WO2003008588A2 - Method for producing 2-keto-l-gulonic acid and vitamin c while using l-sorbose dehydrogenase and l-sorbosone dehydrogenase - Google Patents

Abstract

The invention relates to polypeptides having an L-sorbose dehydrogenase (SDH) or L-sorbosone dehydrogenase (SNDH) activity and nucleic acid sequences that code for these polypeptides. The invention also relates to transgenic expression contructs, vectors and transgenic organisms containing these nucleic acid sequences, and to methods for producing 2-keto-L-gulonic acid or ascorbic acid while using the same.

Description

Process for the preparation of 2-eto-L-gulonic acid and vitamin C.

description

The present invention relates to polypeptides with L-sorbose dehydrogenase (SDH) or L-sorbosone dehydrogenase (SNDH) activity and nucleic acid sequences coding for these polypeptides. The invention further relates to transgenic expression constructs, vectors and transgenic organisms which contain these nucleic acid sequences and to processes for the preparation of 2-keto-L-gulonic acid or ascorbic acid using the same.

2-Keto-L-gulonic acid (2-KLG) is an important intermediate for the synthesis of ascorbic acid (vitamin C). Humans and various animal species have lost the ability to synthesize vitamin C, so the supply of vitamin C through food is essential. The worldwide consumption of vitamin C is increasing. However, the vitamin C available from natural, vegetable sources is limited and would not be sufficient to meet the need.

It is known that numerous microorganisms produce 2-keto-L-gulonic acid starting from sorbitol or L-sorbose, for example microorganisms of the genera Acetobacter and Pseudomonas (see, for example, Japanese Patent Publication 40, 154; 1976). Various compounds have become known as intermediates in the synthesis of 2-KLG (Okazaki et al. (1969) Agr Biol Che 33: 207-211).

Ascorbic acid can be chemically synthesized using 2-KLG starting from D-sorbitol using the Reichstein method known to the person skilled in the art (Helv Chim Acta 17: 311-328; 1934). The synthesis of ascorbic acid by this process requires the bioconversion of sorbitol followed by a 7-step chemical process. The process is expensive.

Biological methods for the production of 2-KLG, which can subsequently be converted chemically to ascorbic acid, are described (see, inter alia, Lazarus et al., "Vitamin C: Bioconversion via a Recombinant DNA approach", Genetics and Molecular Biology of Industrial Microorganis s, American Society for Microbiology, Washington DC, editor CL Hershberger). The synthesis usually proceeds from glucose via D-sorbitol, L-sorbose, L-sorbosone to 2-keto-L-gulonic acid (Makover et al. (1975) Biotechnol and Bioeng 17: 1485-1514). The conversion of glucose to sorbitol and the subsequent cyclization of 2-KLG to ascorbic acid is usually carried out by classic chemical reactions in a manner familiar to the person skilled in the art. Various methods, some of them only for individual synthetic steps, using microorganisms have been described 5 (EP 0 366 922, EP 0 278 447, EP 0 518 136, EP 0 790 301). Individual or mixed cultures of microorganisms from the Acetobacter, Gluconobacter, Escherichia, Bacillus, Candida or Pseudomonas strains are used (Considine, "Ascorbic Acid" Van Nostrand's Scientific Encylopedia, Vol. 1, pp. 237-238, 10 1989) ,

Fermentation of other microorganisms such as algae is also described, e.g. in Chlorella pyrenoidosa (US 5,001,059). Production in plants is also described (WO 99/64618).

15

The production of 2-KLG using recombinant microorganisms is described. Saito et al. report the production of an expression system for the production of 2-KLG from D-sorbitol using L-sorbose dehydrogenase (SDH)

20 and L-sorbosone dehydrogenase (SNDH) genes (Saito Y et al. (1997) Appl Environ Microbiol 63 (2): 454-60).

Nucleic acid sequences coding for certain polypeptides with SDH and SNDH activity as well as methods for the production

25 of 2-KLG using the same are described (US

5,861,192, US 5,753,481). EP 0 373 181 discloses DNA sequences and methods for producing 2-KLG from sorbosone using a coenzyme-independent L-sorbosone dehydrogenase. Despite the progress made in the production of 2-KLG and

30 Ascorbic acid still has a need to improve and increase 2- LG productivity to meet the increasing need for ascorbic acid and to make production more efficient. The task was therefore to further improve the 2-KLG productivity and alternative processes for the

35 To provide 2-KGL or vitamin C. This object has been achieved by the present invention.

A first subject of the invention relates to processes for the production of vitamin C or 2-keto-L-gulonic acid, thereby

40 characterized in that an organism or an enzyme preparation obtained from it is used, the organism containing at least one transgenic nucleic acid sequence coding for a polypeptide according to SEQ ID NO: 2 or 4 or a functional equivalent thereof.

45 In a preferred embodiment, the process according to the invention comprises at least one of the following reaction steps:

a) conversion of L-sorbose to L-sorbosone using an organism or an enzyme preparation obtained therefrom, the organism containing at least one transgenic nucleic acid sequence coding for a polypeptide with sorbose dehydrogenase activity according to SEQ ID NO: 2 or a functional equivalent thereof, or

b) conversion of L-sorbosone to 2-KLG using a transgenic organism or an enzyme preparation obtained therefrom, the organism containing at least one transgenic nucleic acid sequence coding for a polypeptide with sorbosone dehydrogenase activity according to SEQ ID NO: 4 or a functional equivalent thereof.

Both of the reaction steps a) and b) mentioned are preferably used in the process according to the invention. The process steps can be carried out in parallel or successively.

With respect to a nucleic acid sequence, an expression cassette, a vector or an organism, “transgene” means all such constructions which have been obtained by genetic engineering methods and in which either

a) at least one of the nucleic acid sequences to be expressed,

(for example a nucleic acid sequence coding for a polypeptide according to SEQ ID NO: 2 or 4 or a functional equivalent thereof) or

b) at least one of the genetic control elements which control the expression of said nucleic acid sequence to be expressed, or

c) (a) and (b)

are not in their natural, genetic environment (for example at their natural chromosomal locus) or have been modified by genetic engineering methods, the modification may include, for example, substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. In a particularly preferred embodiment, the organism used (or the organism used for the production of the enzyme preparation used) contains at least one transgenic nucleic acid sequence coding for

i) a polypeptide according to SEQ ID NO: 2 or a functional equivalent thereof and

ii) a polypeptide according to SEQ ID NO: 4 or a functional equivalent thereof.

2-KLG can be converted into vitamin C in a manner familiar to the person skilled in the art. Sorbose is available from glucose.

In the process according to the invention, the organisms used for the production of 2-KLG are grown in a medium which enables these organisms to grow. This medium can be a synthetic or a natural medium. Depending on the organism, media known to the person skilled in the art are used. For the growth of the microorganisms, the media used contain a carbon source, a nitrogen source, inorganic salts and possibly small amounts of vitamins and trace elements.

Advantageous carbon sources are, for example, sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose, starch hydrolysates, complex sugar sources such as molasses, sugar phosphates how

Fructose-1,6-bisphosphate, sugar alcohols such as mannitol, polyols such as glycerol, alcohols such as methanol or ethanol, carboxylic acids such as citric acid, lactic acid or acetic acid, fats such as soybean oil or rapeseed oil, amino acids such as a mixture of amino acids, for example so-called casa acids (Difco ) or individual amino acids such as glycine or aspartic acid or aminosugar, the latter can also be used simultaneously as a nitrogen source.

Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials that contain these compounds. Examples are ammonium salts such as NH ₄ CI or (H ₄ ) ₂ S0 _{4 /} nitrate, urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soybean meal, wheat gluten, yeast extract, meat extract, casein hydrolyzate, yeast or potato protein, which are often also used simultaneously can serve as a nitrogen source. Examples of inorganic salts are the salts of calcium, magnesium, sodium, cobalt, nickel, molybdenum, manganese, potassium, zinc, copper and iron. The chlorine, sulfate and phosphate ions are particularly worth mentioning as the anion of these salts. An important factor 5 for increasing productivity in the process according to the invention is the control of the Fe ^{2 + _} or Fe ³⁺ ion concentration in the production medium.

If necessary, further growth factors 10 are added to the nutrient medium, such as vitamins or growth promoters such as biotin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids such as alanine, cysteine, proline, aspartic acid, glutamine, serine, phenylalanine, ornithine or valine, carboxylic acids such as citric acid, formic acid, pimelic acid 15 or lactic acid, or substances such as dithiothreitol.

The mixing ratio of the nutrients mentioned depends on the type of fermentation and is determined in each individual case. The medium components can all be introduced at the beginning of the fermentation 20, after they have been sterilized separately if necessary or sterilized together, or else they can be added continuously or discontinuously during the fermentation as required.

