced per hour, for example the amount of vanillin, and L of fermentation broth per gram of biomass. The amount of wet cell mass, denoted as WCW, represents the amount of biologically active microorganisms in a biochemical reaction. This value is expressed as g product per WCW per hour (i.e., g / g WCW -1 h -1 ). Alternatively, the amount of biomass may be expressed as the amount of dry cell mass, denoted as DCW. Furthermore, biomass concentration can be determined more easily by measuring the optical density at 600 nm (OD 600 ) and using experimentally determined correlation coefficients to estimate the corresponding wet cell or dry cell mass, respectively.

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

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

[0051] The “enzymatic catalysis” or “biocatalysis” method means carrying out the method under the catalytic action of an enzyme, including an enzyme variant, as defined herein. Accordingly, the method may be carried out in the presence of an isolated (purified, concentrated) or crude enzyme, or in the presence of a cell line, in particular a natural or recombinant microorganism containing the active form of the enzyme and capable of catalyzing the conversion reaction disclosed herein.

[0052] The terms “selectively converting” or “increasing selectivity” generally mean that a particular stereoisomer, such as the E-form of an unsaturated hydrocarbon, is converted at a higher rate or in a higher quantity (in molar terms) than the corresponding Z-form during the entire course of the reaction (i.e., between the start and end of the reaction), at a specific point in time in the reaction, or during “intervals” in the reaction. In particular, such selectivity may be observed during “intervals” corresponding to conversions 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 the substrate. The higher rates or quantities mentioned above may be, for example, from the following perspectives: - Higher maximum yield of isomers observed throughout the entire reaction process or during intervals thereafter; - Higher relative amounts of isomers at a defined percentage of substrate conversion rate; and / or - The same relative amount of isomers at a higher percentage of the conversion value; These may be expressed as follows, each of which is preferably observed in comparison to a reference method, the reference method being carried out under the same conditions as other known chemical or biochemical means.

[0053] "E-stereoselectivity" or "E-selectivity" refers to the ability to produce E-isomers of a particular C=C-double bond in an E-isomerically pure, essentially pure, or concentrated form, or the ability to specifically or essentially specifically convert an E-isomer from multiple other isomers at the aforementioned specific position of the double bond or from a mixture of E-isomers and Z-isomers by the enzymatic catalysis methods described herein.

[0054] In general, according to the present invention, all "isomers" of the compounds described herein, such as constituent isomers, and in particular stereoisomers and mixtures thereof, such as optical isomers or geometric isomers, such as E isomers and Z isomers, and combinations thereof. Where several asymmetric centers are present in the molecule, the present invention includes all combinations of different stereostructures of these asymmetric centers, such as pairs of enantiomers, or any mixture of stereoisomers.

[0055] Where this disclosure describes features, parameters, and their ranges of different priorities (including, in general, features, parameters, and their ranges that are not clearly preferred features), any combination of two or more such features, parameters, and their ranges is included in this disclosure, regardless of their respective priorities, unless otherwise specified.

[0056] "Polycistronic" refers to a nucleic acid molecule, particularly mRNA or its corresponding cDNA, that can independently 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 can be modified by introducing one or more identical or different regulatory sequences at appropriate locations, such as promoters, RBSs and / or terminators, in order to affect or regulate the transcription and / or translation of at least one coding sequence contained in the nucleic acid molecule.

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

[0059] 1. An expression system for recombinant expression of polypeptides having isoeugenol oxidation 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 that has isoeugenol oxidation activity, particularly in the presence of oxygen, especially molecular oxygen, to form vanillin and acetaldehyde from isoeugenol, and b. At least one, for example, one, two, or three, preferably two, helper polypeptides encoded by the nucleotide sequence (A), which, alone or preferably in cooperation, encode a helper polypeptide that assists in the functional expression of the polypeptide, particularly in the correct folding of the polypeptide expressed herein. The expression system comprising and providing simultaneous expression of the nucleotide sequences (A) and (B).

[0060] 2. An expression system according to Embodiment 1, a. A combination of at least two nucleic acid constructs, preferably acting or functioning independently of each other, each possessing the nucleic acid sequence (A) and the at least one nucleic acid (B), and b. A single nucleic acid construct having the nucleic acid sequence (A) and the at least one nucleic acid (B), which is ultimately transcribed into a single mRNA molecule containing, for example, a nucleic acid construct or vector having multiple cloning sites, or more preferably a polycistronic nucleic acid construct. An expression system selected from the above. The single nucleic acid construct possessing the nucleic acid sequence (A) and the at least one nucleic acid (B) may also be derived from polycistronic nucleic acid constructs. These may be derived from them by introducing one or more identical or different regulatory sequences at appropriate locations, e.g., promoters, RBS and / or terminators, to affect or regulate the transcription and / or translation of at least one coding sequence of (A) and (B) contained in the nucleic acid molecule, as further illustrated in the experimental paragraphs.

[0061] 3. An expression system according to Embodiment 1 or 2, wherein the nucleotide sequence (A) is a. A polypeptide having isoeugenol oxidative activity and containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2 (i.e., IEM1 isolated from the original Pseudomonas nitroredusens Jin1), or b. A polypeptide comprising an amino acid sequence having isoeugenol oxidation activity and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4 (i.e., IEM2 isolated from the original Pseudomonas ptyida IE27), wherein the sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with respect to SEQ ID NO: 4. The amino acid sequence having % or 100% identity includes at least one mutation at an amino acid sequence position selected from T52, Q74, and D440, and optionally includes at least one, for example, one, two, three, four, or five further mutations at an amino acid sequence position selected from N120, T121, F281, M298, and L470, particularly N120I, T121P, F281Q, M298K, and L470S, the polypeptide The expression system that encodes the aforementioned expression system.

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

[0063] 5. An expression system according to Embodiment 4, wherein the mutant is the following 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) Selected from, During the ceremony, X1 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either P, K, or M, or other natural or non-natural, particularly natural, vanillin-producing enzyme variants, preferably X1 is P or M, most preferably P. X2 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either H or A, or other natural or non-natural, particularly natural, and most preferably X2 is H. X3 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either 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, particularly natural, vanillin-producing enzyme variants, 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 aforementioned expression system.

[0064] Examples of such dual mutants expressed by such an expression system without restrictions include the following: (T52X1, Q74X2): In the formula, X1 is P and X2 is either H or A; or X1 is K and X2 is either H or A; or X1 is M, and X2 is either H or A. (T52X1, D440X3) In the formula, 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) In the formula, 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] The preferred double mutants are as follows: (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), where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0066] Examples of such triple mutants expressed by such an expression system without restrictions include the following: (T52X1, Q74X2, D440X3) In the formula, 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] The preferred triple mutant is as follows: (T52P, Q74H, D440X3), where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0068] The aforementioned single, double, or triple mutants may be further modified by at least one, for example, one, two, three, four, or five, preferably one, two, or three, most preferably one, further mutation at the amino acid sequence position of SEQ ID NO: 4, which may be optionally selected from N120, T121, F281, M298, and L470, and particularly selected from mutants N120I, T121P, F281Q, M298K, and L470S.

[0069] 6. An expression system described in any one of Embodiments 1 to 5, wherein at least the nucleotide sequence (B) comprises nucleotide sequences (B1) and (B2), a. (B1) encodes a polypeptide having chaperonin activity that contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 10 (GroES). The aforementioned expression system.

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

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

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

[0073] 8. A polypeptide having isoeugenol oxidative activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 4 comprises at least one mutation at an amino acid sequence position selected from T52, Q74, and D440, and further mutations at an amino acid sequence position optionally selected from N120, T121, F281, M298, and L470, particularly 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. A polypeptide according to 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) Selected from, During the ceremony, X1 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either P, K, or M, or other natural or non-natural, particularly natural, vanillin-producing enzyme variants, preferably X1 is P or M, most preferably P. X2 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either H or A, or other natural or non-natural, particularly natural, and most preferably X2 is H. X3 is an amino acid substitution resulting in an enzyme variant that functionally produces vanillin, either 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, particularly natural, vanillin-producing enzyme variants, preferably X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y, and most preferably N. Polypeptide.

[0076] For examples of such double and triple variants of the present invention that are not limited, please refer to Embodiment 5 above.

[0077] The single, double, or triple mutants may be further modified by at least one, for example, one, two, three, four, or five, preferably one, two, or three, most preferably one, further mutation at the amino acid sequence position of SEQ ID NO: 4, which is optionally selected from N120, T121, F281, M298, and L470, and particularly selected from N120I, T121P, F281Q, M298K, and L470S.

[0078] 11. Recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide described in any one of Embodiments 8 to 10.

[0079] 12. Recombinant nucleic acids comprising a nucleotide sequence or its reverse complement having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less than 100%, for example 99.9%, sequence identity with respect to SEQ ID NOs: 1, 3, or 5.

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

[0081] Specific examples of recombinant nucleic acids include those having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less than 100%, for example 99.9%, sequence identity to SEQ ID NO: 3 or 5, and containing a nucleotide sequence encoding the IEM2 variant, or its reverse complement, as described above for Embodiment 5.

