DE102010023749A1 - Cell and process for the preparation of resveratrol - Google Patents

Abstract

The invention relates to a cell which is able to form resveratrol, the cell having at least one activity of an enzyme E1 which is reduced in comparison to its wild type and which is involved in a cataplerotic metabolic pathway in relation to malonyl-coenzyme A, and a process for the production of resveratrol.

Description

Field of the invention

The invention relates to a cell which is capable of forming resveratrol, wherein the cell has at least one reduced activity of an enzyme E ₁ , which is involved in a cataplerotic pathway related to malonyl-coenzyme A, compared to its wild-type , as well as a process for the preparation of resveratrol.

State of the art

Resveratrol (or 3,4,5-trihydroxystilbene) is a plant-derived secondary metabolite that is of significant commercial interest because of its antioxidant properties and biological activity as an ingredient in cosmetic formulations and dietary supplements.

Resveratrol is a phytophenol belonging to the stilbene phytoalexin family that provides an active defense mechanism in plants in response to infection or other stress events. Stilbene Phytoalexins have the skeleton of stilbene derivatives (trans-1,2-diphenylethylene) as a common basic structure; this can be supplemented by substitution with other groups ( Hart, JH (1981) Annu. Rev. Phytopathology 19, 437-458 and Hart, JH, Shrimpton, DM (1979) Phytopathology 69, 1138-1143 ).

Stilbenes have been detected in certain trees (covered and naked), but also in some herbs (in species of the Myrtaceae, Vitaceae and Leguminosae families). Stilbenes are toxic to pests, especially fungi, bacteria and insects. However, only a few plants are able to synthesize stilbenes in concentrations that confer sufficient resistance to pests.

Nevertheless, resveratrol is currently only obtained by extractive methods from plants, especially Polygonum cuspidatum and Vitis vinifera.

Disadvantage of the extractive process is the low yield at high solvent and plant levels.

Alternative methods of preparation of resveratrol have been described both in the form of chemical syntheses (e.g. US 6552213 . WO 2005023740 . US 7235324 ) as well as biotechnological processes in microorganisms ( WO 06089898 . WO 06124999 ).

The microorganisms used in biochemical processes are usually recombinant, since the biosynthetic path to resveratrol is merely realized in some plants as already mentioned above and this metabolic pathway has to be transferred into microorganisms.

Resveratrol is synthesized a.) By three or b.) Four enzymes. For the enzyme combination a.) Resveratrol synthesis takes place from the metabolites tyrosine and malonyl-coenzyme A (malonyl-CoA), which occur in all microorganisms instead. The three enzymes are i) tyrosine ammonium lyase (TAL), which converts tyrosine into 4-coumaric acid by deamination, ii) 4-coumaryl CoA ligase, which catalyzes the activation of coumaric acid to 4-coumaryl CoA, and finally, iii) the stilbene synthase (resveratrol synthase, EC: 2.3.1.95), which condenses three molecules of malonyl-CoA with one molecule of 4-coumaryl-CoA to the resveratrol in three steps. For the enzyme combination b.) Resveratrol synthesis is synthesized by four enzymes from the metabolites phenylalanine and malonyl-CoA, which are found in all microorganisms. The four enzymes are i) phenylalanine ammonium lyase (EC: 4.3.1.5) (PAL), which converts phenylalanine into trans-cinnamic acid by deamination, ii) cinnamate-4-hydroxylase (EC: 1.14.13.11), which the hydroxylation of trans-cinnamate to 4-coumarate catalyzes iii) 4-coumaryl CoA ligase, which catalyzes the activation of coumaric acid to 4-coumaryl CoA, and finally, iii) the stilbene synthase (resveratrol synthase, EC : 2.3.1.95), which condenses three molecules of malonyl-CoA with one molecule of 4-coumaryl-CoA into resveratrol in three steps.

If coumaric acid is fed as a precursor molecule, it is also possible to produce resveratrol by biotechnological means only using the last two enzymes, 4-coumaryl-CoA ligase and stilbene synthase.

Functional expression of two of the enzymes mentioned (4-coumaryl-CoA ligase and stilbene synthase) or of the entire metabolic pathway has been described both in bacteria ( Beekwilder et al., 2006 ; Watts et al., 2006 . Katsuyama et al., 2007 . WO 05084305 ) as well as yeasts ( Becker et al., 2003 ; Beekwilder et al., 2006 . WO 06089898 ) already shown. It was possible to prepare resveratrol either from the precursor molecule coumaric acid and glucose (as carbon and energy source for the production of malonyl-CoA as a second precursor molecule) or else exclusively from glucose.

WO 06124999 describes a method to increase the intracellular availability of malonyl-CoA using an acetyl-CoA carboxylase (synthesis of malonyl-CoA by carboxylation of acetyl-CoA) or a malonyl-CoA synthetase (synthesis of malonyl-CoA from malonate and CoA) is expressed recombinantly. In addition to malonyl-CoA synthetase, malonate (substrate of malonyl-CoA synthetase) is designed to co-express a malonate transporter that promotes the transport of extracellular malonate across the plasma membrane into the cell. This approach has the great disadvantage that heterologous expression of complex enzyme systems such as acetyl-CoA carboxylase and the combination of malonate transporter and malonyl-CoA synthetase is not trivial.

The acetyl-CoA carboxylase from E. coli and other Gram-negative bacteria is a heterotetramer, and the stoichiometry of the subunits is very important for both the activity and growth of the expression or production host. Additional load on the cell arises from heterologous recombinant protein expression.

With regard to the combination of malonate transporter and malonyl-CoA synthetase, in particular the expression and functionality of the malonate transporter is problematic since it is a transmembrane protein, as well as the added burden of heterologous recombinant protein expression.

Disadvantage of all biotechnological methods described above is that despite massive overproduction of the resveratrol synthesizing enzymes and other auxiliary enzymes, the product could be achieved only in a concentration of 100 mg / l based on the culture broth. This is very far removed from concentrations that make biotechnological production meaningful.

The object of the invention was therefore to provide a method which overcomes at least one of the described disadvantages of the prior art.

Description of the invention

Surprisingly, it has been found that the cells described below according to claim 1 solve the object underlying the invention.

The present invention therefore relates to cells as described in claim 1, a process for the preparation of resveratrol using the cells according to the invention as well as the resveratrol prepared with the cells according to the invention or the process according to the invention.