25 The breeding conditions are determined in such a way that the organisms grow optimally and that the best possible yields are achieved. Preferred cultivation temperatures are 15 ° C to 40 ° C. Temperatures between 25 ° C and 37 ° C are particularly advantageous. Preferably the pH is in one

30 range from 3 to 9 recorded. PH values between 5 and 8 are particularly advantageous. In general, an incubation period of a few hours to a few days, preferably 8 hours to 21 days, particularly preferably 4 hours to 14 days, is sufficient. The maximum accumulates within this time

35 amount of product in the medium.

How media can be advantageously optimized, the expert can, for example, the textbook Applied Microbiol ^Physiology, A Practical Approach (Eds. PM Rhodes, PF Stanbury, IRL Press,

40 1997, pp. 53-73, ISBN 0 19 963577 3). Advantageous media and growing conditions for Bacillus and other organisms are, for example, the document EP-A-0 405 370, specifically Example 9, for Candida, the document WO 88/09822, especially Table 3, and for Ashbya, the letter from Schmidt et al. (Micro-

45 biology, 142, 1996: 419-426). The process according to the invention can be carried out continuously or batchwise in batch or fed-batch fashion.

The method can preferably be implemented using a host organism which is itself already capable of 2-KLG or vitamin C biosynthesis. Depending on how high the initial productivity of the organism used is, the 2-KLG productivity can be increased by the method according to the invention to different extents. As a rule, productivity can advantageously be increased by at least 5%, preferably by at least 10%, particularly preferably by 20%, very particularly preferably by at least 100% in each case compared to the starting organism.

Alternatively, the reaction can also be carried out with enzyme preparation derived from one of the transgenic organisms. The organisms are unlocked. The enzyme preparation with sorbosone and / or sorbose dehydrogenase activity can be used unpurified or completely or partially purified. Immobilization, for example on a solid support material, is also preferred.

Another object of the invention relates to polypeptides with L-sorbose dehydrogenase (SDH) activity according to SEQ ID NO: 2 and L-sorbosone dehydrogenase (SNDH) activity according to SEQ ID NO: 4 and their functional equivalents.

The invention further relates to nucleic acid sequences coding for the polypeptide according to SEQ ID NO: 2, preferably the nucleic acid sequence according to SEQ ID: 1 or nucleic acid sequences which can be derived from the polypeptide sequence according to SEQ ID NO: 2 due to the degeneracy of the genetic code. Functional equivalents of said nucleic acid sequences are also included.

The invention further relates to nucleic acid sequences coding for the polypeptide according to SEQ ID NO: 4, preferably the nucleic acid sequence according to SEQ ID NO: 3 or nucleic acid sequences which can be derived from the polypeptide sequence according to SEQ ID NO: 4 due to the degeneracy of the genetic code. Functional equivalents of said nucleic acid sequences are also included.

“Functional equivalents” means in particular natural or artificial mutations of the polypeptides according to SEQ ID NO: 2 or 4 as well as homologous polypeptides from other organisms which still have essentially the same properties. Prefers are homologous polypeptides from the preferred prokaryotic and eukaryotic organisms suitable as hosts described below, homologs from microorganisms are very particularly preferred.

The same properties refer to functional equivalents to polypeptides according to SEQ ID NO: 2, those which have a sorbose dehydrogenase activity. The same properties refer to functional equivalents to polypeptides according to SEQ ID NO: 4, those which have a sorbosone dehydrogenase activity.

An activity is essentially said to be the same if the conversion of a specific, suitable substrate under the action of a specific, functional equivalent to a polypeptide according to SEQ ID NO: 2 or 4 under otherwise unchanged conditions is at least 10%, preferably at least 30%, particularly preferably at least 50%, very particularly preferably at least 70%, most preferably at least 90% in comparison to a conversion obtained using one of the polypeptides described by SEQ ID NO: 2 or 4. Sorbose can be used as a suitable substrate, for example in the case of functional equivalents to polypeptides according to SEQ ID NO: 2. Sorbosone can be used as a suitable substrate, for example, in the case of functional equivalents to polypeptides according to SEQ ID NO: 4.

The activity can differ both downwards and upwards compared to the comparison value. Preference is given to those sequences whose activity, measured on the basis of the conversion of the suitable substrate, under otherwise unchanged conditions, quantitatively by no more than 50%, preferably 25%, particularly preferably 10%, of a comparison value obtained with a by SEQ ID NO: 2 or 4 described polypeptides differs. Particularly preferred are those sequences whose activity, measured on the basis of the conversion of the suitable substrate, under otherwise unchanged conditions, quantitatively by more than 50%, preferably 100%, particularly preferably 500%, very particularly preferably 1000%, is compared with a value obtained by SEQ ID NO: 2 or 4 polypeptides described. Corresponding methods for determining sorbose dehydrogenase or sorbosone dehydrogenase activity are given in Examples 4 and 7.

Otherwise unchanged conditions means that all framework conditions such as type of host organism, culture or breeding conditions, assay conditions (such as buffer, temperature, Substrates etc. ) are kept identical between the activities to be compared and the approaches differ only in the sequence of the polypeptides to be compared.

Mutations include substitutions, additions, deletions, inversions, or insertions of one or more amino acid residues. Thus, for example, the present invention also includes those polypeptides which are obtained by modifying a polypeptide according to SEQ ID NO: 2 or 4. The aim of such a modification can be to further narrow down the sequence contained therein, to remove unnecessary sequences or to add further sequences, for example sequences which facilitate the purification or detection of the polypeptides.

Homology between two nucleic acid sequences is understood to mean the identity of the nucleic acid sequence over the entire total length of the sequence, which is determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25: 3389ff) using the following parameters:

Gap Weight: 50 Length Weight: 3

Average Match: 10 Average Mismatch: 0

By way of example, a sequence which has a homology of at least 80% based on nucleic acid with the sequence SEQ ID NO: 1 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set, has a homology of has at least 80%.

Homology between two polypeptides is understood to mean the identity of the amino acid sequence over the entire entire length of the sequence, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters is calculated:

Gap Weight: 8 Length Weight: 2

Average Match: 2,912 Average Mismatch: -2, 003 By way of example, a sequence which has a homology of at least 80% on a protein basis with the sequence SEQ ID NO: 2 is understood to mean a sequence which, when compared with the sequence SEQ ID NO .: 2, has homology according to the above program algorithm with the above parameter set of at least 80%.

Functional equivalents, derived from the polypeptide according to SEQ ID NO: 2 by substitution, insertion or deletion, have a homology of at least 85%, preferably 90%, preferably at least 95%, particularly preferably at least 97%, very particularly preferably at least 99%, and are characterized by essentially the same properties as the polypeptide according to SEQ ID NO: 2.

Functional equivalents, derived from the polypeptide according to SEQ ID NO: 4 by substitution, insertion or deletion, have a homology of at least 45%, preferably 60%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95% , and are characterized by essentially the same properties as the polypeptide according to SEQ ID NO: 4.

Functional equivalents, derived from the nucleic acid sequence according to the invention according to SEQ ID NO: 1 by substitution, insertion or deletion, have a homology of at least 75%, preferably 80%, preferably at least 85%, particularly preferably at least 90%, very particularly preferably at least 95% , and code for polypeptides with essentially the same properties as the polypeptide according to SEQ ID NO: 2. Functional equivalents, derived from the nucleic acid sequence according to the invention according to SEQ ID NO: 3 by substitution, insertion or deletion, have a homology of at least 50%, preferably 60%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, and code for polypeptides with essentially the same properties as the polypeptide according to SEQ ID NO: 4.

Mutations are preferably implemented at the level of the nucleic acid sequence coding for the polypeptide. Where insertions, deletions or substitutions, such as transitions and transversions, come into question, techniques known per se, such as in vitro mutagenesis, "primer repair", restriction or ligation can be used. Manipulations such as restriction, "chewing-back" or filling in overhangs for "blunt ends" can provide complementary ends of the fragments for the ligation. Analogous results can also be obtained using the polymerase chain reaction (PCR) come using specific oligonucleotide primers. A method for producing functional equivalents according to the invention preferably comprises the introduction of mutations into a nucleic acid sequence SEQ ID NO: 1 or 3. Mutagenesis can be carried out in an undirected ("random") manner, the properties of the mutagenized sequences then being followed by a "trial-by-error""Procedure to be screened. Particularly advantageous selection criteria include, for example, the enzyme activity of the polypeptide encoded by the nucleic acid. Alternatively, non-essential sequences can be deleted without significantly impairing the properties mentioned. Methods for mutagenizing nucleic acid sequences are known to the person skilled in the art and include, for example, the use of oligonucleotides with one or more mutations compared to the region to be mutated (for example in the context of a "site-specific mutagenesis"). Typically, primers with approximately 15 to approximately 75 nucleotides or more are used, preferably approximately 10 to approximately 25 or more nucleotide residues being located on both sides of the sequence to be changed. The details and implementation of said mutagenesis methods are familiar to the person skilled in the art (Kunkel et al. (1987) Methods Enzyol 154: 367-382; Tomic et al. (1990) Nucl Acids Res 12: 1656; Upender, Raj, Weir (1995) Bio - techniques 18 (l): 29-30; US 4,237,224, Glover DM et al. (1995) DNA Cloning Vol.l, IRL Press (ISBN 019-963476-9), chapter 6, p. 193 ff). Mutagenesis can also be achieved by treating, for example, vectors which contain one of the nucleic acid sequences according to the invention with mutagenizing agents such as hydroxylamine. Spee et al. describe a PCR method using dITP for random mutagenesis (Spee et al. (1993) Nucl Acids Res 21 (3): 777-778). The use of an "in vitro" recombination technique for molecular evolution has been described (Stemmer et al. (1994) Proc Natl Acad Sei USA 91: 10747-10751). The combination of the PCR and recombination method is also described (Moore et al. (1996) Nature Biotechnology 14: 458-467). The modified nucleic acid sequences are then brought back into the organisms via vectors.