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

[0083] 13. Recombinant nucleic acid constructs comprising recombinant nucleic acids of Embodiment 11 or Embodiment 12, or their reverse complements.

[0084] 14. The following, a. A recombinant expression system according to any one of Embodiments 1 to 6, or one of the partial nucleic acid constructs, or their reverse complements, b. Nucleic acids as defined in Embodiment 11 or 12, or c. Structure described in Embodiment 13 An expression vector containing this vector.

[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 described above may be such that a monocistronic mRNA or, in particular, a polycistronic mRNA encoding the isoeugenol oxidase of the present invention and one or more helper polypeptides described herein is transcribed upon expression.

[0087] 16. Arbitrarily and stably incorporated into its genome, a. One of the recombinant expression systems or partial nucleic acid constructs described in any one of Embodiments 1 to 6, or b. A nucleic acid or its reverse complement as defined in Embodiment 11 or 12, or c. The structure described in Embodiment 13, or d. Vector according to Embodiment 14 or 15 Non-human host organisms or host cells, including those mentioned above.

[0088] 17. A non-human host organism or host cell according to Embodiment 16, selected from prokaryotic or eukaryotic microorganisms, plants, or cells derived therefrom.

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

[0090] 19. The non-human host organism or host cell according to Embodiment 18, wherein the bacteria are selected from the genus Escherichia, particularly from the Escherichia species, and the yeast is selected from the genus Saccharomyces or Pichia, particularly 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, for example substantially simultaneous and particularly simultaneous, of the polypeptide having isoeugenol oxidation activity with at least one helper polypeptide encoded by an expression system defined in any of Embodiments 1 to 6 in a host cell line, and optionally isolation of the polypeptide having isoeugenol oxidation activity.

[0092] "Co-expression" or "simultaneous expression" should be broadly understood as the functional expression of a polypeptide having isoeugenol oxidation activity (IEM), in particular in a manner that facilitates the correct folding of the expressed IEM polypeptide by a helper polypeptide. Simultaneous or substantially simultaneous co-expression of both polypeptides is one unrestricted alternative. Other unrestricted alternatives may be seen in the timely sequential expression of both polypeptides, beginning with the expression of the helper polypeptide, followed by the expression of the IEM polypeptide. Other unrestricted alternatives may be seen in the timely overlapping co-expression of both polypeptides, in which only the helper polypeptide is expressed in the initial stage, and both polypeptides are expressed in the overlapping stage. Other alternatives may be developed by skilled readers without inventive effort.

[0093] 21. The method according to Embodiment 20, for producing 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. A step of contacting isoeugenol with a polypeptide having isoeugenol oxidation activity as defined in any one of Embodiments 7 to 10 or a polypeptide having isoeugenol oxidation activity produced by the method of any one of Embodiments 20 to 22 in the presence of oxygen, particularly molecular oxygen, to produce vanillin and acetaldehyde, and b. Optionally, a step to isolate the vanillin produced in step a. A method for producing vanillin, including the method described above.

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

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

[0098] Vanillin produced by any of the methods described herein may be converted to derivatives such as, but are not limited to, hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals, or ketals.

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

[0100] Alternatively, vanillin derivatives may be obtained using biochemical methods by contacting vanillin with enzymes, such as, but not limited to, oxidoreductases, monooxygenases, dioxygenases, and transferases. The biochemical transformation 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 defined in any one of Embodiments 1 to 6 to express their coding nucleotide sequences (A) and (B).

[0102] 25. The method according to Embodiment 23 or 24, wherein isoeugenol is contacted with host cells, cell lysates of host cells, or culture media containing said host cells and / or polypeptides having isoeugenol oxidation activity as defined in any one of Embodiments 7 to 10, isolated from host cells, cell lysates, or culture media.

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

[0104] 26. The method according to Embodiment 25, wherein vanillin is produced by fermentation using the non-human host organism or host cell.

[0105] 27. The method according to Embodiment 25, wherein vanillin is produced enzymatically by the conversion of isoeugenol in an isolated polypeptide having isoeugenol oxidation 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 the steps of chemically or biochemically isomerizing eugenol to isoeugenol prior to step a, and optionally isolating isoeugenol from the reaction medium.

[0107] 29. A method for producing a mutant polypeptide having isoeugenol oxidation activity, a. A step of selecting nucleic acids according to Embodiment 11 or 12, b. A step of modifying the selected nucleic acid to obtain at least one mutant nucleic acid, c. Providing a mutant nucleic acid sequence to a host cell or single-celled organism to express a polypeptide encoded by the mutant nucleic acid sequence, d. A step of screening for at least one mutant polypeptide that is active in the oxidation of isoeugenol, e. Optionally, if the mutated polypeptide does not have the desired activity, repeat process steps a. to d. until a polypeptide with the desired activity is obtained, and f. Optionally, if a mutant polypeptide with desired activity is identified in step d. or e., the step of isolating the corresponding mutant nucleic acid. The method, including the method described above.

[0108] Further aspects and embodiments of the present invention described above will be explained in subsequent paragraphs with reference to certain preferred embodiments.

[0109] b. Polypeptides applicable according to the present invention In the contents of this document, the following definitions apply.

[0110] The interchangeable terms "polypeptide" or "peptide" refer to a natural or synthetic linear or continuous chain of peptide-linked amino acid residues containing approximately 10 to over 1000 residues. Short-chain 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 the polypeptides represents the "primary structure" of the protein. The amino acid sequence also predetermines the "secondary structure" of the protein through the formation of special structural elements within the polypeptide chain, such as alpha-helices and beta-sheet structures. The arrangement of multiple such secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. When a protein contains one or more polypeptide chains, these chains are spatially arranged to form the "quaternary structure" of the protein. The correct spatial arrangement or "folding" of a protein is a requirement for protein function. Denaturation or unfolding destroys protein function. If such destruction is reversible, protein function can be restored by refolding.

[0112] A typical protein function referred to herein is "enzymatic function," where a protein acts as a biocatalyst for a substrate, such as a chemical compound, and catalyzes the conversion of the substrate into a product. Enzymes may exhibit high or low substrate and / or product specificity.

[0113] Therefore, in this specification, a "polypeptide" said to have a specific "activity" implicitly refers to a correctly folded protein exhibiting the indicated activity, such as specific enzymatic activity.

[0114] Therefore, unless otherwise specified, the term "polypeptide" also includes the terms "protein" and "enzyme."

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

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

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

[0118] The present invention also relates to “functional equivalents” (also referred to as “analogs” or “functional variants”) of polypeptides specifically described herein.

[0119] For example, “functional equivalent” means a polypeptide that exhibits at least 1–10%, at least 20%, at least 50%, at least 75%, or at least 90% higher or lower IME activity than each polypeptide as specifically defined herein, in tests used to measure enzymatic isoeugenol oxidation activity, or more precisely, IEM activity.

[0120] A “functional equivalent” may, for example, be derived from the functional expression of another, preferably enzymatically active, polypeptide, and more particularly from the aforementioned helper polypeptide that assists in the correct folding of the expressed polypeptide, such as a polypeptide having isoeugenol oxidation activity or, more precisely, IEM activity. Such a modified helper polypeptide may be considered functional insofar as it improves the correct expression or folding of the 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] The “functional equivalents” according to the present invention also include specific variants having an amino acid at at least one sequence position of the amino acid sequences listed herein that is different from the specifically listed amino acids but exhibits one of the aforementioned biological activities, such as enzymatic activity. Therefore, “functional equivalents” include variants obtained by the addition, substitution, especially conservative substitution, deletion and / or inversion of one or more amino acids, for example, 1 to 20, particularly 1 to 15 or 5 to 10, and the listed changes can occur at any sequence position, insofar as they result in a variant having the characteristic profile according to the present invention. Functional equivalents are particularly important when the activity patterns qualitatively match between the variant and the unchanged polypeptide, i.e., when the interaction with the same agonist or antagonist or substrate is different (i.e., EC 50 or IC 50 It is also provided (either expressed as a value or by other parameters appropriate to the current technical field). Examples of suitable (conservative) amino acid substitutions are shown in the following table: [Table 1]

[0122] In the sense described above, "functional equivalent" also refers to the "precursors" of polypeptides, as well as the "functional derivatives" and "salts" of polypeptides described herein.

[0123] In this case, the "precursor" is a natural or synthetic precursor of a polypeptide that may or may not have the desired biological activity.

[0124] The term "salt" in this invention refers to salts of the carboxyl group of the amino group of a protein molecule and salts of acid addition. Salts of the carboxyl group may be produced by known methods and include inorganic salts, such as sodium, calcium, ammonium, iron, and zinc salts, and salts with organic bases, such as amines, such as triethanolamine, arginine, lysine, and piperidine. Salts of acid addition, 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 this invention.

[0125] The "functional derivatives" of polypeptides according to the present invention may be produced using known techniques on or at the N-terminus or C-terminus of functional amino acids. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtained by reaction with ammonia or 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" naturally include polypeptides obtained from other organisms, as well as naturally occurring variants. For example, the range of homologous sequence regions may be established by sequence comparison, and equivalent polypeptides may be determined based on the specific parameters of the present invention.