The advantage of the cells according to the invention is a considerably simplified handling, since no additional enzymes synthesizing malonyl-CoA have to be prepared in the production strain. In the method according to the invention, unlike additionally introduced genes, no selection pressure has to be exerted on the culture.

Another advantage of certain embodiments of inventive cells or methods is that the desired effect can be induced; such as in the case of the use of specific inhibitors.

Yet another advantage of certain embodiments of cells or methods according to the invention is that the desired effect can both be induced and set off again and thus be completely time-controlled; such as in the case of the use of strains that have temperature-sensitive malonyl-CoA as substrate accepting enzymes.

An object of the present invention is therefore a cell which is capable of forming resveratrol, which cell has at least one reduced activity of an enzyme E ₁ , which is involved in malonyl-coenzyme A cataplerotic pathway, compared to its wild-type.

Another object of the invention is a process for the preparation of resveratrol.

By the term "resveratrol" is meant not only the compound cis- / trans-3,4,5-trihydroxystilbene, but also oligomers of resveratrol such as trans-ε-viniferin, Gnetin H, suffructicosol B, upunaphenol B / C / D / E, or glycosylated forms such as Piceid / Polydatin, resveratrol based prodrugs (eg RT-A) or mixtures thereof understood.

By the term "reduced activity of an enzyme" is preferably used a factor of at least 0.5, more preferably at least 0.1, more preferably at least 0.01, even more preferably at least 0.001 and most preferably understood as having at least 0.0001 reduced activity. The reduction of the activity of a particular enzyme can be carried out, for example, by targeted or undirected mutation, by the addition of competitive or non-competitive inhibitors or by other measures known to those skilled in the art for reducing the synthesis of a particular enzyme.

The term "a malonyl-coenzyme A cataplerotic pathway" as used herein refers to the naturally occurring metabolic pathways in a cell that require malonyl-CoA to drain and consume it. As an example, fatty acid biosynthesis may be mentioned here.

In this context, the term "directly cataplerotic" is understood to mean that an enzyme directly converts malonyl-CoA, where the term "indirectly cataplerotic" refers to an enzyme involved in the malonyl-coenzyme A catabolic pathway , but not directly malonyl-CoA but implements its derivatives.

The term "activity of an enzyme which participates in a metabolic pathway" refers to an enzyme activity which has a direct influence on the genesis and / or formation rate of the metabolic pathway product.

"CoA" is used synonymously for "coenzyme A" below.

In the following, "ACP" is used synonymously for "acyl carrier protein".

All percentages (%) are percentages by weight unless otherwise specified.

A preferred cell according to the invention is characterized in that it has been genetically engineered such that it has at least one reduced activity of an enzyme E ₁ compared to its wild-type.

A cell which is preferred according to the invention has an activity which is reduced in comparison to its wild-type, of at least one of the enzymes E ₁ which is selected from the group comprising, preferably consisting of:
an enzyme E _1a , which is the reaction
catalysed by malonyl-CoA to malonic acid semialdehyde;
an enzyme E _1b , which is the reaction
catalysed by malonyl-CoA to acetyl-CoA;
an enzyme E _1c , which is the reaction
of holo- [ACP] + malonyl-CoA catalyses malonyl- [ACP] + coenzyme A;
an enzyme E _{1d having} polyketide synthase activity; (Polyketide synthases (PKSs) produce secondary metabolites and are found in the genome of a variety of organisms, such as bacteria, fungi, marine organisms, and plants Polyketide synthases are grouped into 3 classes: Type I, II, and III, Type I PKSs are large multidomain enzymes, type II PKSs have many single-function subunits, and type III PKSs consist of only one protein, most commonly arranged as a homodimer, using as substrate both acetyl-CoA and malonyl-CoA);
an enzyme E _1e , which is the reaction
from acetyl-CoA + malonyl- [ACP] to acetoacetyl- [ACP] and / or
of holo- [ACP] + acetyl-CoA catalyzes to acetyl- [ACP];
an enzyme E _1f , which is the reaction
from trans -Δ ³ -cis-Δ ⁵ -dodecenoyl- [ACP] to cis-Δ ⁵ -dodecenoyl- [ACP] and / or
from 2,3,4-saturated acyl- [ACP] to trans-Δ ² -enoyl-acyl- [ACP] catalyzed;
an enzyme E _1g , which is the reaction
from octanoyl- [ACP] + malonyl- [ACP] to 3-oxo-decanoyl- [ACP] + holo- [ACP] and / or
of decanoyl- [ACP] + malonyl- [ACP] to 3-oxo-dodecanoyl- [ACP] + holo- [ACP] and / or
from dodecanoyl- [ACP] + malonyl- [ACP] to 3-oxo-meristoyl- [ACP] + holo- [ACP] and / or
from myristoyl [ACP] + malonyl- [ACP] to 3-oxo-palmitoyl- [ACP] + holo- [ACP] and / or
of butyryl [ACP] + malonyl [ACP] to 3-oxo-hexanoyl- [ACP] + holo- [ACP] and / or
of hexanoyl- [ACP] + malonyl- [ACP] to 3-oxo-octanoyl- [ACP] + holo- [ACP] and / or
of cis-Δ ³ -deenoyl- [ACP] + malonyl- [ACP] to β-keto-cis-Δ ⁵ -dodecenoyl- [ACP] + holo- [ACP] and / or
of malonyl [ACP] + acetyl- [ACP] to holo- [ACP] + acetoacetyl- [ACP] and / or
from malonyl [ACP] + 2,3,4-saturated acyl- [ACP] to holo- [ACP] + β-ketoacyl- [ACP] and / or
catalyzed by malonyl [ACP] to acetyl [ACP] and / or by palmitoleoyl [ACP] + malonyl
[ACP] to 3-oxo-cis-vaccenoyl- [ACP] + holo- [ACP] and / or
of malonyl [ACP] + acetyl- [ACP] to holo- [ACP] + acetoacetyl- [ACP] and / or
of malonyl [ACP] + 2,3,4-saturated acyl- [ACP] to holo- [ACP] + β-ketoacyl [ACP] catalyzed;
an enzyme E _1h , which is the reaction
from β-keto-cis-Δ ⁵ -dodecenoyl- [ACP] to β-hydroxy cis Δ ⁵ -dodecenoyl- [ACP] and / or
catalysed by (3R) -3-hydroxyacyl [ACP] to β-ketoacyl [ACP];
an enzyme E _1i , which is the reaction
from β-hydroxy cis Δ ⁵ -dodecenoyl- [ACP] to trans-Δ ³ -cis-Δ ⁵ -dodecenoyl- [ACP] and / or
from (R) -3-hydroxydecanoyl- [ACP] to trans-Δ ² -decenoyl- [ACP] and / or
from (3R) -3-hydroxyacyl- [ACP] to trans-Δ ² -enoyl-acyl- [ACP] and / or
from trans-Δ ² -decenoyl- [ACP] to cis-Δ ³ -deenoyl- [ACP] catalyzed;
an enzyme E _1j , which is the reaction of
apo- [ACP] catalyzes adenosine 3 ', 5'-ice phosphate + holo- [ACP];
an enzyme E _{1k having} fatty acid synthase activity;
an enzyme E _1j , which is the reaction
catalysed by pantothenate + ATP to D-4'-phosphopantothenate + ADP;
an enzyme E _1m , which is the reaction of
4'-phosphopantetheine + ATP catalyses dephospho-CoA + diphosphate;
an enzyme E _1n , which is the reaction of
Dephospho-CoA + ATP catalyzed to coenzyme A + ADP and
an enzyme E _1o , which is the reaction of
D-4'-phosphopantothenate + L-cysteine + CTP to diphosphate + CMP + R-4'-phosphopantothenoyl-L-cysteine and / or
R-4'-phosphopantothenoyl-L-cysteine catalyzed to 4'-phosphopantetheine.