The aim of mutagenesis of the nucleic acid sequences according to the invention can, for example, be to increase the enzyme activity.

Functional equivalents also include truncated sequences, single-stranded DNA and promoter variants. The promoters that precede the specified nucleotide sequences together or individually can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), without however affecting the functionality or effectiveness of the promoters. are pregnant. Furthermore, the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.

Non-functional equivalents are also understood to mean those sequences whose nucleotide sequence has been changed before the start codon in such a way that the gene expression and / or the protein expression is changed, preferably increased.

For optimal expression of heterologous genes in organisms, it is advantageous to change the nucleic acid sequences in accordance with the specific "codon usage" used in the organism. The "codon usage" can easily be determined on the basis of computer evaluations of other known genes of the organism in question. Corresponding codon-adapted nucleic acid sequences are also included under the term functional equivalents.

The invention further relates to chimeric enzymes consisting of two or more of the polypeptides according to the invention, manifestations in which the polypeptides according to the invention are present in the form of homo- and / or heterodimers, and fusion proteins from the polypeptides according to the invention with other amino acid sequences. By way of example, but not by way of limitation, the amino acid sequences which are advantageously used in the fusion proteins should be mentioned:

a) a signal or transit peptide which directs the fusion protein to the desired site of action (e.g. the plastids), or

b) an antigenic polypeptide sequence which can be used to detect expression (e.g. myc-tag or his-tag), or

c) a polypeptide sequence with the aid of which purification of the fusion protein is made possible (e.g. tags from several histidine residues such as, for example, hexa-His tag, GST tag, etc.)

d) another enzyme, preferably an enzyme which increases vitamin C biosynthesis.

The invention further relates to transgenic expression cassettes which contain the nucleic acid sequences according to the invention. In these, the nucleic acid sequence to be expressed is functionally linked to at least one genetic control sequence, preferably a promoter, which controls the transcription and / or translation of said nucleic acid sequence guaranteed. Furthermore, said expression constructs can contain further genetic control sequences and / or functional elements.

A functional link is generally understood to mean an arrangement in which a genetic control sequence can perform its function in relation to the nucleic acid sequence to be expressed. Function can, for example, control expression, i.e. Mean transcription and / or translation of the nucleic acid sequence. Control includes, for example, the initiation, increase, control or suppression of expression, i.e. Transcription and, if necessary, translation. The control, in turn, can take place in a tissue-specific or time-specific manner, for example. It can also be inducible, for example, by certain chemicals, stress, temperature etc.

A functional link is understood to mean, for example, the sequential arrangement of a promoter, the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator such that each of the regulatory elements can fulfill its function in the expression of the nucleic acid sequence.

This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically is positioned behind the sequence which acts as a promoter, so that both sequences are covalently linked to one another. The distance between the promoter sequence and the nucleic acid sequence to be expressed is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

Various ways are known to the person skilled in the art to arrive at an expression cassette according to the invention. An expression cassette according to the invention is preferably produced, for example, by direct fusion of a nucleic acid sequence functioning as a promoter with a nucleotide sequence to be expressed. A functional link can be established using common recombination and cloning techniques, such as those described in T Maniatis, EF Fritsch and J Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley InterScience (1987). However, further sequences can also be positioned between the two sequences, which for example have the function of a linker with certain restriction enzyme interfaces or a signal peptide. The insertion of sequences can also lead to the expression of fusion proteins.

However, an expression cassette can also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example a promoter, for example by means of homologous recombination or also by random insertion. Such constructions are also to be understood as expression cassettes in the context of the invention. For example, in order to increase the enzyme activities, modified promoter regions can be placed in front of the natural genes, so that the expression of the genes is increased and thus the activity is ultimately increased. Sequences can also be introduced at the 3 'end which, for example, increase the stability of the mRNA and thereby enable increased translation. This also leads to higher enzyme activity. Further gene copies of the nucleic acid sequences according to the invention are preferably introduced into the cell. These gene copies can be subject to natural regulation, a changed regulation, the natural regulatory regions being changed in such a way that they enable increased expression of the genes, or regulatory sequences of foreign or foreign genes can be used. A combination of the above methods is particularly advantageous.

Furthermore, an expression cassette according to the invention means constructs in which the endogenous SDH or SNDH genes are changed. The change can take place in the coding (e.g. the open reading frame) or non-coding region (e.g. the promoter region). The aim of such changes can be, for example, the expression of enzymes with increased activity compared to the starting enzymes. A

Enzyme activity can be increased, for example, by increasing the substrate turnover by changing the catalytic centers or by canceling the action of enzyme inhibitors. This means that they have an increased specific activity or their activity is not inhibited. In a further advantageous embodiment, increased enzyme activity can also be increased by increasing the enzyme synthesis take place in the cell, for example by switching off factors that repress enzyme synthesis or by increasing the activity of factors or regulatory elements that promote enhanced synthesis, or preferably by introducing additional gene copies. These measures increase the overall activity of the gene products in the cell without changing the specific activity. A combination of these methods can also be used. That means increasing specific activity as well as increasing overall activity. The changes described can be implemented with the methods of mutagenesis described above in a manner familiar to those skilled in the art, the activity of the enzymes or host organisms obtained in each case containing them being analyzed using suitable test systems. Corresponding test systems for determining a sorbose dehydrogenase or sorbosone dehydrogenase activity are known to the person skilled in the art and are described, inter alia, in Examples 4 and 7.

The skilled worker is also aware that nucleic acid molecules can also be expressed using artificial transcription factors of the zinc finger protein type (Beerli RR et al. (2000) Proc Natl Acad Sei USA 97 (4): 1495-500). These factors can be adapted to any sequence region and allow expression independent of certain promoter sequences.

The term “genetic control sequences” is to be understood broadly and means all those sequences which have an influence on the formation or the function of the expression cassette according to the invention. Genetic control sequences ensure, for example, transcription and, if necessary, translation in prokaryotic or eukaryotic organisms. The expression cassettes according to the invention preferably comprise a promoter 5 'upstream of the respective nucleic acid sequence to be expressed transgenically and a terminator sequence 3' downstream as an additional genetic control sequence, as well as, if appropriate, other customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be expressed.

Genetic control sequences are described, for example, in "Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)" or "Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton , Florida, eds.:Glick and Thompson, Chapter 7, 89-108 "and the citations cited therein. Examples of such control sequences are sequences to which inducers or repressors bind and thus regulate the transgenic expression of the nucleic acid. In addition to these new control sequences or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. However, the expression cassette can also have a simpler structure, that is to say no additional regulatory signals are inserted in front of the genes mentioned above and the natural promoter with its regulation is not removed. Instead, the natural control sequence is mutated so that regulation no longer takes place and gene expression is increased. These modified promoters can also be placed in front of the natural genes to increase activity.

Depending on the host organism or starting organism described in more detail below, which is converted into a genetically modified or transgenic organism by introducing the expression cassettes or vectors, different control sequences are suitable.

Advantageous control sequences for the expression cassettes or vectors according to the invention are, for example, in

Promoters such as cos, tac, trp, tet, lpp, lac, laclq, T7, T5, T3, gal, trc, ara, SP6, tuf, 1-PR - Or contained in the 1-PL promoter, which are advantageously used in gram-negative bacteria.

Further advantageous control sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], SSU, OCS, LEB4, USP, STLS1, B33, NOS; FBPaseP (WO 98/18940) or contained in the ubiquitin or phaseolin promoter.

Vectors such as the TK promoter, the RSV 3 'LTR promoter, the CMV promoter, the SV40 "early" or late "promoter are suitable for expression in vertebrates, preferably in mammals. Other promoters are known to the person skilled in the art Promoters suitable for use in vertebrates, preferably in mammals, include, for example, the tet promoter / repressor inducible or repressible by tetracycline or derivatives, the dexamethasone inducible MMTV-LTR promoter, the Drosophila minimal heat shock promoter inducible by Ecdysone or the analog Ponasterone A (as part of the pVgRXR expression system; Invitrogen, Inc.).