[0127] "Functional equivalents" include "fragments" of the polypeptide according to the present invention, particularly individual domains or sequence motifs, or N-terminal and C-terminal cleavage forms, which may or may not exhibit the desired biological function. Preferably, such "fragments" retain at least the desired biological function qualitatively.

[0128] A “functional equivalent” is a fusion protein having one of the polypeptide sequences or functional equivalents derived therefrom listed herein and at least one further functionally different heterologous sequence at the N-terminal or C-terminal association (i.e., without substantial mutual dysfunction of the fusion protein portion). Examples of these heterologous sequences, not limited to these, include signal peptides, histidine anchors, or enzymes.

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

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

[0131] In the case of possible protein glycosylation, the “functional equivalents” according to the present invention include the polypeptides described herein in the form of deglycosylation or glycosylation, and in modified forms obtained by changing the glycosylation pattern.

[0132] Functional equivalents or homologs of polypeptides according to the present invention can be generated by mutagenesis, for example, by point mutation, elongation or shortening of proteins, or as described in more detail below.

[0133] Functional equivalents or homologs of polypeptides according to the present invention can be identified by screening a combinatorial database of mutants, for example, truncated mutants. 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 many methods that can be used to generate a database of potential homologs from denatured oligonucleotide sequences. Degenerate gene sequences may be chemically synthesized using an automated DNA synthesizer, and the synthetic genes may be ligated into a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture and encode a desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art.

[0134] Several techniques are known for screening gene products in combinatorial databases generated by point mutations or shortenings, and for screening cDNA libraries of gene products having selected characteristics. These techniques can be adapted for rapid screening of gene banks generated by combinatorial mutagenesis of homologs according to the present invention. The most frequently used techniques for screening large gene banks based on high-throughput analysis include cloning the gene bank with replicable expression vectors, transforming suitable cells in the resulting vector database, and expressing the combined gene under conditions where detection of desired activity facilitates the isolation of the vector encoding the detected gene. Recurrent ensemble mutation (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] The 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. Applicable coding nucleic acid sequences according to the present invention In the contents of this document, 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 a single-stranded or double-stranded deoxyribonucleotide or a ribonucleotide of any length, and includes coding and non-coding sequences of genes, complementary sequences of exons, introns, sense and antisense, 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 will notice that the nucleic acid sequences of RNA are identical to the sequences of DNA in which various thymine (T) atoms are replaced by uracil (U). The term “nucleotide sequence” should also be understood to include polynucleotide molecules or oligonucleotide molecules in the form of separate fragments or as components of a larger nucleic acid.

[0138] "Isolated nucleic acids" or "isolated nucleic acid sequences" refer to nucleic acids or nucleic acid sequences that are in an environment different from naturally occurring nucleic acids or nucleic acid sequences and that may include those substantially free from endogenous contamination. The term "naturally occurring" as used herein, in reference to nucleic acids, means nucleic acids that are naturally found in the cells of living organisms and that have not been intentionally modified by humans in a laboratory.

[0139] A “fragment” of a polynucleotide or nucleic acid sequence refers, in particular, to a sequence of nucleotides having a length of at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp, and / or at least 60 bp of the polynucleotide of the embodiments herein. In particular, a fragment of a polynucleotide includes at least 25, more specifically at least 50, more specifically at least 75, more specifically at least 100, more specifically at least 150, more specifically at least 200, more specifically at least 300, more specifically at least 400, more specifically at least 500, more specifically at least 600, more specifically at least 700, more specifically at least 800, more specifically at least 900, and more specifically at least 1000 of the polynucleotide of the embodiments herein. Without limitation, fragments of polynucleotides herein may be used as PCR primers and / or probes, or for silencing or RNAi of antisense genes.

[0140] As used herein, the terms “hybridization” or “hybridize” under specific conditions are intended to mean hybridization and washing conditions in which nucleotide sequences that are significantly identical or homologous to each other remain bound together. These conditions may be such that sequences that are at least about 70%, for example, at least about 80%, and at least about 85%, 90%, or 95% identical remain bound together. Definitions of low-stringency, moderate-stringency, and high-stringency hybridization conditions are provided below herein. Appropriate hybridization conditions can also be selected by those skilled in the art with minimal experiments, as described in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Furthermore, 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).

[0141] A "recombinant nucleic acid sequence" is a nucleic acid sequence resulting from the use of experimental methods (e.g., molecular cloning) to produce or modify nucleic acid sequences that do not occur naturally and are otherwise not found in biological organisms, together with genetic material from one or more sources.

[0142] "Recombinant DNA technology" refers to molecular biological methods for generating recombinant nucleic acid sequences, such as those 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 containing a transcribed region in an RNA molecule, such as mRNA in a cell, which is operably ligated to a suitable regulatory region, such as a promoter. A gene, therefore, includes several operably ligated sequences, such as a 5' leader sequence containing a promoter, such as a sequence included at the start of translation, a coding region of cDNA or genomic DNA, introns, exons, and / or a 3' untranslated sequence containing, for example, a transcription termination site.

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

[0145] A “chimeric gene” typically refers to any gene not found naturally in a species, particularly a gene containing one or more nucleic acid sequences that are not naturally related to each other. For example, a promoter is not naturally related to some or all of a transcription region or to other regulatory regions. The term “chimeric gene” is understood to include expression constructs in which a promoter or transcriptional regulatory sequence is manipulably ligated to one or more coding sequences or to one antisense sequence (i.e., the reverse complementary strand of the sense strand), or to reverse repeat sequences (sense and antisense, thereby causing RNA transcription to form double-stranded RNA for transcription). The term “chimeric gene” also includes genes obtained by combining parts of one or more coding sequences to produce a new gene.

[0146] The "3'UTR" or "3' untranslated sequence" (also called the "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of the coding sequence of a gene, including, for example, the transcription termination site and (almost, but not all, eukaryotic mRNA) polyadenylation signals, such as AAUAAA or their variants. After transcription termination, mRNA transcription may be cleaved downstream of the polyadenylation signal and may be joined at the site of translation, for example, during the transport of mRNA into the cytoplasm, where the poly(A) terminus is included.

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

[0148] The term "selectable marker" refers to any gene that may be used to select one or more cells containing the selectable marker for expression. Examples of selectable markers are listed below. Those skilled in the art know that selectable markers for various antibiotics, fungicides, nutrient requirements, or herbicides are applicable to various target species.

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

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

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

[0152] The present invention also relates to nucleic acids having a certain degree of "identity" with the sequences specifically disclosed herein. The "identity" between the two nucleic acids means the identity of the nucleotides throughout the entire length of the nucleic acid in each case.

[0153] The "identity" between two nucleotide sequences (and similarly for peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) that are identical in the two sequences when an alignment occurs between them. Identical residues are defined as residues that are identical in the two sequences at a given position in the alignment. The percentage of sequence identity is calculated from the best 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, as used herein. The best alignment is the alignment that is likely to have the highest percentage of identity. Gaps may be introduced in one or both sequences at one or more positions in the alignment to obtain the best alignment. These gaps are taken into account as non-identical residues for the calculation of the percentage of sequence identity. Alignments for the purpose of determining the percentage of sequence identity of amino acids or nucleic acids may be obtained in various ways using computer programs and publicly available computer programs, for example, available on the web.

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

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

[0156] Alternatively, identity may be measured according to the webpage 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 (single-stranded and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) listed herein can be produced by known methods, for example, by degeneracy of individual overlapping complementary nucleic acid components of a double helix through chemical synthesis from nucleotide components. The chemical synthesis of oligonucleotides can be carried out by known methods, for example, the amidite phosphite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and gap filling by Klenow fragment and ligation reactions of DNA polymerase, as well as general cloning techniques, are described in Sambrook et al. (1989) (see below).

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

[0159] The present invention further relates to nucleic acid molecules that are complementary to a nucleotide sequence or segment thereof as specifically described.

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

[0161] "Isolated" nucleic acid molecules are separated from other nucleic acid molecules present in the natural source of nucleic acids, and furthermore, if produced by recombinant technology, they do not require substantially other cell material or culture medium, or if chemically synthesized, they do not require chemical precursors or other chemical substances.

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

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

[0164] Nucleic acid sequences or derivatives thereof, homologs or parts thereof, according to the present invention can be isolated, for example, by conventional hybridization techniques or PCR techniques from other bacteria, for example, by decomposing a genome or cDNA library. These DNA sequences hybridize with the sequences according to the present invention under standard conditions.

[0165] "Hybridizing" refers to the ability of polynucleotides or oligonucleotides to bind to nearly complementary sequences under standard conditions; however, non-specific binding does not occur between non-complementary partners under these conditions. Therefore, sequences may be 90-100% complementary. The property of complementary sequences to bind specifically to each other is utilized, for example, in Northern blotting or Southern blotting, or in primer binding in PCR or RT-PCR.