Particularly preferred in this context are cells in which the activity of the abovementioned enzymes is reduced in the following combinations: E _1a , E _1b , E _1c , E _1d , E _1e , E _1f , E _1g , E _1h , E _1i , E _1j , E _1k , E _1l , E _1m , E _1n , E _1o ,

E _1a E _1b E _1c E _1d E _1j E _1f , E _1a E _1b E _1c E _1j E _1f , E _1a E _1c E _1d E _1j E _1f , E _1a E _1b E _1d E _1j E _1f , E _1a E _1b E _1j E _1f , E _1a E _1c E _1j E _1f , E _1a E _1d E _1j E _1f , E _1b E _1c E _1d E _1j E _1f , E _1b E _1c E _1j E _1f , E _1b E _1d E _1j E _1f , E _1c E _1d E _1j E _1f , E _1a E _1j E _1f , E _1b E _1j E _1f , E _1c E _1j E _1f , E _1d E _1j E _1f ,

E _1a E _1b E _1c E _1d E _1f , E _1a E _1b E _1c E _1f , E _1a E _1c E _1d E _1f , E _1a E _1b E _1d E _1f , E _1a E _1b E _1f , E _1a E _1c E _1f , E _1a E _1d E _1f , E _1b E _1c E _1d E _1f , E _1b E _1c E _1f , E _1b E _1d E _1f , E _1c E _1d E _1f , E _1a E _1f , E _1b E _1f , E _1c E _1f , E _1d E _1f , E _1a E _1b E _1c E _1d E _1j , E _1a E _1b E _1c E _1j , E _1a E _1c E _1d E _1j , E _1a E _1b E _1d E _1j , E _1a E _1b E _1j , E _1a E _1c E _1j , E _1a E _1d E _1j , E _1b E _1c E _1d E _1j , E _1b E _1c E _1j , E _1b E _1d E _1j , E _1c E _1d E _1j , E _1a E _1j , E _1b E _1j , E _1c E _1j , E _1d E _1j ,

E _1a E _1b E _1c E _1d , E _1a E _1b E _1c , E _1a E _1c E _1d , E _1a E _1b E _1d , E _1a E _1b , E _1a E _1c , E _1a E _1d , E _1b E _1c E _1d , E _1b E _1c , E _1b E _1d , E _1c E _1d , in particular E _1a , E _1b , E _1c E _1d
and most preferably E _1c and E _1f .

In this context, it is further preferred that the enzyme
E _{1a is} a malonate semialdehyde dehydrogenase (EC: 1.2.1.15 or EC: 1.2.1.18), for example known and well characterized from L. casei, P. aeruginosa, P. fluorescens and C. aurantiacus,
E _{1b represents} a malonyl-CoA decarboxylase (EC: 4.1.1.9), for example known and well characterized from P. fluorescens, P. putida, Rhizobium leguminosarum and S. cerevisiae,
E _{1c represents} an [acyl carrier protein] S malonyltransferase (EC: 2.3.1.39), for example, known and well characterized from E. coli (FabD), M. tuberculosis (FabD2), H. pylori, P. aeruginosa,
E _{1e represents} an [acyl carrier protein] -S-acetyltransferase (EC: 2.3.1.38) or a [myelin proteolipid] -O-palmitoyltransferase (EC: 2.3.1.100), for example, known and well characterized from E. coli (FabH), E. gracilis and S. oleracea,
E _{1f is} an enoyl [acyl carrier protein] reductase (EC: 1.3.1.9), for example, known and well characterized from E. coli (Fabl), M. tuberculosis, B. anthracis, S. aureus, S pneumoniae, H. pylori, A. thaliana, E. tenella and B. subtilis,
E _{1g is} a beta-ketoacyl-acyl-carrier-protein synthase I or II (EC: 2.3.1.41/179), for example known and well characterized from E. coli (FabB / F), S. aureus, S. oleracea, L. lactis, S. pneumoniae, M. tuberculosis and A. thaliana,
E _{1h is} a 3-oxoacyl [acyl carrier protein] reductase (EC: 1.1.1.100), for example, known and well characterized from E. coli (FabG), P. putida, M. tuberculosis, P. aeruginosa , S. pneumoniae and L. lactis,
E _{1i is} a 3-hydroxydecanoyl [acyl carrier protein] dehydratase or 3-hydroxyoctanoyl [acyl carrier protein] dehydratase (EC: 4.2.1.60 or EC: 4.2.1.59) or a crotonoyl [acyl] Carrier protein] hydratase (EC: 4.2.1.58) or a trans-2-decenoyl [acyl carrier protein] isomerase (EC: 5.3.3.14), for example, is known and well characterized from E. coli ( FabA / Z),
E _{1j represents} a holo [acyl carrier protein] synthase (EC: 2.7.8.7), for example, known and well characterized from E. coli (Acps), B.subtilis, S. aureus, P. aeruginosa, and S. cerevisiae,
E _{1k represents} a fatty acid synthase or fatty acid-acyl-CoA synthase (EC: 2.3.1.85 or EC: 2.3.1.86),
E _{1l represents} a pantothenate kinase (EC: 2.7.1.33), for example known and well characterized from E. coli (CoaA), S. aureus, B. anthracis, B. subtilis, M. tuberculosis, P. aeruginosa, S. oleracea, H. pylori and A. thaliana,
E _{1m is} a panthenol-phosphate adenylyltransferase (EC: 2.7.7.3), for example known and well characterized from E. coli (CoaD), T. thermophilus, M. tuberculosis, H. pylori and A. thaliana,
E _{1n is} a dephospho-CoA kinase (EC: 2.7.1.24), for example, known and well characterized from E. coli (CoaE) and T. thermophilus, and
E _{1o is} a phosphopantothenoylcysteine decarboxylase (EC: 4.1.1.36), for example known and well characterized from E. coli (CoaBC), M. tuberculosis and A. thaliana, or a phosphopantothenoylcysteine ligase (6.3.2.5).