Also suitable as promoters are those which can control the expression of genes, in particular foreign genes, in plants. Promoters which allow constitutive expression in plants are preferred (Benfey et al., EMBO J. 8 (1989) 2195-2202). In particular, a plant promoter or a promoter derived from a plant virus is preferably used. The promoter of the 35S- is particularly preferred.

Cauliflower mosaic virus transcripts (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol. Biol. 6, 221-228) or the 19S CaMV promoter (US 5,352,605 and WO 84/02913). Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028). Another example of a suitable promoter is the LeguminB promoter (Accession No. X03677). Further preferred constitutive promoters are, for example, the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649) , the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991).

The expression cassettes can also contain a chemically inducible promoter (Rewiew: Gatz (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time. Such promoters, e.g. the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible (WO 95/19443), a benzenesulfonamide-inducible (EP-A-0388186) , an inducible by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), an inducible by abscisic acid (EP-A 335528), an by

Salicylic acid inducible (WO 95/19443) or an ethanol or cyclohexanone inducible (WO 93/21334) promoter can also be used.

Also preferred are promoters that are induced by biotic or abiotic stress, such as the pathogen-inducible promoter of the PRPl gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp80 promoter from tomato ( No. 5,187,267), the cold-inducible alpha-amylase promoter from the potato (WO 96/12814) or the wound-induced pinII promoter (EP375091). Ascorbic acid can act as a protective agent under these stress conditions. An inducible induction would therefore be advantageous to achieve an increased stress tolerance.

Also preferred are promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds.

Examples of seed-specific promoters that may be mentioned include the promoter of phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53), of 2S albumingen (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), the legumin (Shirsat A et al. (1989) Mol Gen Genet 215 (2): 326-331), the USP (unknown seed protein; Bäumlein H et al. (1991) Molecular & General Genetics 225 (3): 459-467), the Napin gene (US 5,608,152; Stalberg K, et al. (1996) L Planta 199: 515-519), the sucrose binding protein (WO 00/26388) or the legumin B4 Promoter (LeB4;

Baumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baeumlein H et al. (1992) Plant Journal 2 (2): 233-239; Fiedler U et al. (1995) Biotechnology (NY) 13 (10): 1090), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). The promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can be used advantageously Prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the kasirin gene or the secalin gene).

Further suitable promoters are, for example, specific promoters for tubers, storage roots or roots, such as, for example, the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato, the promoter of the starch synthase (GBSS1) or the sporamine promoter and fruit-specific promoters, such as the fruit-specific promoter from tomato (EP-A 409 625).

Promoters which are also suitable are those which ensure leaf-specific expression. These include the promoter of the cytosolic FBPase from potatoes (WO 98/18940), the SSU promoter (small subunit) from Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potatoes (Stockhaus et al. (1989) EMBO J 8: 2445-245). Promoters which control expression in seeds and plant embryos are also preferred.

Further suitable promoters are, for example, fruit-ripening-specific promoters, such as the fruit-ripening-specific promoter from tomato (WO 94/21794), flower-specific promoters, such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene ( WO 98/22593) or specific plastid or chromoplast promoters, such as the RNA poly erase promoter (WO 97/06250) or the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max (see also Genbank Accession number U87999) or another node-specific promoter as in EP-A 249676 can be used advantageously.

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.

Furthermore, plastid-specific promoters are preferred for the targeted expression in the plastids. Suitable promoters are described, for example, in WO 98/55595. These include the rpo B promoter element, the atoB promoter element, the clpP promoter element (see also WO 99/46394) or the 16SrDNA promoter element. Viral promoters are also suitable (WO 95/16783, WO 97/06250).

Targeted plastid expression can also be achieved if, for example, a bacterial or bacteriophage promoter is used, the resulting expression cassette is inserted into the plastid DNA and the expression is then expressed by a fusion protein consisting of a bacterial or bacteriophage polyerase and a plastid transit peptide. A corresponding method is described in US 5,925,806.

Genetic control sequences also include the 5 'untranslated region, introns or the non-coding 3' region of genes. It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. They can also promote tissue specificity (Rouster J et al., Plant J. 1998, 15: 435-440.). Conversely, the 5 'untranslated region of the opaque-2 gene suppresses expression. Deletion of the corresponding region leads to an increase in gene activity (Lohmer S et al., Plant Cell 1993, 5: 65-73).

The expression cassette can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically. The transgene to be expressed Nucleic acid sequences can be contained in one or more copies in the gene construct.

Genetic control sequences also mean sequences which code for fusion proteins consisting of a signal peptide sequence.

The expression of the target gene is in any desired cell compartment, e.g. the endomembrane system, the vacuole and the chloroplasts possible. By using the secretory path, desired glycosylation reactions, special folds, etc. possible. Secretion of the target protein to the cell surface or secretion into the culture medium, for example when using suspension-cultured cells or protoplasts, is also possible. The target sequences required for this can be taken into account both in individual vector variations and can be introduced into the vector together with the target gene to be cloned by using a suitable cloning strategy. Both target genes, if available, or heterologous sequences can be used as target sequences. Additional, heterologous sequences preferred but not limited to the functional linkage are further targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments; and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al. (1987) Nucl. Acids Res. 15: 8693-8711) and the like. The process of specifically transporting proteins that are not located in the plastids per se into the plastids is described (Klosgen RB and Weil JH (1991) Mol Gen Genet 225 (2): 297-304; Van Breusegem F et al. (1998) Plant Mol Biol. 38 (3): 491-496). Preferred sequences are:

a) Small subunit (SSU) of ribulose bisphosphate carboxylase (Rubisco ssu) from pea, corn, sunflower

b) transit peptides derived from genes of vegetable fat biosynthesis such as the transit peptide of the plastid "acyl carrier protein" (ACP), the stearyl-ACP desaturase, β-keto-acyl-ACP synthase or the acyl-ACP thioesterase.

c) the transit peptide for GBSSI ("Starch Granule Bound Synthase I")

d) LHCP II genes. Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome. Methods such as cre / lox technology allow tissue-specific, possibly inducible removal of the expression cassette from the genome of the host organism (Sauer B. Methods. 1998; 14 (4): 381-92). Here certain flanking sequences are added to the target gene (lox sequences), which later enable removal using the cre recombinase.

Polyadenylation signals suitable as genetic control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium turne faciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. ( 1984) EMBO J. 3: 835ff) or functional equivalents thereof. Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.

The expression cassettes according to the invention and the vectors derived from them can contain further functional elements.

The term functional element is to be understood broadly and means all those elements which have an influence on the production, multiplication or function of the expression cassettes according to the invention or of vectors or organisms derived therefrom. Examples include, but are not limited to:

a) Selection markers are usually required in order to successfully select homologously recombined or transformed cells. The selectable marker introduced with the expression construct gives the successfully recombined or transformed cells resistance to a biocide (for example a herbicide such as phosphinothricin, glyphosate or bromoxynil), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or Antibiotic, such as Kanamycin, G 418, bleomycin, hygromycin). The selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5: 81-84). Particularly preferred selection markers are those which confer resistance to herbicides. Examples of selection markers are: DNA sequences which code for phosphinothricin acetyltransferases (PAT), which acetylate the free amino group of the glutamine synthase inhibitor phosphinothricin (PPT) and thus detoxify the PPT (de Block et al. (1987) EMBO J. 6: 2513-2518) ( also called Bialophos ^® resistance gene (bar))

5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which confer resistance to Glyphosat® (N- (phosphonomethyl) glycine),

the gox gene (glyphosate oxidoreductase) coding for the glyphosate-degrading enzyme,

- the gene (coding for a dehalogenase which inactivates Dalapon ^® ),

Sulfonylurea and imidazolinone inactivating aceto-lactate synthases

bxn genes which code for bromoxynil-degrading nitrilase enzymes

the kanamycin or G418 resistance gene (NPTII). The NPTII gene codes for a neomycin phosphotransferase, which reduces the inhibitory effect of kanamycin, neomycin, G418 and paromomycin through a phosphorylation reaction.

- Nucleic acid sequences confer resistance to tetracycline, spectinomycin, ampecillin or chloramphenicol.

the D0G ^R 1 gene. The gene D0G ^R 1 was isolated from the yeast Saccharomyces cerevisiae (EP 0 807 836). It codes for a 2-deoxyglucose-6-phosphate phosphatase that confers resistance to 2-D0G (Randez-Gil et al. 1995, Yeast 11, 1233-1240).