[0166] Short oligonucleotides of conserved regions are advantageously used for hybridization. However, it is also possible to use longer fragments of nucleic acids or the entire sequence for hybridization according to the present invention. These "standard conditions" vary depending on the nucleic acid used (oligonucleotide, longer fragment, or entire sequence) or 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 specific nucleic acid, standard conditions mean a temperature of 42-58°C in a buffered aqueous solution at a concentration of 0.1-5×SSC (1×SSC = 0.15M NaCl, 15mM sodium citrate, pH 7.2) or further in the presence of 50% formamide, for example, 42°C in 5×SSC and 50% formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1×SSC and a temperature of about 20°C-45°C, preferably about 30°C-45°C. Advantageously, the hybridization conditions for DNA:RNA hybrids are 0.1×SSC and a temperature of about 30°C-55°C, preferably about 45°C-55°C. These aforementioned hybridization temperatures are examples of melting temperature values ​​calculated for nucleic acids having a length of about 100 nucleotides and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, e.g., Sambrook et al. (1989), and can be calculated using formulas known to those skilled in the art, depending on factors such as nucleic acid length, hybrid type, or 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 can be performed under particularly stringent conditions. Such hybridization conditions are described, for example, 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 terms hybridization or hybridize under specific conditions are intended to refer to hybridization and washing conditions in which nucleotide sequences that are significantly identical or homologous to each other remain bound to one another. Such conditions may be those in which sequences that are at least about 70%, for example, at least about 80%, and at least about 85%, 90%, or 95% identical remain bound to one another. Definitions of low stringency, moderate, and high stringency hybridization conditions are provided herein.

[0170] Appropriate hybridization conditions can be selected by those skilled in the art with minimal experiments, as described in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Furthermore, 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 for low stringency are as follows: A filter containing DNA is pretreated at 40°C for 6 hours in a solution containing 35% formamide, 5×SSC, 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 carried out in the same solution with the following modifications: using 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and a 5-20 × 10⁶ 32P labeled probe. The filter is incubated in the hybridization mixture at 40°C for 18-20 hours and then washed at 55°C for 1.5 hours. Wash in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Replace the washing solution with a fresh solution and incubate at 60°C for a further 1.5 hours. Absorb and dry the filter and expose it for autoradiography.

[0172] As used herein, the defined conditions for moderate stringency are as follows: A filter containing DNA is pretreated at 50°C for 7 hours in a solution containing 35% formamide, 5×SSC, 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 carried out in the same solution with the following modifications: using 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and a 5-20 × 10⁶ 32P labeled probe. The filter is incubated in the hybridization mixture at 50°C for 30 hours and then washed at 55°C for 1.5 hours. Wash in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Replace the washing solution with a fresh solution and incubate at 60°C for a further 1.5 hours. Absorb and dry the filter and expose it for autoradiography.

[0173] As used herein, the defined conditions for high stringency are as follows: Pre-hybridization of the DNA-containing filter is performed at 65°C for 8 hours to overnight in a buffer consisting of 6×SSC, 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. The filter is hybridized at 65°C for 48 hours in a pre-hybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5 to 20 × 10⁶ cpm of 32P-labeled probe. The filter is washed at 37°C for 1 hour in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Subsequently, it is washed at 50°C for 45 minutes with 0.1×SSC.

[0174] Other conditions of low, moderate, and high stringency known in the art (e.g., those used in interspecific hybridization) may be used when the aforementioned conditions are unsuitable (e.g., when used in interspecific hybridization).

[0175] A detection kit for nucleic acid sequences encoding a polypeptide according to the present invention may include primers and / or probes specific to the nucleic acid sequence encoding the polypeptide, and associated protocols for using the primers and / or probes to detect the nucleic acid sequence encoding the 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 the sequence encoding the polypeptide.

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

[0177] The present invention also relates to a nucleic acid sequence specifically disclosed or a derivative of an inducible nucleic acid sequence.

[0178] Accordingly, further nucleic acid sequences according to the present invention may be derived from the sequences specifically disclosed herein and may differ from them by the addition, substitution, insertion or deletion of one or more (e.g., 1 to 10) nucleotides, for example, 1 to 20, particularly 1 to 15, or 5 to 10, and may further encode polypeptides having a profile of desired properties.

[0179] The present invention also includes nucleic acid sequences that contain or are modified with so-called silent mutations compared to the sequences specifically cited, depending on the use of a particular protozoan or host organism codon.

[0180] According to specific embodiments of the present invention, variant nucleic acids may be prepared to adapt their nucleotide sequences to specific expression systems. 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 genetic coding, more than one codon may encode the same amino acid sequence, and multiple nucleic acid sequences may encode the same protein or polypeptide; all of these DNA sequences are included in the embodiments of the present invention. Where appropriate, nucleic acid sequences encoding 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 specifically for the host for improved expression.

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

[0182] The allele variant has at least 60% homology at the induced amino acid level, preferably at least 80% homology across the entire sequence range, and very preferably at least 90% homology (for homology at the amino acid level, see the details provided above for polypeptides). Advantageously, the homology may be higher than that of a subregion of the sequence.

[0183] The present invention also relates to sequences obtained by conservative nucleotide substitutions (i.e., as a result thereof, the amino acid in question is replaced with an amino acid of the same charge, size, polarity, and / or solubility).

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

[0185] Furthermore, derivatives should also be understood as homologs of nucleic acid sequences according to the present invention, for example, homologs of animals, plants, fungi, or bacteria, truncated sequences, coding and non-coding DNA sequences, single-stranded DNA or single-stranded RNA. For example, homologs have at least 40%, preferably at least 60%, particularly preferably at least 70%, and very particularly preferably at least 80% homology at the DNA level across a given DNA region in the sequence specifically disclosed herein.

[0186] Furthermore, derivatives should be understood, for example, as fusion products with promoters. Promoters attached to the given nucleotide sequences may be modified by at least one nucleotide exchange, at least one insertion, inversion, and / or deletion, without impairing the functionality or efficiency of the promoter. Moreover, the efficiency of promoters may be increased by altering their sequences, or they may be completely replaced with more efficient promoters, even in organisms of different genera.

[0187] d. Generation of functional polypeptide variants Furthermore, those skilled in the art are familiar with methods for generating functional variants encoded by nucleic acid molecules comprising nucleotide sequences 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 with any of the amino acids related to the SEQ ID NOs disclosed herein, and / or nucleotide sequences having at least 70% sequence identity with any of the amino acids related to the SEQ ID NOs disclosed herein, or their reverse complements.

[0188] Depending on the technique used, those skilled in the art can introduce completely random or more targeted mutations into genes or non-coding nucleic acid regions (e.g., those important for regulating expression) and subsequently generate a gene library. The molecular biological methods necessary for this purpose are known to those skilled in the art 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 therefore methods for modifying polypeptides encoded by genes, have long been known to those skilled in the art, for example, as follows: - Site-directed mutagenesis in which individual nucleotides or several nucleotides of a gene are substituted in a specified manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey). - Saturated mutagenesis that allows for the exchange or non-exchange of any amino acid codon at any location in a 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) - Error-prone DNA polymerase, which mutates nucleotide sequences via error-prone polymerase chain reaction (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739); - SeSaM method (sequence saturation method) that inhibits desirable conversion by polymerase, Schenk et al. (Biospektrum, Vol. 3, 2006, 277-279) - For example, a defect in the DNA repair mechanism increases the rate of nucleotide sequence mutations, or the passage of genes in mutagenic strains (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 (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747) involves forming and digesting a pool of closely related genes, and then using these fragments as templates for polymerase chain reactions that ultimately generate full-length mosaic genes through repeat strand separation and recombination.

[0190] Using so-called directed evolution (in particular, as described 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), those skilled in the art can produce functional variants on a large scale in a directed manner. For this purpose, in the first step, a gene library of each polypeptide is first generated, for example, using the aforementioned method. The gene library is then expressed in a suitable manner, for example, by bacteria or by a phage display system.

[0191] The relevant genes of a host organism expressing a functional mutant with characteristics that adequately correspond to the desired traits may follow other mutation cycles. The mutation and selection or screening steps may be repeated until the existing functional mutants possess the desired traits to a sufficient degree. Using this iterative procedure, a limited number of mutations, e.g., 1, 2, 3, 4, or 5 mutations, may be performed stepwise and evaluated and selected for their effect on the activity in question. The selected mutants are then sent to further mutation steps in the same manner. This significantly reduces the number of individual mutants that need to be investigated.

[0192] The results obtained according to the present invention also provide important information regarding the structure and sequence of the relevant polypeptides, which is necessary for generating 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 regarding the position of amino acid sequences can also be estimated, and mutations that likely have little effect on activity may occur in those regions, potentially designing them as "silent mutations."

[0194] e. Constructs for expressing the polypeptide of the present invention In the contents of this document, the following definitions apply.

[0195] "Gene expression" encompasses "heterogenetic expression" and "overexpression," and includes gene transcription and translation of mRNA into proteins. Overexpression refers to the production of gene products, as measured by levels of mRNA, polypeptide, and / or enzyme activity in transgenic cells or transgenic organisms, that exceed the levels of products in non-transformed cells or non-transformed organisms of the same genetic background.

[0196] As used herein, "expression vector" means a nucleic acid molecule designed using molecular biological methods and recombinant DNA techniques for introducing foreign or exogenous DNA into host cells. An expression vector typically contains the sequence required for proper transcription of a nucleotide sequence. The coding region usually codes for the protein of interest, but may also code for RNA, such as antisense RNA or siRNA.