The term "EC:" here stands for the Enzyme Commission Number according to the systematic nomenclature of the Enzyme Commission of the International Union of Biochemistry (IUB) and thus designates the affiliation of the enzyme concerned to the corresponding class of enzymes.

It is straightforward for one skilled in the art having the above information and current knowledge, especially with regard to bioinformatics, to identify the gene encoding a considered enzyme in the cell to be manipulated and to reduce its activity by the genetic engineering methods described below

Preferred cells for the purposes of the present invention are microorganisms, in particular those selected from the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Hansenula, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia and Clostridium, wherein Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha, Ralstonia eutropha H16, Schizosaccharomyces pombe, Pichia pastoris and Pichia ciferrii are particularly preferred, especially E. coli.

Since for the synthesis of resveratrol also malonyl-CoA as a substrate accepting enzymes are needed, their activity should be as low as possible, preferably not at all diminished in the cells according to the invention; Therefore, it is preferred according to the invention that an enzyme which catalyzes the conversion of 4-coumaryl coenzyme A to resveratrol and is also described below as enzyme E ₅ is excluded as enzyme E ₁ .

This is relevant, for example, when inhibitors are used which highly specifically inhibit one or more of the enzymes E _1a to E _1o , but nevertheless exert an undesired, slight inhibitory side effect on the activity of an enzyme E ₅ .

To reduce the activity of an enzyme E ₁ , all inhibitors known to those skilled in the art which inhibit enzymes which are involved in a cataplerotic pathway related to malonyl-coenzyme A are suitable.

Exemplary representatives of suitable inhibitors and the enzyme activities to be reduced therewith are, for example Zhang et. al. (2006) Inhibiting bacterial fatty acid synthesis JBC 281 (26) pp. 17,541 to 17,544 . Wright, H.T., and Reynolds, KA Antibacterial Targets in Fatty Acid Biosynthesis Current Op. Microbiol., 10, 447-453, 2007 and the table below. enzyme Inhibitor (s) AcpS Sch 538415 4H-oxazole-5-one anthranilic acid FabD Corytuberin FabH HR12, RWJ-3981 indole analogs, SB418001,) 1,2-dithiols-3-one benzoylaminobenzoic acids FabH / F Platensimycin, platencin phomallic acid FabB / F Cerulenin thiolactomycin BABX phomaleric acids FabG polyphenols FabA Allenic acids 3-decynoyl-NAC FabZ NAS-91, NAS-21 Fab Isoniazid triclosan diazoborin aminopyridine FASII Bishloroanthrabenzoxocinone, BABX FabK aminopyridine CoaA Pantothenamide Coad specifically selected dipeptides

Likewise suitable are the inhibitors listed in the following table, which are described in detail in Georgopapadakou et al. (1986) Effect of Antifungal Agents on Lipid Biosynthesis and Membrane Integrity in Candida albicans ACC 31 (1) pp. 46-51 ) to be discribed. inhibitor Inhibitory effect on: clotrimazole Fatty acid biosynthesis / ergosterol biosynthesis econazole Fatty acid biosynthesis / ergosterol biosynthesis ketoconazole Fatty acid biosynthesis / ergosterol biosynthesis miconazole Fatty acid biosynthesis / ergosterol biosynthesis naftifine Fatty acid biosynthesis / ergosterol biosynthesis tolnaftate Fatty acid biosynthesis / ergosterol biosynthesis Azaterol Fatty acid biosynthesis / ergosterol biosynthesis cerulenin Fatty acid biosynthesis / ergosterol biosynthesis

Another suitable inhibitor is, for example for FabD, the 3-HMG-CoA, whose use in Kizer et. al. (2008) Application of functional genomics to pathway optimization for increased isoprenoid production. Applied and Environmental Microbiology 74 (10) pp. 3229-3241 is described in detail.

In order to reduce the activity of an enzyme E ₁ by genetic manipulation, targeted mutations, for example directed knockouts, in which, for example, promoter deletes active sequences of the target gene, active (necessary for the catalytic function of the enzyme) regions of the target gene or the entire target gene, with foreign DNA substituted or disrupted with foreign DNA.

Furthermore, various techniques of gene silencing are known to those skilled in the art, which specifically reduce or even completely prevent the transcription or translation of individual genes via antisense nucleic acids or small-interfering RNA (siRNA). Depending on the chosen system, this suppression may be transient, inducible or constitutive.

Likewise, those skilled in methods are known with which one can selectively set individual point mutations in the target gene.

Particularly preferred are cells in which the activity of the enzyme E ₁ can be reduced in a conditioned manner.

In particular, cells in which the activity of the enzyme E ₁ can be conditioned by temperature changes have been found to be advantageous according to the invention.

The expert will find instructions for influencing protein properties by temperature changes in the following literature, the teaching of which can be transferred to the influence of the activity of the enzyme E ₁ :
In Wintrode et al. (2000) Cold Adaptation of a mesophilic subtilisin-like protease by laboratory evolution, J. Bio. Chem. 275 (41), pp. 31635-31640 ), the mesophilic serine protease (SSII) from Bacillus sphaericus was transformed into its psychrophile counterpart by direct evolution. The half-life of the SSII at 70 ° C could be reduced by 3.3 times compared to the wild type.