Reporter genes that code for easily quantifiable proteins and a via self-color or enzyme activity

Ensure evaluation of the transformation efficiency, the expression location or time. This reporter gene should allow easy detection via a growth, fluorescence, chemo- or bioluminescence assay or via a photometric measurement. Examples include reporter genes, hydrolase genes, fluorescence protein genes, bioluminescence genes, glucosidase genes, peroxidase genes or biological Synthetic genes such as the 2-KLG synthetic genes, the luciferase gene, ß-galactosidase gene, gfp gene, lipase gene, esterase gene, peroxidase gene, ß-lactamase gene, acetyl-, phospho- or adenyl-transferase gene called. These genes make it easy to measure and quantify the transcription activity and thus the expression of the genes. It can be used to identify genome sites that show up to a factor of 2 different productivity. In the event that the biosynthesis genes themselves enable easy detection, an additional reporter gene can be dispensed with. Genes coding for reporter proteins are very particularly preferred (see also Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (l): 29-44) such as

"Green fluorescence protein" (GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23 (5): 912-8, 1997; Sheen et al. ( 1995) Plant Journal 8 (5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sei USA 94 (6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sei USA 93 (12) : 5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267-271; WO 97/41228).

chloramphenicol,

- Luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414; Ow et al. (1986) Science, 234: 856-859); allows bioluminescence detection.

β-galactosidase, encoded for an enzyme for which various chromogenic substrates are available.

β-glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the uidA gene, which encodes an enzyme for various chromogenic substrates.

- R-Locus gene product: protein that regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus enables a direct analysis of the promoter activity without the addition of additional auxiliaries or chromogenic substrates (Dellaporta et al.,

In: Chromosome Structure and Function: Impact of New Concepts, 18 ^th Stadler Genetics Symposium, 11: 263-282, 1988). β-lactamase (Sutcliffe (1978) Proc Natl Acad Sei USA 75: 3737-3741), enzyme for various chromogenic substrates (eg PADAC, a chromogenic cephalosporin).

- xylE gene product (Zukowsky et al. (1983) Proc Natl Acad Sei USA 80: 1101-1105), catechol dioxygenase, which can convert chromogenic catechols.

Alpha amylase (Ikuta et al. (1990) Bio / technol. 8: 241-242).

Tyrosinase (Katz et al. (1983) J Gen Microbiol 129: 2703-2714), enzyme that oxidizes tyrosine to DOPA and dopaquinone, which consequently form the easily detectable melanin.

Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268) can be used in calcium-sensitive bioluminescence detection.

c) Origins of replication, which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli. Examples are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, ^2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

d) Elements, for example "border sequences", which represent an agrobacterium-mediated transfer in plant cells for the

Enables transmission and integration into the plant genome, such as the right or left border of the T-DNA or the vir region.

e) Multiple cloning regions (MCS) allow and facilitate the insertion of one or more nucleic acid sequences.

The invention further relates to vectors which comprise the nucleic acid sequences or expression cassettes according to the invention. The introduction of a nucleic acid sequence or expression cassette according to the invention into cells can advantageously be implemented using vectors into which these nucleic acid sequences or cassettes are inserted. Vectors can be, for example, plasmids, cosmids, phages, viruses, retroviruses or also agrobacteria. As vectors for expression

a) pQE70, pQE60 and pQE-9 (QIAGEN, Inc.) are preferred in E. coli; pBluescript vectors, Phagescript vectors, pNH8A, pNHlδa, pNHlδA, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -Bl, λgtll or pBdCI. So-called “broad host range” vectors, such as pBHR1, pBBR122 or pRS201, are particularly preferred,

b) in Streptomyces, pIJlOl, pIJ364, pIJ702 or pIJ361 are preferred,

c) in Bacillus pUBllO, pC194 or pBD214 are preferred, in Corynebacterium pSA77 or pAJ667,

d) pALSl, pIL2 or pBBH6 are preferred in fungi, 2μM, pAG-1, YEp6, YEpl3 or pEMBLYe23 in yeasts

e) plants are preferably pLGV23, pGHlac ⁺ , pBINl9, pAK2004 or pDH51,

f) in eukaryotes v.a. Mammals are preferred pWLNEO, pSV2CAT, pOG44, pXTl and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG and pSVL (Pharmacia Biotech, Inc.). Examples of inducible vectors are pTet-tTak, pTet-Splice, pcDNA4 / T0, pcDNA4 / TO / LacZ, pcDNA6 / TR, pcDNA4 / T0 / Myc-His / LacZ, pcDNA4 / T0 / Myc-His A, pcDNA4 / T0 / Myc -His B, pcDNA4 / T0 / Myc-His C, pVgRXR (Invitrogen, Inc.) or the pMAM series (Clontech, Inc .; GenBank Accession No.: U02443). These already provide the inducible regulatory control element, for example for chemical, inducible expression,

g) in yeast, for example pYES2, pYDl, pTEFl / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2, PHIL-Sl, PPIC3SK, pPIC9K, and PA0815 (Invitrogen, Inc.),

or derivatives of the plasmids mentioned above. The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ) can be removed. Suitable plant vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119. In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. Preferred vectors are those which enable stable integration of the expression cassette into the host genome.

If several genes (for example the sorbose dehydrogenase and sorbosone dehydrogenase genes according to the invention) are to be introduced together into one organism, then all of them together with a reporter gene or selection marker in a single vector or each individual gene with a reporter gene or selection marker in a vector in each Organism are introduced, wherein the different vectors can be introduced simultaneously or successively.

The invention further relates to organisms containing one of the nucleic acid sequences according to the invention, expression constructs or plasmids.

Organism preferably means microorganisms, animal or plant organisms or cells derived therefrom (for example mammalian cells or plant cells), as well as tissues, parts, organs or reproductive material (seeds or fruits) of the aforementioned.

Suitable organisms or host organisms for the process according to the invention are preferably organisms which are capable of synthesizing L-sorbose, L-sorbosone, 2-KLG or ascorbic acid. Organisms that can naturally synthesize L-sorbose, L-sorbosone, 2-KLG or ascorbic acid are preferred. However, organisms which are able to synthesize 2-KLG due to the introduction of the complete 2-KLG synthesis genes are also suitable for the process according to the invention.

Organisms such as bacteria, yeasts, fungi, vertebrates or invertebrates (as well as cells derived therefrom such as mammalian cells) or plants are suitable for the method according to the invention.

Are included by way of example but not by way of limitation

a) Mushrooms such as Aspergillus, Eremotheciurα, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6 described mushrooms. The filamentous Hemiascomycet Ashbya gossypii or Eremothecium ashbyii is particularly preferred.

b) yeasts such as Candida, Saccharomyces, Hansenula or Pichia, Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178) are particularly preferred, c) Plants such as arabidopsis, tomato, potato, corn, soybean, rapeseed, barley, wheat, rye, rice, millet, cotton, sugar beet, sunflower, flax, hemp, canola, oats, tobacco, alfalfa, lettuce, rose hip or the various Tree, nut and wine types,

d) Vertebrates and invertebrates. Particularly preferred vertebrates are non-human mammals such as in dogs, cats, sheep, goats, chickens, mice, rats, cattle or horses. Preferred animal cells include CHO, COS, HEK293 cells. Preferred invertebrates include insect cells such as Drosophila S2 and Spodoptera Sf9 or Sf21 cells,

e) prokaryotic organisms such as gram-positive or gram-negative bacteria such as Acetobacter, Gluconobacter, Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanibacter, Escherichia (especially Escherichia coli), Serratia, Staphylococcus, Aerobacter, Alcaligenes, Penicillium Called pseudomonas or Klebsieila.

Organisms of the genus and species Acetobacter liquefaciens, Acetobacter aceti, Acetobacter pasteurianus, Acetobacter hansenii, Gluconobacter oxidans, Ashbya gossypii, Eremothecium ashbyii, Saccharomyces cerevisiae, Candida flaveri, Candida famata ammonium bacilli, or Corynebacterium, are particularly preferred. Maize, soybean, rapeseed, barley, wheat, potato and tomato are particularly preferred as plants.

The production of a transformed organism or cell requires that the corresponding DNA

(for example one of the nucleic acid sequences, expression cassettes or vectors according to the invention) is introduced into the corresponding host cell. A large number of methods are available for this process, which is referred to as transformation (see also Keown et al. 1990 Methods in Enzymology 185: 527-537). For example, the DNA can be introduced directly by microinjection, electroporation or by bombardment with DNA-coated microparticles (biolistic method with the gene gun "particle bombardment"). The cell can also be chemically permeable, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion. The DNA can also be obtained by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes. Electroporation is another suitable method for introducing DNA in which the cells are reversibly permeabilized by an electrical pulse. Preferred general methods should be mentioned Calcium phosphate mediated transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection. Such methods are familiar to the person skilled in the art and are described, for example, 5 in Davis et al., Basic Methods In Molecular Biology (1986). Another option for introducing DNA into microorganisms is conjugation, with both biparental and triparental conjugation being possible.

10 For microorganisms, the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA cloning

15 Vol.l, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. See Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press. For example, methods such as insertion are advantageous

20 of the DNA via homologous or heterologous recombination, for example using the ura-3 gene and / or using the REMI method described below (= "Restriction Enzyme-Mediated Integration"), or expression using expression vectors ,

25

The REMI technique is based on the co-transformation of a linear DNA construct that was cut at both ends with the same restriction endonuclease, together with the restriction endonuclease used for this restriction of the DNA construct.