[0197] The “expression vector” as used herein includes, but is not limited to, any linear or circular recombinant vector, including 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 includes the nucleic acid of the embodiment herein, which includes at least one “control sequence” that controls transcription, translation, initiation, and termination, for example, a transcription promoter, operator, or enhancer, or an mRNA ribosome binding site, and optionally at least one selective marker. The nucleotide sequence is “operably linked” if the control sequence is functionally relevant to the nucleic acid of the embodiment herein.

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

[0199] As used herein, the terms “amplifying” and “amplifying” 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 present invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo-dT primers) for amplifying naturally expressed nucleic acids (e.g., genomic DNA or mRNA) or recombinant nucleic acids (e.g., cDNA) of the present invention in vivo, ex vivo, or in vitro (e.g., by polymerase chain reaction (PCR)).

[0200] A "control sequence" refers to a nucleic acid sequence that can determine the expression level of the nucleic acid sequence in the embodiments described herein and control the transcription rate of the nucleic acid sequence operably linked to the control sequence. The control sequence includes promoters, enhancers, transcription factors, promoter elements, and the like.

[0201] The terms “promoter,” “promoter-active nucleic acid,” or “promoter sequence” are understood to mean a nucleic acid that, when functionally linked to a nucleic acid to be transcribed according to the present invention, regulates the transcription of said nucleic acid. “Promoter” specifically refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and, but not limited to, other factors required for proper transcription, including transcription factor binding sites, repressors, and activating protein binding sites. The meaning of the term promoter also includes the term “promoter-controlled sequence.” Promoter-controlled sequences may include upstream and downstream elements that can affect transcription, RNA processing, or the stability of the associated coding nucleic acid sequence. Promoters include naturally occurring and synthetic sequences. The coding nucleic acid sequence is typically located downstream of the promoter in relation to the direction of transcription, starting at the transcription initiation site.

[0202] In this description, “functional” or “operable” linkage is understood to mean, for example, a single contiguous arrangement of nucleic acids having a regulatory sequence. For example, promoter activity, the nucleic acid sequence to be transcribed, and optionally further regulatory elements, such as sequences that ensure nucleic acid transcription, such as terminators, are linked in such a way that each regulatory element can perform its function during transcription of the nucleic acid sequence. This does not necessarily require direct linkage in a chemical sense. Gene regulatory sequences, such as enhancer sequences, can exert their function on the target sequence from a more distant location or from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is located after the promoter sequence (i.e., at the 3' end) and the two sequences are covalently linked together. The distance between the promoter sequence and the nucleic acid sequence to be expressed by recombination may be less than 200 base pairs, less than 100 base pairs, or less than 50 base pairs.

[0203] In addition to promoters and terminators, other regulatory elements may include: target sequences, enhancers, polyadenylation signals, selection markers, amplification signals, and origins of replication. 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 enables the sequential transcription of manipulably linked nucleic acid sequences.

[0205] The term “operable linking” refers to the linking of functionally related polynucleotide elements. A nucleic acid is “operable linking” when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operable linking to a coding sequence if it affects the transcription of that coding sequence. Operationally linking means that the linked DNA sequences are typically adjacent. 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 synthesized whole or partially. 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 being bound to the polypeptide of the embodiments herein. The associated nucleic acid may encode a protein that is desired to be expressed or repressed throughout the organism, or in a specific tissue, cell, or cellular compartment, at all times or, instead, at specific times. Such nucleotide sequences encode proteins that confer desired phenotypic features to host cells or organisms modified or transformed with them. More specifically, the associated nucleotide sequences lead to the production of vanillin in cells or organisms. In particular, the nucleotide sequence codes for IEM.

[0206] In this specification, the nucleotide sequences described herein may be part of an expression cassette. The terms “expression cassette” and “expression construct” are used synonymously. An expression construct (preferably recombinant) comprises a nucleotide sequence that encodes a polypeptide according to the present invention and 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," particularly a recombinant expression vector.

[0208] In accordance with the present invention, "expression unit" is understood to mean a nucleic acid having expression activity that modulates expression after functionally binding to a nucleic acid or gene to be expressed, i.e., transcription and translation of the nucleic acid or gene, and including a promoter as defined herein. Therefore, in this context, it is also referred to as a "regulatory nucleic acid sequence." In addition to the promoter, other regulatory elements, such as enhancers, may also be present.

[0209] The terms "expression cassette" or "expression construct" are understood to mean an expression unit functionally linked to a nucleic acid or gene to be expressed in accordance with the present invention. Therefore, in contrast to an expression unit, an expression cassette includes not only nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are to be expressed as proteins as a result of transcription and translation.

[0210] In the context of this invention, the terms “expression” or “overexpression” refer to the generation or increase of the intracellular activity of one or more polypeptides in a microorganism encoded by the corresponding DNA. For this purpose, for example, it is possible to introduce a gene into an organism, replace an existing gene with another gene, increase the copy number of a gene, use a strong promoter, or use a gene encoding a corresponding polypeptide with high activity, and optionally, these measurements can be combined.

[0211] Preferably, such a construct according to the present invention includes a 5'-upstream promoter and a 3'-downstream terminator sequence of each code sequence, and optionally other conventional regulatory elements, each functionally linked to the code sequence.

[0212] The nucleic acid constructs according to the present invention particularly include sequences encoding polypeptides derived from amino acids or their reverse complements related to the sequence numbers described herein, for example, or derivatives and homologs thereof, and nucleic acid sequences operationally or functionally linked to one or more regulatory signals to advantageously control, for example, increase gene expression.

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

[0214] A preferred nucleic acid construct also advantageously includes one or more of the aforementioned “enhancer” sequences functionally linked to the promoter, which enable 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 present invention may be present in the construct. In the construct, other markers, such as genes complementing nutritional requirements or antibiotic resistance, may optionally be present for construct selection.

[0215] Examples of suitable regulatory sequences include promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, and 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 present in the promoters and are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, 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, and 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 a host organism, nucleic acid constructs are favorably inserted into vectors, such as plasmids or phages, thereby enabling optimal gene expression in the host. The term "vector" can also be understood to mean, in addition to plasmids and phages, all other vectors known to those skilled in the art, namely, viruses, such as SV40, CMV, baculoviruses and adenoviruses, transposons, IS elements, phasmids, cosmids, and linear or circular DNA or artificial chromosomes. These vectors can be automatically replicated within a host organism or on chromosomes. These vectors represent a further development of the present invention. Binary or CPO integrated vectors can also be applied.

[0217] Suitable plasmids include, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, and pIN-III. 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 of the genus Streptomyces, pUB110, pC194 or pBD214 of the genus Bacillus, pSA77 or pAJ667 of the genus Corynebacterium, pALS1, pIL2 or pBB116 of fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 of yeast, or pLGV23 or pGHlac of plants + These plasmids are pBIN19, pAK2004, or pDH51. The aforementioned 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 on cloning vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0218] In further development of vectors, nucleic acid constructs or vectors containing nucleic acids according to the present invention may be advantageously introduced into microorganisms in the form of linear DNA and incorporated into the genome of the host organism via heterologous or homologous recombination. This linear DNA may consist of a linearized vector, such as a plasmid, or solely of a nucleic acid construct or nucleic acid according to the present invention.

[0219] For optimal expression of heterologous genes in organisms, it is advantageous to modify nucleic acid sequences to match specific "codon usages" used in the organism. These "codon usages" can be readily determined by computer evaluation of other known genes in the organism in question.

[0220] The expression cassette according to the present invention is produced 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), 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 enables 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 P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

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

[0223] Altered expression of the polynucleotides provided herein can also result in ectopic expression, which is a distinct expression pattern in the altered organism compared to a control or wild-type organism. Altered expression arises from the interaction of the polypeptide of embodiments herein with an exogenous or endogenous modulator, or as a result of chemical modification of the polypeptide. This term refers to an altered expression pattern of the polynucleotide of embodiments herein, wherein activity is altered below the detection level or completely suppressed.

[0224] In one embodiment, an isolated recombinant or synthetic polynucleotide encoding the polypeptide or variant polypeptide provided herein is also provided herein.

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

[0226] f. Microorganisms to which the present invention applies Depending on the context described herein, the term "microorganism" may mean wild-type microorganisms, genetically modified recombinant microorganisms, or both.

[0227] In one embodiment, a vector according to the present invention can be used to generate a recombinant microorganism that can be transformed with, for example, at least one vector according to the present invention and used to produce a polypeptide according to the present invention. Advantageously, the recombinant construct according to the present invention described above is introduced into a suitable host system and expressed. Preferably, conventional cloning and transfection methods known to those skilled in the art, such as coprecipitation, protoplast fusion, electroporation, retroviral transfection, etc., are used to express a given nucleic acid in their respective expression systems. 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 prokaryotes or eukaryotes may be considered recombinant host organisms for nucleic acids or nucleic acid constructs according to the present invention. Advantageously, microorganisms, such as bacteria, fungi, or yeasts, may be used as host organisms. Advantageously, Gram-positive or Gram-negative bacteria, preferably from the Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, or Nocardiaceae families, and particularly preferably from the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium, or Rhodococcus. The genus Escherichia coli (Escherichia coli) is especially preferred. Furthermore, other advantageous bacteria are found in the groups of alphaproteobacteria, betaproteobacteria, or gammaproteobacteria.