In Merz et al. (2000) Improving the catalytic activity of a thermophilic enzyme at low temperatures, Biochemistry 39, pp. 880-889 The gene trpC from the hyperthermophilic organism Sulfolobus solfataricus, which codes for an indoleglycerol phosphate synthase (IGPS), was mutagenized by DNA shuffling. In this way, it was possible to produce IGPS variants which were distinguished at 37 ° C. by an increased catalytic activity compared with the parent enzyme.

In Lebbink et al. (2000) Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus β-glucosidase CelB by directed evolution, Biochemistry 39 pp. 3656-3665 The activity of thermostable β-glucosidase (CelB) from the hyperthermophilic Pyrococcus furiosus organism was increased 3-fold at low temperatures.

In Reetz et al. (2008) Knowledge-guided laboratory evolution of protein thermolability, Biotechnology & Bioengineering 102 (6), pp. 1712-1717 ) introduced the so-called B-factor iterative test (B-FIT) for the generation of thermostable enzymes.

By means of B-FIT, an increase in the thermolability of the lipase from Pseudomonas aeruginosa could be achieved. In comparison to the wild-type strain, the thermal stability of the PAL could be reduced from 71.6 ° C to 35.6 ° C.

In Knobling et al. (1975) Temperature-Sensitive Mutants of the Yeast Fatty Acid Synthetase Complex Eur. J. Biochem. 59 pp 415-421 By genetic complementation analysis, temperature-sensitive fatty acid synthetase yeast mutants (fas1 (ts) - / fasII (ts) mutants) was generated. With the help of these mutants the dependence of the cell growth on the temperature and the supplementation of the medium with fatty acids was shown. All tested mutants were unable to grow on medium without fatty acid supplementation at non-permissive temperatures (34-37 ° C). In contrast, these mutants showed an identical growth rate in the presence of fatty acids as the yeast-type cells (Saccharomyces cerevisiae,). The wildtype cells also grew fatty acid-independent at all temperatures tested. It has been observed that the growth of the temperature-sensitive mutants in fatty acid-free medium at 5 to 10 ° C lower temperature compared to the wild-type cells or compared to the growth on fatty acid-supplemented medium stopped. Overall, growth on fatty acid-free medium at 37 ° C for all tested mutants comes to a standstill, whereas this temperature on medium supplemented with fatty acids had no effect on the growth of the ts mutants. investigations with the isolated fatty acid synthetase complex (fas) revealed that the fas activity at non-permissive temperature was either completely lost or greatly reduced.

In C. Rieck (2006) Genetic and biochemical characterization of the fatty acid elongase in the yeast Saccharomyces cerevisiae (dissertation) A mutant FasII (ts) was generated by EMS mutagenesis, which was plated on solid medium with and without exogenous fatty acids and grown at 22 ° C and 35 ° C, respectively. It was found that the fasII (ts) mutant could grow equally well at low (permissive) temperature on both media. In contrast, the temperature-sensitive fasII mutant at non-permissive temperatures (35 ° C) was no longer able to grow on fatty acid-free medium. On the other hand, there were no limitations in the growth of the mutant at 35 ° C on fatty acid-containing YEPES medium.

In accordance with the invention preferred and already described cells in which the activity of the enzyme E ₁ can be reduced conditioned by temperature changes include in particular:
E. coli JP1111 containing a temperature-sensitive FabI and described in Egan et al. (1973) Conditional mutations affecting the cell envelope of Escherichia coli K-12. Genet. Res. 21: 139-152 ;
E. coli L48 and E. coli LA2-89 containing temperature sensitive FabD and described in Harder et al. (1972) Temperature-sensitive mutants of Escherichia coli required saturated and unsaturated fatty acids for growth: isolation and properties. Proc. Natl. Acad. Sci. USA 69: 310 , as in Semple et al. (1975) Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 121 (3): 1036-46 , as in Verwoert et al. (1994) Molecular characterization of Escherichia coli mutant with a temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase. FEBS Letters, 348 (3), 311-16 .
E. coli DV62, E. coli DV79 and E. coli ts9, containing temperature-sensitive CoaA and described in U.S. Pat Flaks et al. (1966) Mutations and genetics concerned with the ribosomes. Cold Spr. Harb. Symp. Quant. Biol. 31: 623-631 , as in Song et al. (1992) coaA and rts are allelic and located at kilobase 3532 on the Escherichia coli physical map. J. Bacteriol. 174: 1705-1706 , as in Vallari at al. (1987) Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli. J. Bacteriol. 169: 5795-5800 , as in Vallari at al. (1988) Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli. J. Bacteriol. 170: 3961-3966 ;
E. coli M5 containing a temperature-sensitive FabB Broekman et al. (1974) Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J. Bacteriol. 117 (3): 971-7. );
E. coli M8, E. coli PAM155, E. coli CY53, E. coli CY55 and E. coli CY50, containing temperature sensitive FabA and described in U.S. Pat Broekman et al. (1973) Growth in high osmotic medium of fatty acid auxotroph of Escherichia coli K-12. J. Bacteriol. 116 (1): 285-9 , as in Broekman et al. (1974) Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J. Bacteriol. 117 (3): 971-7 , as in Johnson et al. (1975) Mapping of sul, the suppressor of ion in Escherichia coli. J. Bacteriol. 122 (2): 570-4 , as in Cronan et al. (1972) Mapping of the fabA locus for fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 112: 206-211 ) and
E. coli CL37, E. coli CL48 and E. coli CL104 containing temperature-sensitive FabG and described in EP-A Lai et al. (2004) Isolation and characterization of .beta.-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar typhimurium. Journal of Bacteriology, 186 (6), 1869-1878 ,

It is easy for the skilled person to isolate the here-listed themolabile enzymes in the form of their genes and to transfer the underlying mechanism on the basis of about the detected point mutations compared to the wild-type gene on other cells. This can be done, for example, in the form that the wild-type gene of the cell to be changed is replaced by the foreign gene coding for the thermolabile enzyme.

Since only a few naturally occurring cells are capable of producing resveratrol, or do so only to a limited extent, it is preferred in accordance with the invention to genetically modify the cell to produce resveratrol, or more, than ever occurring in nature. Therefore, a preferred cell according to the invention is characterized in that the cell is genetically engineered such that it can already form more resveratrol compared to its wild type without the reduced activity of an enzyme E ₁ .