30 was turned into an organism. The restriction endonuclease then cuts the genomic DNA of the organism into which the DNA construct was introduced together with the restriction enzyme. This leads to an activation of the cell's own repair mechanisms. These repair mechanisms repair those through the

35 endonuclease-induced strand breaks in the genomic DNA and at a certain frequency also incorporate the cotransformed DNA construct into the genome. As a rule, the restriction sites at both ends of the DNA are preserved. This technique was developed by Bölker et al. (Mol Gen Genet, 248, 1995:

40 547-552) for the insertion mutagenesis of fungi. Schiestl and Petes (Proc. Natl. Acad. Sei. USA, 88, 1991: 7585-7589) used the method to clarify whether there is a heterologous recombination in Saccharomyces. For stable transformation and regulated expression of a

45 inducible reporter gene was the method of Brown et al. (Mol. Gen. Genet. 251, 1996: 75-80). Using the REMI method, the nucleic acid fragments according to the invention can be ducked be placed at transcriptionally active sites in the genome. In principle, all known restriction enzymes are suitable for the described method for integrating biosynthesis genes into the genome of organisms. Restriction enzymes that only recognize 4 base pairs as a restriction site are less preferred because they cut too frequently in the genome or in the vector to be integrated; preference is given to enzymes that recognize 6, 7, 8 or more base pairs as an interface, such as BamHI, EcoRI, Bglll, SphI , Spei, Xbal, Xhol, Ncol, Sall, Clal, Kpnl, Hindlll, Sacl, PstI, Bpnl, Notl, Srfl or Sfil to name just a few of the possible enzymes. It is advantageous if the enzymes used no longer have interfaces in the DNA to be introduced, this increases the efficiency of the integration. As a rule, 5 to 500 U, preferably 10 to 250, particularly preferably 10 to 100 U of the enzymes are used in the REMI approach. The enzymes are advantageously used in an aqueous solution, the substances for osmotic stabilization such as sugar such as sucrose, trehalose or glucose, polyols such as glycerol or polyethylene glycol, a buffer with an advantageous buffering in the range from pH 5 to 9, preferably 6 to 8 , particularly preferably 7 to 8 such as Tris, MOPS, HEPES, MES or PIPES and / or substances for stabilizing the nucleic acids, such as inorganic or organic salts of Mg, Cu, Co, Fe, Mn or Mo. If appropriate, further substances may also be present be like EDTA, EDDA, DTT, ß-mercaptoethanol or nuclease inhibitors. However, it is also possible to carry out REMI technology without these additives. The process is carried out in a temperature range from 5 to 80 ° C., preferably from 10 to 60 ° C., particularly preferably from 20 to 40 ° C. All known methods for destabilizing cell membranes such as, for example, electroporation, fusion with loaded vesicles or destabilization via various alkali or alkaline earth metal salts such as lithium, rubidium or calcium salts are suitable for the process, the lithium salts being preferred.

Plants can also be transformed by bacterial infection using transgenic Agrobacterium tumefaσiens or Agrobacterium rhizogenes strains. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant after Agrobacterium infection. Part of this plasmid, called T-DNA (transferred DNA), is integrated into the genome of the plant cell. The nucleic acid sequences or expression cassettes according to the invention are preferably integrated into special plasmids, either into an intermediate vector (English: shuttle or intermediate vector) or a binary vector. Binary vectors can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene (for example, the nptll gene, which confers resistance to kanamycin) and a linker or polylinker flanked by the right and left T-DNA restriction sequences. They can be transformed directly into Agrobacterium (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The use of Agrobacterium tumefaciens for the transformation of plants using tissue culture explants has been described by Horsch et al. (Horsch RB (1986) Proc Natl Acad Sei USA 83 (8): 2571-2575), Fraley et al. (Fraley et al. (1983) Proc Natl Acad Sei USA 80: 4803-4807) and Bevans et al. (Bevans et al. (1983) Nature 304: 184-187). Various binary vectors are known and some are commercially available, for example pBIN19 (Clontech Laboratories, Inc. USA). The methods mentioned are described, for example, in B Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by SD Kung and R Wu, Academic Press (1993), 128-143; Höfgen and Willmitzer (1988) Nucl. Acid Res. 16: 9877 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225). Wounded leaves or leaf pieces are bathed in an agrobacterial solution and then cultivated in suitable media. The genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned documents.

For expression in plants, the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res 12: 8711). The Agrobacterium mediated transformation is best suited for dicotyledonous plant cells, whereas the direct transformation techniques are suitable for every cell type.

Plasmids which are replicated autonomously in the host cell are preferably used as the vector (for example for plasmid microorganisms which carry the origin of replication of the 2 μ plasmid from S. cerevisiae). Pendulum vectors and "broad host range" vectors are particularly preferred which enable the replication of the vector in more than one organism.

However, linear expression cassettes can also be used which are integrated into the genome of the host. This integration can take place via hetero- or homologous recombination. However, as mentioned, preferably via homologous recombination (Steiner et al. (1995) Genetics 140: 973-987). The nucleic acid sequences or expression cassettes according to the invention can be present individually in the genome at different locations or on different vectors or together in the genome or on one vector. Transformed cells, ie those which contain the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed cells if a selectable marker is part of the introduced DNA. Any gene that can confer resistance to antibiotics or herbicides can act as a marker, for example. Transformed cells that express such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type. Various positive and negative selection markers are described above. Examples are the bar gene that confers resistance to the herbicide phosphinothricin (Rathore KS et al. (1993) Plant Mol Biol. 21 (5) .871-884), the nptll gene that confers resistance to kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP gene which confers resistance to the herbicide glyphosate.

Once a transformed plant cell has been made, a complete plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.

According to the invention, cells, cell cultures, parts derived from the transgenic organisms described above - such as roots, leaves, etc., for example in the case of transgenic plant organisms - and transgenic propagation material such as seeds or fruits.

The invention further relates to enzyme preparations produced using one of the proteins, nucleic acid molecules, expression cassettes, vectors or organisms which contain at least one of the polypeptides according to the invention. An enzyme preparation preferably contains one of the polypeptides according to the invention as shown in SEQ ID NO: 2 or 4 or one of its functional equivalents. The enzyme preparation can contain the polypeptides according to the invention in unpurified, partially purified or purified form. The content of the total amount of protein in the polypeptide according to the invention, for example in a partially purified enzyme preparation, is at least 1%, preferably at least 10%, particularly preferably at least 50%, very particularly preferably at least 70%, most preferably at least 90%. Such preparations are available, for example, from

Culturing one of the organisms according to the invention (preferably a microorganism), smashing the cells and, if appropriate Isolation and purification of the polypeptides according to the invention from the cell-free extract of the broken cell material. Methods known to those skilled in the art, such as ion exchange chromatography, liquid chromatography, gel filtration, gel electrophoresis, salting out and dialysis, and combinations of the aforementioned methods, can be used to purify the polypeptides according to the invention. In a further preferred embodiment, the enzyme preparation can be immobilized on a water-soluble (for example polyacrylate) or water-insoluble carrier material (for example polystyrene). Various suitable carrier materials are known to the person skilled in the art, to which the enzyme preparations or polypeptides according to the invention can be bound covalently or via adsorption. Celite, silica gel, amberlite, carrier materials made of various polymers (eg polypropylenes, polystyrene, polyurethane, polyacrylates) or sol gels are suitable as solid carriers.

Genetically modified plants according to the invention that can be consumed by humans and animals can also be used, for example, directly or after preparation known per se as food or feed.

Another object of the invention relates to the use of the above-described proteins, nucleic acid sequences, expression cassettes, vectors, enzyme preparations, organisms and the cells, cell cultures, parts (such as roots, leaves, etc.) derived from them and transgenic plant organisms transgenic propagation material (such as seeds or fruits) for the production of food or feed, pharmaceuticals or fine chemicals.

Fine chemicals means enzymes, vitamins, amino acids, sugar,

Fatty acids, natural and synthetic flavors, aromas and colors. Fine chemicals are also to be understood as meaning aldehydes, ketones and carboxylic acids, preferably sorbosone or 2-keto-L-gulonic acid. The production of pharmaceuticals, such as antibodies or vaccines, has been described (Hood EE, Jilka JM. (1999) Curr Opin Biotechnol. 10 (4): 382-386; Ma JK and Vine ND (1999) Curr Top Microbiol Immunol. 236 : 275-92).