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

[0230] g. Recombination generation of polypeptides according to the present invention The present invention further relates to a method for recombinantly producing polypeptides or functionally biologically active fragments thereof according to the present invention, wherein a polypeptide-producing microorganism is cultured, polypeptide expression is optionally induced by applying at least one inducer-induced gene expression, and the expressed polypeptide is isolated from the culture. Polypeptides may be produced on an industrial scale by this method if desired.

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

[0232] The culture medium used must appropriately meet the requirements of each strain. Descriptions of various microbial culture media are provided in the American Society for Bacteriology's manual, "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981).

[0233] These culture media usable in accordance with the present invention typically 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. Excellent carbon sources include, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars may be added to the culture medium via complex compounds, such as molasses, or other by-products of sugar refining. Adding mixtures of different carbon sources may also be advantageous. Other possible carbon sources include 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] Nitrogen sources are typically organic or inorganic nitrogen compounds or materials 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. Nitrogen sources may be used alone or in mixtures.

[0236] Inorganic salt compounds that may be present in the culture 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, and sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, may be used as sulfur sources.

[0238] Phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salt may be used as a phosphorus source.

[0239] Chelating agents may be added to the culture medium to maintain 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 in accordance with the present invention typically includes other growth factors, such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine, as well as vitamins or growth promoters. The growth factors and salts are often derived from components of complex media, such as yeast extract, molasses, and corn steep liquor. 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 described in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media may be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0241] All components of the culture medium are sterilized by either heating (1.5 bar and 121°C for 20 minutes) or sterile filtration. These components may be sterilized together or individually as needed. All components of the culture medium may be present at the start of the culture or added continuously or in batches.

[0242] The culture temperature is usually 15°C to 45°C, preferably 25°C to 40°C, and can be changed or maintained 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 an acidic compound, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or a basic compound, such as phosphoric acid or sulfuric acid. An antifoaming agent, such as fatty acid polyglycol ester, may be used to control foaming. In order to maintain plasmid stability, a suitable selective substance, such as an antibiotic, 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. Cultivation is continued until the maximum desired product is formed. This objective is normally achieved within 10 hours to 160 hours.

[0243] Next, the fermentation broth is further processed. If desired, 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, cells may be disrupted by high-frequency ultrasound, under high pressure, such as in a French press, by osmotic lysis, by the action of detergents, lytic enzymes or organic solvents, by a homogenizer, or by a combination of several of the above methods.

[0245] Polypeptides may be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography, and hydrophobic chromatography, as well as by 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 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 cDNA by a defined nucleotide sequence and thus encode modified polypeptides or fusion proteins that facilitate easier purification, for example. Suitable modifications of this type include so-called "tags" that function as anchors, such as modifications known as hexahistidine anchors, or epitopes that can be recognized as antigens of antibodies (e.g., as described in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors may be provided for attaching proteins to solid carriers, such as polymer matrices, which may be used, for example, as packing material in a chromatography column or on a microtiter plate or some other carrier.

[0247] Simultaneously, these anchors may also be used for protein recognition. For protein recognition, conventional markers that form detectable reaction products after reaction with a substrate, such as fluorescent dyes, enzyme markers, or radioactive markers, may also be used, either alone or in combination with anchors for protein induction.

[0248] For the expression of the mutant according to the present invention, refer to the description of the expression of the wild-type enzyme EbN1, and the expression systems available therein in the explicitly referenced International Publication Nos. 2005 / 108590 (WO2005 / 108590) and International Publication Nos. 2006 / 094945 (WO2006 / 094945).

[0249] h. Immobilization of polypeptides The enzymes or polypeptides according to the present invention may be used free or immobilized in the methods described herein. An immobilized enzyme is an enzyme immobilized on an inert carrier. Suitable carrier materials and enzymes immobilized thereon are known from European Patent Application Publication No. 1149849 (EP-A-1149849), European Patent Application Publication No. 1069183 (EP-A-1069183), German Patent No. 100193773 (DE-OS 100193773), and the references cited herein. In this regard, the entire disclosure is incorporated by reference. Suitable carrier materials include, for example, clay, 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 produce supported enzymes, the carrier material is usually used in the form of finely ground particles, and a porous form is preferred. The particle size of the carrier material is usually 5 mm or less, particularly 2 mm or less (particle size distribution curve). Similarly, when using dehydrogenase as a whole-cell catalyst, free or immobilized forms can be selected. Examples of carrier materials include calcium alginate and carrageenan. The enzyme and cells may also be directly crosslinked with glutaraldehyde (crosslinking to CLEA). Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin, "Immobilization of Enzymes," and K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information regarding in vivo conversion and bioreactors for carrying out the methods according to the present 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 At least one polypeptide / enzyme present in the method of the present invention or in each step of the multi-step method as defined herein may be present naturally or recombinantly in living cells producing one or more enzymes, in harvested cells, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form. At least one enzyme may be present in solution or as an enzyme immobilized on a carrier. One or more enzymes may be present simultaneously in soluble and / or immobilized forms.

[0251] The method according to the present invention may be carried out in a general reactor known to those skilled in the art, and on a range of scales, e.g., from laboratory scale (reaction volume of a few milliliters to tens of liters) to industrial scale (reaction volume of a few liters to several thousand cubic meters). A chemical reactor may be used when polypeptides are used in the form of a somewhat purified cell extract, or in a purified form, encapsulated by non-viable, optionally permeabilized cells. A chemical reactor typically allows control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature, and circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the method would be fermentation. In this case, biocatalyst generation may be carried out in a bioreactor (fermenter), where parameters necessary for growth conditions suitable for living cells (e.g., culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, etc.) may be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, procedures for scaling chemical or biotechnological methods from laboratory to industrial scale, or procedures for optimizing process parameters, and these are extensively described in the literature (for biotechnological methods, see, for example, Crueger 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, lysating enzymes and surfactants present in the medium, or a combination of such methods. Examples of surfactants include digitonin, n-dodecyl maltoside, octyl glycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonate), Nonidet® P40 (ethylphenol poly(ethylene glycol ether), etc.).

[0253] Instead of living cells, biomass from dead cells containing the necessary biocatalysts may also be applied to the in vivo conversion reaction of the present invention.

[0254] When immobilizing at least one enzyme, the enzyme is attached to the aforementioned inert carrier.

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

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

[0257] Aqueous or aqueous organic media may contain a buffer suitable for adjusting the pH to a range of 5 to 11, for example, 6 to 10.

[0258] In aqueous organic media, organic solvents that are miscible, partially miscible, or immiscible with water may be used. Examples of suitable solvents, though not limited to those listed below, are also included. Further examples include monohydric or polyhydric aromatic or aliphatic alcohols, particularly polyhydric aliphatic alcohols, such as glycerol.

[0259] The non-aqueous medium contains substantially no water, i.e., contains about 1% by mass or less than 0.5% by mass of water.

[0260] The biocatalytic method 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 compounds, and ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, or dibutyl ether; or mixtures thereof.

[0261] The reactant / substrate concentrations may be adjusted to suit optimal reaction conditions, which may depend on the specific enzyme being applied. For example, the initial substrate concentration may be, for example, 0.1–0.5 M or 10–100 mM.

[0262] The reaction temperature may be adjusted to suit optimal reaction conditions, which may depend on the specific enzyme being applied. For example, the reaction may be carried out at temperatures in the range of 0 to 70°C, e.g., 20 to 50°C or 25 to 40°C. Examples of reaction temperatures include approximately 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, and 60°C.

[0263] This process may be continued until equilibrium is reached between the substrate and the product, but it may also be stopped earlier. Typical processing times range from 1 minute to 25 hours, particularly from 10 minutes to 6 hours, and for example, from 1 hour to 4 hours, especially from 1.5 hours to 3.5 hours. These parameters are examples of suitable process conditions, but are not limiting.

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

[0265] k. Isolation of the product The methodology of the present invention may further include a step of recovering the final product or intermediate product in a substantially pure form, optionally stereoisomerically or enantiomerically. The term “recover” includes extracting, collecting, isolating, or purifying a compound from a culture or reaction medium. The 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, nonionic adsorption resins, etc.), conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH modification, solvent extraction (e.g., conventional solvents, e.g., alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze-drying, etc.

[0266] The isolated product may be identified and purified by known techniques, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g., IR, UV, NMR), colorimetric chromatography, TLC, NIRS, enzyme assay, or microbial assay (e.g., 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, pp. 89-90, 521-540, 540-547, 559-566, 575-581 and 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, Vol. 17.).

[0267] l. Production by fermentation of vanillin This invention also relates to a method for producing vanillin by fermentation.

[0268] The fermentation used in accordance with the present invention may be carried out, for example, in a stirred fermenter, a bubble tower, and a loop reactor. A comprehensive overview of possible method types, including the type of agitator and geometric design, can be found in "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". Typical variations available in the method of the present invention are those known to those skilled in the art or 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, good yields (YP / S) can be achieved by spraying air, oxygen, carbon dioxide, hydrogen, nitrogen, or a suitable gas mixture.