The phrase "that it is able to produce more resveratrol compared to its wild type" also applies to the case where the wild type of the genetically modified cell is unable to form resveratrol, or at least no detectable amounts of these compounds, and detectable only after the genetic modification Quantities of these components can be formed.

A "wild type" of a cell is preferably referred to as a cell whose genome is in a state as naturally evolved. The term is used for both the entire cell and for individual genes. The term "wild-type" therefore does not include, in particular, those cells or genes whose gene sequences have been altered at least in part by humans by means of recombinant methods.

These cells are preferably genetically engineered to produce more resveratrol from a carbon source compared to their wild type. Suitable carbon sources are, for example, sugars, hexoses, pentoses, disaccharides, oligosaccharides, polysaccharides, fats, triglycerides, glycerol, fatty acids, alkanes, methane, Methanol, ethanol, alcohols, carbon dioxide, carbon monoxide, lactate, pyruvate, acetate, propionate, butyrate or organic acids as well as synthesis gas and exhaust gases.

It is preferred in accordance with the invention for the genetically modified cell to be genetically modified in such a way that it is particularly preferred within a defined time interval, preferably within 2 hours, more preferably within 8 hours, and most preferably within 24 hours, at least 2 times at least 10 times, more preferably at least 100 times, more preferably at least 1,000 times, and most preferably at least 10,000 times more resveratrol than the wild type of the cell. The increase in product formation can be determined, for example, by culturing the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then the amount Target product (Resveratrol) is determined in the nutrient medium.

In this connection, it is preferred that the cell has an increased activity of at least one of the following enzymes compared to its wild type:
an enzyme E ₂ which catalyzes the conversion of tyrosine to 4-coumarate or the conversion of L-phenylalanine to trans-cinnamate;
an enzyme E ₃ , which catalyzes the conversion of trans-cinnamate to 4-coumarate;
an enzyme E ₄ which catalyzes the conversion of 4-coumarate to 4-coumaryl coenzyme A;
an enzyme E ₅ , which catalyzes the conversion of 4-coumaryl coenzyme A to resveratrol.

The term "an activity of an enzyme Ex" which is increased in comparison to its wild-type is preferably always a factor of at least 2, more preferably of at least 10, more preferably of at least 100, moreover more preferably at least 1,000 and most preferably at least 10,000 increased activity of the respective enzyme E _x . Furthermore, the cell according to the invention which "has an increased compared to their wild type activity of an enzyme E _x ", in particular a cell whose wild type has no or at least no detectable activity of this enzyme E _x and which only after increasing the enzyme activity, for example by overexpression, a detectable activity of this enzyme E _x shows. In this context, the term "overexpression" or the expression "increase in expression" used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Expression of the enzyme E _{x is} induced.

In this context, particularly preferred cells are those in which the activity of the following enzyme or the following enzymes is increased: E ₂ , E ₃ , E ₄ , E ₅ , E ₂ + E ₄ , E ₂ + E ₅ , E ₄ + e _5, e ₂ + e ₄ + e _5, e ₃ + e _4, e ₃ + e _5, e ₃ + e ₄ + e _5, e ₂ + e ₃ + e ₄ + e 5, wherein e ₄ + E _{5 is} particularly preferred.

Cells in which the activity of one or more of these enzymes has been increased are, for example, in WO-A-2006/089898 and WO-A-2005/084305 The disclosure content of these publications with regard to recombinant microorganisms in which the activity of one or more of the enzymes E ₂ , E ₃ , E ₄ , E _{5 is} increased is hereby introduced as a reference and forms part of it the disclosure of the present invention.

Furthermore, it is preferred according to the invention that it is
in the enzyme E ₂ to a tyrosine ammonium lyase (EC: 4.3.1.23), in particular from yeast or bacteria, in particular from Rhodotorula rubra or Rhodobacter capsulatus, or to a phenylalanine-ammonium-lyase (EC: 4.3.1.25), especially from plants such as Arabidopsis, Brassica, Citrus, Phaseolus, Pinus, Populus, Solanum, Prunus, Vitus, Zea, Agastache, Pineapple, Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia, Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago, Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea Petroselinum, Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Quercus, Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria, Stylosanthes, Triticum, Trifolium, Triticum, Vaccinium, Vigna, Zinnia, in particular
A. thaliana, B. napus, B. rapa, C. reticulata, C. clementinus, C. limon, P. coccineus, P. vulgaris, P. banksiana, P. monticola, P. pinaster, P. sylvestris, P. Taeda, P. balsamifera, P. deltoides, P. Canadensis, P. kitakamiensis, P. tremuloides, S. tuberosum, P. avium, P. persica, Vitus vinifera, Z. mays
or from fungi such as Agaricus, Aspergillus, Ustilago, in particular A. bisporus, A. oryzae, A. nidulans, A. fumigatus, U. maydis,
or from bacteria such as Rhodobacter, in particular R. capsulatus,
or from yeast such as Rhodotorula, in particular R. rubra.
acting;
in the enzyme E ₃ to a cinnamate-4-hydroxylase (EC: 1.14.13.11) in particular from plants such as Arabidopsis, Citrus, Phaseolus, Pinus, Populus, Solanum, Vitus, Zea, Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine , Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, Vigna, in particular from A. thaliana, C. sinensis, C. paradisi, P. vulgaris, P. taeda, P. deltoides, P. tremuloides, P. trichocarpa, S tuberosum, Vitus vinifera, Z. mays,
or from fungi such as Aspergillus, in particular A. oryzae
acting;
in the case of the enzyme E ₄ , a 4-coumarate-CoA ligase (EC: 6.2.1.12), in particular from plants such as Abies, Arabidopsis, Brassica, Citrus, Larix, Phaseolus, Pinus, Populus, Solanum, Vitus, Zea, Agastache, Amorpha, Cathaya r Cedrus, Crocus, Festuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Pelargonium, Petroselinum, Physcomitrella, Picea, Prunus, Pseudolarix, Pseudotsuga Rosa, Rubus, Ryza, Saccharum, Suaeda, Thielliella, Triticum, Tsuga, in particular A. beshanzuensis, B. firma, B. holophylla, A. thaliana, B. napus, B. rapa, B. oleracea, C. sinensis, L decidua, L. gmelinii, L. griffithiana, L. himalaica, L. kaempferi, L. laricina, L. mastersiana, L. occidentalis, L. potaninii, L. sibirica, L. speciosa, P. acutifolius, P. coccineus , P. armandii P. banksiana, P. pinaster, P. balsamifera, P. tomentosa, P. tremuloides, S. tuberosum, Vitus vinifera or
from a fungus such as Aspergillus, Neurospora, Yarrowia, Mycosphaerella, especially A. flavus, A. nidulans, A. oryzae, A. fumigatus, N. crassa, Y. lipolytics, M. graminicola,
from a bacterium such as Mycobacterium, Neisseria, Streptomyces, Rhodobacter, in particular M. bovis, M. leprae, M. tuberculosis, N. meningitidis, S. coelicolor, R. capsulatus or
from a nematode such as Ancylostoma, Caenorhabditis, Haemonchus, Lumbricus, Meilodogyne, Strongyloidus, Pristionchus, in particular A. ceylanicum, C. elegans, H. contortus, L. rubellus, M. hapla, S. rattii, S. stercoralis, P. pacificus most preferably one encoded by the At4Cl1 gene from Arabidopsis thaliana;
in the case of the enzyme E ₅ , a stilbene synthase, preferably a resveratrol synthase (EC: 2.3.1.95),
especially from plants such as Arachis, Rheum, Vitus, Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus, Cissus, Calochortus, Polygonum, Gnetum, Artocarpus, Nothofagus, Phoenix, Festuca, Carex, Veratrum, Bauhinia, Pterolobiumm, in particular A. glabatra, A Hypogaea, R. tataricum, V. labrusca, V. riparaia, V. vinifera,
most preferably one encoded by the gene VvSTS of Vinis vinifera.