Aldehydes, ketones and carboxylic acids preferably mean sugar alcohols, ketoses, aldoses and corresponding sugar acids such as D-glucose, D-mannose, D-mannitol, L-sorbose, D-fructose, D-sorbitol, L-sorbosone, L-gulose, 2 -Keto-D-gluconic acid, L-idose, glycerol, D-gluconic acid, D-mannonic acid, L-idonic acid, 5-keto-D-gluconic acid, 5-keto-D-mannonic acid, D-glucosones. L-Sorboson and 2-KLG are particularly preferred. Another object of the invention relates to processes for the preparation of aldehydes, ketones or carboxylic acids starting from the corresponding alcohols or aldehydes, characterized in that the alcohol or aldehyde in the presence of one of the polypeptides according to the invention, a transgenic organism, cell cultures, parts, tissues, organs or oxidized reproductive material of the same or an enzyme preparation. The production of 2-keto-L-gulonic acid by oxidation of sorbose and / or L-sorbosone is preferred. The production of L-ascorbic acid using the 2-keto-L-gulonic acid prepared by the above process is also particularly preferred.

sequences

1. SEQ ID No. 1: Nucleic acid sequence coding for the

Sorbose dehydrogenase from Acetobacter liquefaciens ATCC14835

2. SEQ ID No. 2: Amino acid sequence for sorbose dehydrogenase from Acetobacter liquefaciens ATCC14835

3. SEQ ID No. 3: Nucleic acid sequence coding for the sorbosone dehydrogenase from Acetobacter liquefaciens ATCC14835

4. SEQ ID No. 4: Amino acid sequence for sorbosone dehydrogenase from Acetobacter liquefaciens ATCC14835

5. SEQ ID No. 5: Nucleic acid sequence coding for the genomic clone 17 from Acetobacter liquefaciens ATCC14835, which completely comprises the nucleic acid sequence coding for the sorbose dehydrogenase and partially encoding the nucleic acid sequence for the sorbosone dehydrogenase.

6. SEQ ID No. 6: Amino acid sequence for the sorbosone dehydrogenase from Acetobacter liquefaciens ATCC14835 (partial)

7. SEQ ID No. 7: Amino acid sequence for sorbose dehydrogenase from Acetobacter liquefaciens ATCC14835

8. SEQ ID NO. 8: oligonucleotide primer KEI9 5 '-GCTCTAGATGCCCTACAACCCTGACTTCAACG-3'

9. SEQ ID NO. 9: KEI10 5 '-CGGGATCCCGCGCCGCCCTCAACCACGTTGGA-3' oligonucleotide primer

10. SEQ ID NO. 10: KEI376 oligonucleotide primer 5 '-GCTCTAGAGGATCGCCAACGACACCGTCTACG-3' 11. SEQ ID NO. 11: KEI377 oligonucleotide primer 5 '-CGGGATCCTCATGATGCGATCCAGTGCGTGCG-3'

12. SEQ ID NO. 12: KEI390 5 5 '-ACTGCGCGTCCATGGGCTGGAAGG-3' oligonucleotide primer

13. SEQ ID NO. 13: KEI720 5 '-AAAACATATGAAGATCCATGCA-3' oligonucleotide primer

10 14. SEQ ID NO. 14: KEI721 oligonucleotide primer 5 '-TATCTGGATCCTCATGATGCGAT-3'

Examples

15 General nucleic acid methods such as cloning,

Restriction cleavages, agarose gel electrophoresis, linking of DNA fragments, transformation of microorganisms, cultivation of bacteria and sequence analysis of recombinant DNA were, unless otherwise described, as described by Sambrook et al. (1989)

20 (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). The sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sei USA 74: 5463-5467). Resulting fragments

25 from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.

The strain Acetobacter liquefaciens (ATCC14835) was used.

30

Example 1: Construction of a genomic bank of Acetobacter liquefaciens ATCC14835

With the help of the Qiagen Blood & Cell Culture DNA Kit, 35 genomic DNA was prepared. Using known methods, the gene bank was then constructed using the zero background cloning kit (Invitrogen) in the vector pZerO-2 (Invitrogen). For this, the genomic DNA was digested with Sau3A and ligated into pZerO-2 vector linearized with BamHI. The mean size of the 40 insert was approximately 2.7 kb. The gene bank represented more than 99% of the bacterial genome. The individual clones were transferred to nylon membranes, lysed and prepared for hybridization.

5 Example 2: Preparation of the probes and hybridization

An internal 500 bp fragment of the Gluconobacter oxydans SDH gene was used as the probe. The probe was labeled using the DIG PCR Labeling Kit (Röche Diagnostics). The nucleotide sequence of the primers used can be found in Table 1.

Table 1: Nucleotide sequence of the primers used

Several clones were identified which gave a positive signal with the probe derived from the sorbose dehydrogenase gene. The insert of some clones were sequenced. The sequence of the insert from clone 17 is given in SEQ ID No: 5. Clone 17 contains a DNA fragment comprising 2541 nucleotides, which carries the complete gene of the SDH from Acetobacter liquefaciens (SEQ ID No: 1). This gene consists of 1827 nucleotides that code for 609 amino acids (SEQ ID No: 2).

Example 3: Sequence comparison of Acetobacter liquefaciens sorbose dehydrogenase

A BLAST search was carried out against Genbank (Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs; Nucleic Acids Res. 25: 3389ff) with the sequence from SEQ ID No: 1. Homology to the sequence of a sorbose dehydrogenase from Gluconobacter oxydans (Sequence 5, patent US 5,834,263; program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) Gap weight 50, Length weight 3 , Average match 10, Average mismatch 0). This resulted in 71.7% DNA sequence similarity with the SDH from G. oxydans. The sequence similarity at the amino acid level is 82.3% (program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) Gap weight 8, Length weight 2, Average match 2,912, Average mismatch -2,003) , Example 4: Expression of Acetobacter liquefaciens sorbose dehydrogenase

The vector pVC-77 was cut using the restriction endonucleases Nde I and BamH I. After electrophoresis on a 1% agarose gel, a piece of gel was cut out, which corresponded to a band size of 1.6 kb. The DNA was isolated from the gel matrix using the QIAquick Gel Extraction Kit (Qiagen), ligated into pT7-7 vector linearized with Nde I and BamHI and transformed into E. coli BL-21. The new plasmid was named pEVC-7.

To confirm that the open reading frame (ORF) is a sorbose dehydrogenase from A. liquefaciens, 100 mL LB medium are cultivated at 37 ° C to an optical density of 0.5. Then 0.3 mM IPTG is added, which induces the T7 DNA polymerase integrated chromosomally under the control of the lac promoter. After 2 hours, the cells are harvested.

Approximately 500 mg of bio-moist mass are digested with the RiboLyser Cell Disrupter (Tristar, Thermo Hybaid, UK). According to the literature, the solubilized membrane fraction is purified by DEAE chromatography, confirmed by N-terminal sequencing and the activity of the SDH is determined analogously to the procedure described by Fujisawa (EP 0 758 679; US 5,834,263). In addition, the final sample is examined by HPLC. L-sorbosone is detected as the product. SEQ ID NO: 1 is the gene for a new sorbose dehydrogenase from Acetobacter liquefaciens.

Example 5: Cloning of Acetobacter liquefaciens sorbosone dehydrogenase

Upstream of the sorbose dehydrogenase gene in clone 17 are another 250 nucleotides. This sequence shows similarity to the C-terminus of Gluconobacter oxydans sorbosone dehydrogenase. Therefore, as in G. oxydans in A. liquefaciens, the sorbose and sorbosone dehydrogenase genes are organized one after the other on a DNA fragment as an operon.

The sequence information from the 250 nt was used to isolate the gene of sorbosone dehydrogenase from genomic DNA from Acetobacter liquefaciens.

Genomic DNA from a 30 mL overnight culture in YSM medium (yeast extract 5 g / L, D-sorbitol 50 g / L, mannitol 10 g / L, pH 5-5.5) from Acetobacter liquefaciens ATCC14835 was prepared (Genomic DNA buffer set and Genomic-tip, Qiagen) and digested at 37 ° C for 60 h with the restriction endonuclease Aatll. This was followed by electrophoresis in a 1% agarose gel. Southern blot was used to transfer the DNA to a positively charged nylon membrane (Röche Diagnostics) overnight.

The 250 bp fragment of the Acetobacter liquefaciens SNDH gene from Klonl7 was used as the probe. The probe was labeled using the DIG-High Prime DNA Labeling and Detection Starter II kit (Röche Diagnostics). The nucleotide sequence of the primers used can be found in Table 2. The probe was isolated after electrophoresis in a 1.5% agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and used in the hybridization.

Table 2: Nucleotide sequence of the primers used

The hybridization was carried out under standard conditions (pre-hybridization 37 ° C; hybridization 37 ° C for 60 h, wash at 75 ° C in 0.1 x SSC and 0.1% SDS). A positive signal with a band size of 3.6 kb could be detected by the DIG Luminescent Detection Kit (Röche Diagnostics).

A piece of gel corresponding to a band size of 3.6 kb was cut out from a parallel run of 1% agarose gel with AatII digested genomic DNA from A. liquefaciens. The DNA was isolated from the gel matrix using the QIAquick Gel Extraction Kit (Qiagen), religated and used as a template in a polymerase chain reaction (inverse PCR). The nucleotide sequence of the primers used can be found in Table 3.

Table 3: Nucleotide sequence of the primers used

After electrophoresis in a 1% agarose gel, an approximately 2 kb long DNA fragment was identified as the PCR product. This was extracted from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen). The fragment was labeled with the restriction endonu cleases BamHI and Ncol digested and ligated into pLitmus28 vector (Invitrogen) linearized with BamHI / NcoI. The transformation was carried out by electroporation into XL-1 blue electrocompetent cells (Stratagene).