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

[0270] These culture 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. Excellent carbon sources include, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars may be added to the culture medium via complex compounds, such as molasses, or other by-products from sugar refining. Adding mixtures of various carbon sources may also be advantageous. Other possible carbon sources include 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] Nitrogen sources are typically organic or inorganic nitrogen compounds or materials 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. Nitrogen sources may be used separately or in mixtures.

[0273] Inorganic salt compounds that may be present in the culture 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, and sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, may be used as sulfur sources.

[0275] Phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salt may be used as a phosphorus source.

[0276] Chelating agents may be added to the culture medium to maintain 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 in accordance with the present invention may also contain other growth factors, such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine, as well as vitamins or growth promoters. The growth factors and salts often originate from the medium, for example, from complex components such as yeast extract, molasses, and corn steep liquor. Furthermore, suitable precursors may be added to the medium. The precise composition of compounds in the medium is highly dependent on the specific experiment and must be determined individually for each specific case. Information on medium optimization is described 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 culture medium are sterilized by either heating (1.5 bar and 121°C for 20 minutes) or sterile filtration. The components may be sterilized together or individually as needed. All components of the culture medium may be present at the start of growth or may be added sequentially or in batches as desired.

[0279] The culture temperature is typically 15°C to 45°C, preferably 25°C to 40°C, and may be maintained constant or varied during the experiment. The pH of the culture 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 plasmid stability, suitable selective substances, such as antibiotics, may be added to the culture 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 typically 20°C to 45°C. The culture is continued until the maximum desired product is formed. This is usually achieved within 1 to 160 hours.

[0280] The method of the present invention may further include a step of recovering vanillin.

[0281] The term "recover" includes extracting, collecting, isolating, or purifying a compound from a culture or medium. The recovery of a compound may be carried out according to any conventional isolation or purification method known in the art, but is not limited to these, including treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH modification, solvent extraction (e.g., conventional solvents, e.g., alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze-drying, etc.

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

[0283] In one embodiment, the fermentation broth may be sterilized or pasteurized. In a further embodiment, the fermentation broth may be concentrated. This concentration may be carried out in batches or continuously, as needed. The pressure and temperature ranges should be selected so as to first avoid damage to the product, and then to ensure minimal use of equipment and energy. In particular, a clever selection of pressure and temperature levels for multi-stage evaporation allows for energy savings.

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

[0285] A number of possible modifications, which will become immediately apparent to those skilled in the art after considering the disclosures provided herein, also fall within the scope of the present invention.

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

[0287] Unless otherwise specified, recombinant proteins should be processed using standard methods, e.g., Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2 ndCloning and expression are performed according to the method described in Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

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

[0289] The gas chromatography program is as follows: 100°C (1 min), 20°C (min). -1 The experiment was conducted using an Agilent Technologies '6850 with an FID detector and DB-WAX columns (30 m, 250 microM, 0.25 microM) at 245°C for 10 minutes. Carrier gas: hydrogen, 75 cm⁻¹ / sec. -1 Spirit = 50, injection volume = 1 μL.

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

[0291] Newly grown colonies of BL21(DE3):pIEM1 strain were taken from LB agar plates and transferred to 20 mL of LB medium containing 100 mg / L ampicillin. The cultures were incubated at 37°C for 20 hours with shaking at 225 rpm. 1 mL of the culture medium was taken and, using a 1 L Erlenmeyer flask, 100 mg / L ampicillin was added. -1 It was inoculated into 200 mL of sterile LB medium containing OD. 600 The culture was continued with shaking at 220 rpm and 37°C until the OD reached 0.4. 600The temperature was lowered to 20°C while the culture was allowed to grow until it reached 0.6. The culture was induced with 1 mM IPTG and incubated for a further 18 hours. Typically, after overnight incubation, OD 600 It reached 5.5-7.

[0292] The cells were centrifuged at 9000g and 4°C for 20 minutes. The supernatant was discarded, and the cells were resuspended in 100mM glycine-NaOH buffer (pH 10.5) containing 10v / v% DMSO (the optimal pH was later found to be approximately pH 9 for P. nitroredusens Jin1 isoeugenol monooxygenase). OD 600 Suspensions of different cell densities corresponding to 10-90 were prepared. 2 mL of the defined optical density cell suspension and 75 mg of isoeugenol (Sigma Aldrich #17206) were added to a 20 mL vial. The reaction was carried out at room temperature (24°C) for 20 hours, stirred at 500 rpm, using a vial stopper made of perforated aluminum membrane for oxygenation. The reaction mixture was acidified with 6 drops of 15% HCl, and 1 g L of tridecane was added as an internal standard before gas chromatography analysis. -1 Extraction was performed with 10 mL of MTBE containing [the specified substance]. A negative control was performed using empty cells in the same manner. The time curve for vanillin formation is shown in Figure 3.

[0293] Example 2: Vanillin production by first-generation isoeugenol monooxygenase (IEM1) Vanillin was produced as follows: Cells of *Escherichia coli* expressing *P. nitroredusens* Jin1 (see Example 1) were collected from ampicillin-containing LB agar plates using 15 g L of glucose as the carbon source. -1 The cells were transferred to 50 mL of ampicillin-containing mineral salt medium and grown at 225 rpm and 37°C for 20 hours with shaking. The inorganic salt medium contained 15 g L of glucose. -1 (NH4)2HPO45g L -1 K2HPO4 16g L -1 2g citric acid L -1 MgSO4 1g L-1 CaCl230mg L -1 , trace elements, vitamins, and ampicillin 100 mg L -1 It was composed of the following. Then, 0.4 mL of this culture medium was taken and inoculated into 200 mL of inorganic salt medium of the same composition using a 1 L flask. Several flasks were moved in parallel. The culture was shaken at 37°C and 180 rpm while OD 600 The culture was allowed to grow until it reached 2.5. The temperature was lowered to 20°C. The culture was then treated with OD. 600 In 3, the cells were induced with 1 mM IPTG and then grown at 20°C and 180 rpm for a further 17 hours with shaking. OD at the end of culture. 600 The result was 24. The culture was centrifuged at 4000g at 4°C for 50 minutes. The cells were resuspended in 100mM glycine-NaOH buffer (pH 9) and the final OD was calculated. 600 I changed it to 45.

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

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

[0296] Newly grown cells were isolated from the agar plate, and the optical density (OD) was measured. 600The cultures were grown in 2 mL of LB medium containing ampicillin and chloramphenicol, shaking at 37°C until the culture reached 1. 1 mL of this culture was taken and treated with 100 mg of ampicillin. -1 and Chloramphenicol 30mg L -1 FeCl 35 mg L was inoculated into 200 mL of LB medium containing [the specified ingredient] and placed in a 1000 mL flask. -1 The following was supplemented: Chaperone induction was performed with arabinose 2 mg mL. -1 And 5 ng mL of tetracycline as appropriate. -1 This was performed at the beginning of the culture according to the manufacturer's (Takara Bio Inc.) protocol. 600 The culture was incubated at 37°C with shaking at 180 rpm until the OD reached 0.4. The temperature was then lowered to 20°C. 600 The cells were induced with 1 mM IPTG at a concentration of 0.6. After culturing the cells overnight, they were harvested by centrifugation at 3600 g and resuspended in 0.1 M glycine NaOH (pH 9.5) cold buffer to the desired optical density. Catalytic activity was tested as described in Example 1. The catalytic activity observed with different constructs is shown in Figure 4. Most second-generation catalysts showed higher specific activity than first-generation catalysts (control). Cells co-expressing P. nitroredusense Jin1 isoeugenol monooxygenase and chaperonins GroES and GroEL showed the highest activity. Cells possessing plasmid pKJE7 showed very weak growth and were not tested for activity.

[0297] Example 4: Fed-batch culture of second-generation isoeugenol monooxygenase The bacterial strain BL21(DE3):pIEM1_pGro7 was tested under fed-batch culture conditions. Fed-batch culture was performed in a 3.7 L working volume reactor (Bioengineering, Switzerland) using dissolved oxygen-based glucose supply. 5 g L of yeast extract was used. -1 A mineral salt medium containing carbenicillin and chloramphenicol at pH 7 served as a growth medium using glycerol instead of glucose as the carbon source. The culture temperature was 37°C. OD 600At 48, the temperature was lowered to 25°C and the culture was OD. 600 51g Arabinose 1.5g L -1 It was induced by OD. 600 At 59, the culture was induced with 1 mM IPTG. The supply solution contained 70% sterile aqueous glycerol. The pH was adjusted with NH3 aqueous solution. Fermentation was continued for 43 hours, and the final OD was reached. 600 The number reached 163. Plasmid stability was measured using agar plates with or without antibiotics appropriate for selection. Bacterial cells were collected 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 The reaction, carried out as described in Example 1 with a catalyst addition amount equivalent to 5, was observed by gas chromatography.