It may be advantageous for the cell according to the invention to increase further enzyme activities, as described in the already cited documents WO-A-2006/089898 and WO-A-2005/084305 for example, that of a malonate transporter protein.

• I) In Kontakt Bringen der vorstehend beschriebenen, erfindungsgemäßen Zelle mit einem Nährmedium beinhaltend mindestens eine Kohlenstoffquelle
• II) Kultivieren der Zelle unter Bedingungen, die es der Zelle ermöglichen, aus der Kohlenstoffquelle und oder Vorläufermolekülen von Resveratrol wie Coumarsäure und/oder Zimtsäure Resveratrol zu bilden und gegebenenfalls
• III) Isolierung des gebildeten Resveratrols.

A contribution to the solution of the abovementioned objects is also made by a process for producing resveratrol, comprising the process steps:

• I) contacting the cell of the invention described above with a nutrient medium comprising at least one carbon source
• II) culturing the cell under conditions that allow the cell to form resveratrol from the carbon source and or precursor molecules of resveratrol such as coumaric acid and / or cinnamic acid, and optionally
• III) Isolation of Resveratrol formed.

It is apparent that in the method according to the invention the cells which correspond to the preferred embodiments of the cells according to the invention are preferably used.

Suitable carbon sources for process step I) are, for example, sugars, hexoses, pentoses, disaccharides, oligosaccharides, polysaccharides, fats, triglycerides, glycerol, fatty acids, alkanes, methane, Methanol, ethanol, alcohols, carbon dioxide, carbon monoxide, lactate, pyruvate, acetate, propionate, butyrate or organic acids as well as synthesis gas and exhaust gases.

Cultivation in process step II) comprises both the anaerobic and the aerobic cultivation of the cells; The cells may be under these conditions in growth (existing cell division), but it can also be used so-called "resting cells", which are mitigated or inhibited, for example, by limiting a substance in the nutrient medium.

It is inventively preferred that in step II) a cultivation in two steps IIa) and IIb), which is characterized in that in the first step IIa) cells of the invention are attracted to high cell densities, without the activity of an enzyme E ₁ reduced and in the second step IIb) the reduction of the activity of the enzyme E _{1 is} induced.

By "high cell density" in this context is meant a cell density which corresponds to between 5 and 200 g, preferably between 10 and 200, particularly preferably between 10 and 100 g of cell dry matter per liter of total culture.

According to a further, special embodiment of the method according to the invention, this method additionally includes the process step III) isolation of the resveratrol formed.

In step III) of the process according to the invention, the resveratrol formed by the cells can optionally be isolated from the cells and / or the nutrient medium, all methods known to those skilled in the art for isolating low molecular weight substances from complex compositions being suitable for isolation. By way of example, the precipitation by means of suitable solvents, the extraction by means of suitable solvents, the complexation, for example by means of cyclodextrins or cyclodextrin derivatives, crystallization, purification or isolation by means of chromatographic methods or the conversion of the formed resveratrol into readily separable derivatives may be mentioned at this point ,

A preferred method for isolating the resveratrol is a back-extraction with water from an organic solvent, with which first the resveratrol was extracted from the culture medium; this procedure is in the DE 10 2009 054 984 described in detail.

The resveratrol, after extraction with an organic solvent such as ethyl acetate, can also be isolated by common methods such as evaporation crystallization. Another way to isolate resveratrol from an organic solvent, in which the resveratrol was extracted from the culture medium, is the reduction of the water content in the corresponding solvent. Removing the water lowers the solubility limit of the resveratrol and initiates crystallization. This procedure is in DE 10 2010 028 874 described in detail.

Another method for isolating the Resveratrol is exceeding the solubility limit in the medium, thereby the resveratrol crystallized in the medium and can be separated by conventional separation methods. This is in the patent application WO 2009/016108 described.

In process step III) of the process according to the invention, an isolation of the resveratrol is preferably carried out which corresponds to that in the DE 10 2009 054 984 corresponds to described method.

• C) Abtrennen der wässrigen Phase von dem mit Wasser im Wesentlichen nicht mischbaren Lösungsmittel und gegebenenfalls
• D) Isolierung des Stilbenoids aus der wässrigen Phase.

Thus, a preferred method step III) comprises steps A) extracting the resveratrol from the cells and / or the nutrient medium with at least one water-immiscible solvent, B) contacting the at least one water-immiscible solvent with a aqueous phase,

• C) separating the aqueous phase from the water-immiscible solvent and optionally
• D) Isolation of stilbenoid from the aqueous phase.