Several clones were obtained. The insert of a clone was sequenced. It contains an 1865 nucleotide DNA fragment which carries the complete gene of the SNDH from Acetobacter liquefaciens (SEQ ID No: 3). This gene contains an open reading frame of 1431 nucleotides that code for 477 amino acids (SEQ ID No. 4).

Example 6: Sequence comparison of Acetobacter liquefaciens sorbosone dehydrogenase

A BLAST search was performed against Genbank (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res., 25: 3389ff) with the sequence from SEQ ID No.2 performed. Homology to the sequence of a sorbosone dehydrogenase from Gluconobacter oxydans (Sequence 5, patent US5834263) fell. This resulted in 48.7% DNA sequence similarity with the SNDH from G. oxydans (program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) Gap weight 50, Length weight 3, Average match 10, average mismatch 0). At the amino acid level, the sequence similarity is 42.7% (program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) gap weight 8, length weight 2, average match 2,912, average mismatch -2,003) ,

Example 7: Expression of Acetobacter liquef ciens sorbosone dehydrogenase

The vector pVC-96 clone 3 is used as a template in a polymerase chain reaction. The nucleotide sequence of the primers used can be found in Table 3.

Table 4: Nucleotide sequence of the primers used After electrophoresis in a 1% agarose gel, an approximately 1.6 kb long DNA fragment is identified as the PCR product. This is cut using the restriction endonucleases Nde I and BamH I. After electrophoresis on a 1% agarose gel, a piece of gel is cut out, which corresponds to a band size of 1.6 kb. The DNA is isolated from the gel matrix by the QIAquick Gel Extraction Kit (Qiagen), ligated into pT7-7 vector linearized with Nde I and BamHI and transformed into E. coli BL-21. The new plasmid is called pEVC-9.

To confirm that the ORF is an SNDH from A. liquefaciens, 100 mL LB medium are cultivated at 37 ° C to an optical density of 0.5. Then 0.3 mM IPTG is added, which induces the T7 DNA polymerase integrated chromosomally under the control of the lac promoter. To

The cells are harvested for 2 hours.

Approximately 500 mg of bio-moist mass are digested with the RiboLyser Cell Disrupter (Tristar, Thermo Hybaid, UK). In a modification of the protocol known in the literature (Fujisawa, EP0758679 and US5834263), the crude extract is purified by Q-Sepharose chromatography, confirmed by N-terminal sequencing and the activity of the SNDH is analogous to that described by Fujisawa (EP0758679 (US5834263)) certainly. In addition, the final sample is examined by HPLC. The product 2-keto-L-gulonic acid is detected. Thus SEQ ID NO is:

3 a new sorbosone dehydrogenase from Acetobacter liquefaciens.

Claims

claims

1. A process for the preparation of vitamin C or 2-keto-L-gulonic acid (2-KLG), characterized in that a

Organism that contains at least one transgenic nucleic acid sequence coding for

i) a polypeptide according to SEQ ID NO: 2 or a functional equivalent thereof or

ii) a polypeptide according to SEQ ID NO: 4 or a functional equivalent thereof,

or an enzyme preparation obtained from it is used.

2. The method according to claim 1, characterized in that the organism also contains nucleic acids coding for i) and ii).

3. The method according to any one of claims 1 or 2, characterized in that at least one of the following reaction steps is included:

a) converting L-sorbose to L-sorbosone using an organism or an enzyme preparation obtained therefrom, the organism containing at least one transgenic nucleic acid sequence coding for a polypeptide with L-sorbose dehydrogenase activity according to SEQ ID NO: 2 or a functional equivalent thereof,

b) conversion of L-sorbosone to 2-KLG using a transgenic organism or an enzyme preparation obtained therefrom, the organism containing at least one transgenic nucleic acid sequence coding for a polypeptide with L-sorbosone dehydrogenase activity according to SEQ ID NO: 4 or a functional equivalent thereof ,

4. The method according to claim 3, characterized in that both reaction steps a) and b) are used during the process.

5. Polypeptide according to SEQ ID NO: 2 or its functional equivalents or functionally equivalent parts.

6. Polypeptide according to claim 5 with a homology of at least 85% to the sequence described by SEQ ID NO: 2 and coding for enzymes with L-sorbose dehydrogenase activity.

5 7. Nucleic acid sequences coding for polypeptides according to one of claims 5 or 6.

8. nucleic acid sequences according to claim 7, selected from the group consisting of

10 a) the sequence described by SEQ ID NO: 1,

b) sequences which, on the basis of the degenerate genetic code, result from the back translation of the in

15 SEQ ID NO: 2 deduced amino acid sequence shown

c) Sequences that have a functional equivalent to the nucleic acid sequence represented by SEQ ID NO: 1

20 represent.

9. Nucleic acid sequences according to one of claims 7 or 8 with a homology of at least 75% to the sequence described by SEQ ID NO: 1 and coding for enzymes with L-sorbose

25 dehydrogenase activity.

10. Polypeptides according to SEQ ID NO: 4 or their functional equivalents or functionally equivalent parts.

11. Polypeptides according to claim 10, characterized in that they have a homology of at least 45% to the sequence described by SEQ ID NO: 4 and code for enzymes with L-sorbosone dehydrogenase activity.

35 12. Nucleic acid sequences coding for polypeptides according to one of claims 10 or 11.

13. Nucleic acid sequences according to claim 12, selected from the group consisting of

40 a) the sequence described by SEQ ID NO: 3

b) Sequences which can be derived from the amino acid sequence shown in 45 SEQ ID NO: 4 on the basis of the degenerate genetic code c) Sequences which are a functional equivalent to the nucleic acid sequence represented by SEQ ID NO: 3.

5 1. Nucleic acid sequences according to one of claims 12 or 13 with a homology of at least 50% to the sequence described by SEQ ID NO: 3 and coding for enzymes with L-sorbosone dehydrogenase activity.

15. Transgenic expression cassettes containing at least one nucleic acid sequence according to one of claims 7 to 9 or 12 to 14.

16. Vectors containing at least one nucleic acid sequence

15 according to one of claims 7 to 9 or 12 to 14 or an expression cassette according to claim 15.

17. Transgenic organism containing at least one nucleic acid sequence according to one of claims 7 to 9 or 12 to 14

20 or an expression cassette according to claim 15 or a vector according to claim 16.

18. Transgenic organism according to claim 17 selected from the group consisting of Bacillus, Clostridium, Escherichia,

25 Pichia, Candida, Cyanobacter, Corynebacterium, Brevibacterium, Saccharomyces, Eremothecium and Ashbya.

19. Transgenic organism according to one of claims 17 or 18 selected from the group consisting of Gluσobacter oxidans,

30 Acetobacter xylinium, Acetobacter pasteurianus, Acetobacter aceti, Acetobacter hansenii, Acetobacter suboxidans, Escherischia coli and Pseudomonas putida.

20. Transgenic organism according to claims 17 selected from the group consisting of Arabidopsis, tomato, potatoes, corn,

Rapeseed, wheat, barley, sunflowers, millet, rye, oats, sugar beet, beans and soy.

21. Cell cultures, parts, tissues, organs or transgenic ver

40 reproductive material derived from a transgenic organism according to one of claims 17 to 20.

22. Enzyme preparation with L-sorbose dehydrogenase activity and / or L-sorbosone dehydrogenase activity produced using

45 of a transgenic organism according to one of claims 17 to 20 or of cell cultures, parts, tissues, organs or transgenic propagation material of the same according to claim 21.

23. Use of a polypeptide according to one of claims 5, 6, 5 10 or 11, a transgenic organism according to one of the

Claims 17 to 20, cell cultures, parts, tissues, organs or transgenic propagation material of the same according to claim 21 or an enzyme preparation according to claim 22 for the production for the production of food or feed, pharmaceuticals 10 or fine chemicals.

24. Use according to claim 23, wherein the fine chemical is an aldehyde, ketone or a carboxylic acid.

15 25. Use according to one of claims 23 or 24, wherein the

Fine chemical L-sorbosone, 2-keto-l-gulonic acid or vitamin C.

26. A process for the preparation of aldehydes, ketones or carboxylic acids from the corresponding alcohols or

Aldehydes characterized in that the alcohol or aldehyde in the presence of a polypeptide according to one of claims 5, 6, 10 or 11 of a transgenic organism according to one of claims 17 to 20, of cell cultures, parts, tissues, organs or transgenic propagation material the same according to claim 21 or an enzyme preparation according to claim 22 oxidized.

27. The method according to claim 26 for the preparation of 2-keto-L-

30 gulonic acid or ascorbic acid, characterized in that L-sorbose and / or L-sorbosone is oxidized, and optionally the 2-keto-L-gulonic acid obtained is converted to L-ascorbic acid.

35

40

45