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

[0299] Schematic diagrams of different polycistronic constructs are also shown (Figure 2). Culturing, induction, and activity testing were carried out as described in Example 1. OD 600 Comparative activity tests with a catalytic load of 5 showed that E. coli cells expressing one of the polycistronic constructs (third-generation catalysts) exhibited higher specific catalytic activity compared to the first and second-generation catalysts (Figure 5).

[0300] Example 6: Generation of isoeugenol monooxygenase mutants of P. petida IE27 An artificial mutant of isoeugenol monooxygenase from P. putida IE27 (IEM2) was created using plasmid pJ431 (T7, kan RIt was inserted into a low-copy plasmid (supplier Atum, Newark, CA) and expressed in E. coli BL21(DE3)T1. Alternatively, E. coli cells generating one of the P. putida IE27 mutants were co-transformed with plasmid pGro7 to simultaneously generate the chaperonins GroES and GroEL.

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

[0302] Newly grown E. coli colonies containing the mutant encoded by the plasmid were removed from the LB agar plate and kanamycin 30 mg L -1 The cells were grown overnight (16 hours) at 37°C at 220 rpm in 2 mL of LB liquid culture medium containing the required antibiotics. Then, 150 μL was transferred to a flask containing 50 mL of LB medium containing the required antibiotics, and OD was used. 600 The culture was incubated at 37°C for approximately 2 hours with shaking at 200 rpm until the OD reached 0.45. The culture was continued. 600 The temperature was lowered to 20°C while the culture was grown until the ratio reached 0.6. After induction with 0.5 mM IPTG, the culture was grown for a further 16 hours under the same conditions. E. coli cells containing the P. petida IE27 mutant isoeugenol monooxygenase were collected by centrifugation and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5) containing 10 v / v% DMSO, and finally OD 600 The value was set to 45. For E. coli cells containing isoeugenol monooxygenase from P. nitroredusens Jin1 (used as a positive control), the same procedure was applied using 0.1 M glycine buffer (pH 9.0). In a 20 mL vial, 80 mg of isoeugenol was added to the OD. 600 2 mL of the cell suspension (=45) was added. The vial was closed with a perforated aluminum membrane for air circulation. The reaction was allowed to proceed at room temperature (23°C) for 17 hours, stirred with a magnet at 450 rpm. The reaction mixture was acidified with 3 drops of 15% HCl, and 1 g L of tridecane was added as an internal standard for GC analysis. -1Extraction was performed with 10 mL of MTBE containing [the specified substance]. The vanillin concentrations obtained for each mutant and control are shown in Figure 6. Further mutants of isoeugenol monooxygenase from P. petida IE27 (IEM) were obtained by individual saturation mutagenesis experiments at amino acid positions T52, Q74, or D440, performed by commercial manufacturers. These additional mutants were tested in the same manner as the mutants identified above. As shown in Figure 10, the relative activity of each mutant is expressed as a percentage of the averaged vanillin titer obtained for mutant "C154" (i.e., mutant a154c; corresponding to PQD) under standard assay conditions.

[0303] Example 7: Triple mutant of isoeugenol monooxygenase of P. plutida IE27 co-expressed in GroES and GroEL Colonies of E. coli BL21(DE3)T1 and pGro7 harboring plasmid pIEM2_c154_t222_a1318 (see plasmid map and annotations in Figure 8, and Sequence ID No. 16) were isolated from agar plates and added to 2 mL of LB medium containing 30 mg / L kanamycin and 30 mg / L chloramphenicol. The 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. The cells were then OD 600 The cells were grown at 37°C with shaking at 220 rpm until the OD reached 0.45. The culture was induced with 1.5 g / L arabinose, and the OD was increased. 600 The temperature was lowered to 20°C while shaking until the pH reached 0.6. The culture was induced with 0.5 mM IPTG and then grown at 20°C for 16 hours while shaking at 220 rpm. The cells were harvested by centrifugation and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5). OD 2-45 600Different cell dilutions corresponding to the different cell densities were prepared. 80 mg of isoeugenol and 2 mL of cell suspensions of different optical densities were added to an open 20 mL reaction vial. The reaction was allowed to proceed at room temperature for 17 hours, stirred with a magnetic bar at 500 rpm. The reaction mixture 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 GC analysis. The vanillin concentrations observed in reactions with different cell densities are shown in Figure 7.

[0304] Example 8: Triple mutant of P. putida IE27 isoeugenol monooxygenase co-expressed with GroES and GroEL from different expression constructs derived from the polycistronic construct PC1_triple (SEQ ID NO: 25; including a triple mutant of IEM IE27). Colonies of E. coli cells BL21(DE3)T1 containing different plasmid constructs (constructed according to the general scheme in Figure 11, with nucleotide sequences selected from SEQ ID NOs. 17-24 and corresponding constructs lacking the transcriptional terminator of the IEM coding sequence) for expressing a triple mutant of isoeugenol monooxygenase IE27 together with the chaperonins GroES and GroEL, were collected from kanamycin-containing agar plates and inoculated into 2 mL of inorganic salt medium. The inorganic salt medium consisted of 30 g / L glycerol and 5 g / L (NH4)2HPO4. -1 K2HPO4 16g L -1 , citric acid 2g L -1 MgSO4 1g L -1 CaCl230mg L -1 It contains yeast extract 5g / L, trace elements, and kanamycin 50mg / L.

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

[0306] At the end of the culture, samples were taken to determine the optical density at 600 nm, to estimate the dry mass of the cells using standard mass spectrometry, and for activity testing. For activity testing, 15 mL of the culture was centrifuged at 3500 g for 35 minutes 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) for final OD (Oxygen-Dose) analysis. 600 I set it to 20.

[0307] 80 mg of isoeugenol and 1800 μL of ice-cold 0.1 M glycine buffer (pH 9.5) were added to a 20 mL flask. Cell resuspension (OD) was performed under magnetic agitation. 600 =20) Add 200 μL to the final OD 600 The value was set to 2. The reaction mixture was stirred with a magnetic stirrer at 23°C and 450 rpm for 16 hours.

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

[0309] The content of documents being cross-referenced shall be considered incorporated by reference.

[0310] The sequences referred to herein are as follows: [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 polypeptides having isoeugenol oxidation activity, the following: a. A nucleotide sequence (A) encoding a polypeptide having enzymatic isoeugenol oxidation activity, and b. At least one nucleotide sequence (B) encoding at least one helper polypeptide that assists, alone or in conjunction with, the functional expression of the polypeptide encoded by the nucleotide sequence (A). The present invention comprises a single nucleic acid construct possessing the above, providing simultaneous expression of nucleotide sequences (A) and (B), wherein nucleotide sequence (A) has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and comprises an amino acid sequence containing at least one mutation selected from T52P, Q74H, and D440N, thereby encoding a polypeptide having isoeugenol oxidative activity. The at least one nucleotide sequence (B) comprises nucleotide sequences (B1) and (B2), a. (B1) encodes a polypeptide having chaperonin activity, which contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, and b. (B2) The expression system comprising a polypeptide having chaperonin activity, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

10.

2. The expression system according to claim 1, wherein the expression system is a single polycistronic nucleic acid construct.

3. A polypeptide having isoeugenol oxidation activity, wherein the polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and comprises an amino acid sequence containing at least one mutation selected from T52P, Q74H, and D440N.

4. A polypeptide containing the aforementioned mutation a. Single mutants (T52P), (Q74H), and (D440N) b. Double mutants (T52P, Q74H), (T52P, D440N) and (Q74H, D440N), and c. Triple mutant (T52P, Q74H, D440N) A polypeptide according to claim 3, selected from the following.

5. Recombinant nucleic acid comprising a nucleotide sequence encoding the polypeptide described in claim 3 or 4.

6. A recombinant nucleic acid construct comprising the recombinant nucleic acid described in claim 5.

7. below, a. A single nucleic acid construct included in the recombinant expression system described in claim 1 or 2, or b. Recombinant nucleic acid according to claim 5, or c. Recombinant nucleic acid construct according to claim 6 An expression vector containing [the specified ingredient].

8. A non-human host organism or host cell, in its genome a. A single nucleic acid construct included in the recombinant expression system described in claim 1 or 2, or b. Recombinant nucleic acid according to claim 5, or c. Recombinant nucleic acid construct according to claim 6, or d. The expression vector according to claim 7 Non-human host organisms or host cells, including those stably incorporating the substance.

9. A method for producing an isolated catalytically active polypeptide having isoeugenol oxidation activity, comprising the co-expression of the polypeptide having isoeugenol oxidation activity and at least one helper polypeptide in a host cell line, each encoded by an expression system as defined in claim 1 or 2.

10. The method according to claim 9, further comprising the isolation of the polypeptide having isoeugenol oxidation activity.

11. below, a. A step of producing vanillin by contacting isoeugenol with a polypeptide having isoeugenol oxidation activity as defined in claim 3 or 4, or a polypeptide having isoeugenol oxidation activity produced by the method of claim 9, in the presence of oxygen. A method for producing vanillin, including the method described above.

12. b. A step to isolate the vanillin produced in step a. The method according to claim 11, further comprising:

13. The method according to claim 11, further comprising the step of chemically or biochemically isomerizing eugenol to isoeugenol.

14. The method according to claim 13, further comprising the step of isolating isoeugenol from the reaction medium.

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