In this context, the solvent is preferably an organic solvent, in particular selected from the group consisting of methyl isobutyl ketone, 3-pentanone, ethyl butyrate, myristyl alcohol polypropoxylate, diethyl adipate, ocyl acetate, ethyl acetate, 2-heptanone, 1-octanol, tritolyl phosphate, triacetin, oleyl alcohol, nonanol, Dodecane, dibutyl adipate, diethyl sebacate, polypropylene glycol 1000 to 4000, 2-octanone and 2-undecanone. The aqueous phase preferably contains at least 50% by weight of water, based on the total weight of the aqueous phase; Preferably, it has a pH of greater than 7. The aqueous phase preferably contains alkali metal and / or alkaline earth metal hydroxides in a concentration of 0.1 mol / L to 20 mol / L, based on the total aqueous phase. Preferably, in process step C), the separation of the aqueous phase by the use of separators, centrifuges, decanters and separators. More preferably, in step D) the isolation of the stilbenoid is carried out by evaporation of the solution surrounding the stilbenoid. Preferably, process step D) comprises a step in which the pH of the aqueous phase is adjusted to less than 7, wherein preferably in process step D) the temperature of the aqueous phase is lowered, based on the temperature at the beginning of process step III).

In the examples given below, the present invention is described by way of example, without the invention, the scope of application of which is apparent from the entire description and the claims, to be limited to the embodiments mentioned in the examples.

The following figures are part of the examples:

1 Resveratrol production of E. coli JP1111 and L48-pUC-GAP-VvSTS-At4Cl1 at 30 ° C and 37 ° C.

2 : Resveratrol production based on the biomass (OD ₆₀₀ ) of E. coli JP1111 and L48-pUC-GAP-VvSTS-At4Cl1 at 30 ° C and 37 ° C

3 Resveratrol production and OD ₆₀₀ of E. coli JP1111-pUC-GAP-VvSTS-At4Cl1 at 30 ° C and 37 ° C.

4 Resveratrol production and OD ₆₀₀ of E. coli L48-pUC-GAP-VvSTS-At4Cl1 at 30 ° C and 37 ° C.

5 Resveratrol production and OD ₆₀₀ of E. coli BW27784-pUC-GAP-VvSTS-At4Cl1 (reference) at 30 ° C and 37 ° C.

6 : Increase in resveratrol per OD at a temperature increase from 30 ° C to 37 ° C.

Examples:

Example 1: Cloning of a Vector for Expressing the Coumaryl-CoA Ligase and the Stilbene Synthesis-Coding Genes in Escherichia coli

Amplification of DNA by means of PCR (Polymerase Chain Reaction), restriction digestion, DNA purification, ligation and transformation of chemically competent E. coli cells were carried out in a manner known to the person skilled in the art. All required enzymes were purchased from New England Biolabs, Schwalbach. The synthesis of oligonucleotides was carried out by MWG Biotech, Ebersberg.

First, the lac promoter containing the LacI operator was replaced in the commercially available vector pUC19 (New England Biolabs, Schwalbach) by recloning a synthetic fragment of the lac promoter without operator by analogy Watts et al., "Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli" (BMC Biotechnology 2006, 6:22). , The vector pUC19 (2686 bp) was cut with PvuII and NdeI according to the manufacturer's instructions (New England Biolabs, Schwalbach).

The synthetic fragment of the lac promoter without operator (SEQ ID NO: 1) was cut in the same manner as the modified lac promoter, and the resulting fragment (212bp; SEQ ID NO: 1) after purification on an agarose gel using QIAGEN QlAquick Gel Extraction Kit (Qiagen, Hilden) with the remaining, similarly purified vector backbone of pUC19 (2240 bp) ligated (T4 DNA ligase, New England Biolabs, Schwalbach), resulting in the vector pUCmodII (2452bp) resulted. Ligation and transformation of competent Escherichia coli DH5a were carried out according to the specifications of the manufacturer (New England Biolabs, Schwalbach).

The vector pUCmodII was cut in the next step with XbaI and NotI. Starting from the genomic DNA was determined by PCR using Phusion DNA Polymerase (New England Biolabs, Schwalbach) and the oligonucleotides

VvSTScod-Xba1-fw, SEQ ID NO: 2
5'-tatatatctagaaggaggattacaaaATGGCTTCAGTCGA
GGAAATTAG-3 '

VvSTScod-Not1-rv, SEQ ID NO: 3
5'-tatataGCGGCCGCtttaatttgtaaccatagg-3 '

the gene of stilbene synthase from Vinis vinifera (VvSTS) amplified according to the manufacturer's instructions, resulting in a 1201bp fragment (SEQ ID NO: 4) resulted. This was purified on an agarose gel using QIAGEN QIAquick Gel Extraction Kit (Qiagen, Hilden). The PCR product was then cut analogously to the vector pUCmodII with XbaI and NotI and both fragments were ligated, resulting in the vector pUCmod-VvSTS (3625 bp). The Arabidopsis thaliana Coumaryl CoA ligase At4Cl1 gene was obtained from Arabidopsis thaliana cDNA (United States Biological, Swampscott, USA) by PCR and the oligonucleotides

At4Cl1-Not-fw, SEQ ID NO: 5
5'-tatatagcggccgcaggaggattacaaaatgGCGCCACAAGAAC-3 '

At4Cl1-Not, SEQ ID NO: 6
5'-tatatagcggccgctcacaatccatttgctagtttt-3 '

amplified. The resulting PCR product (SEQ ID NO: 7) was purified on an agarose gel and cut with NotI. The resulting 1708 bp fragment was ligated with the similarly cut vector pUCmod-VvSTS after its dephosphorylation with CIP (New England Biolabs, Schwalbach) according to the manufacturer's instructions, resulting in the vector pUCmod-VvSTS-At4Cl1 (5333bp). The orientation of the insert was chosen so that VvSTS and At4Cl1 are transcribed and translated in the same direction. The promoter of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified by colony PCR with Escherichia coli DH5a cells (New England Biolabs), Phusion DNA polymerase and the oligonucleotides

EcGAP-SapI-fw, SEQ ID NO: 8
5'-TATATAGCTCTTCAAGCgcgtaatgcttaggcacagg-3 '

EcGAP-XbaI-rv, SEQ ID NO. 9
5'-TATATATCTAGAtgaataaaaggttgcctgtaa-3 '

amplified. The resulting PCR product (approximately 110bp; SEQ ID NO: 10) was purified on an agarose gel as previously described and then subjected to SapI and XbaI digestion. The vector pUCmod-VvSTS-At4Cl1 was also cut with XbaI and SapI and the vector backbone (5139bp) ligated to the insert resulting in the vector pUC-GAP-VvSTS-At4Cl1 (5244bp). Cells of the Escherichia coli strain BW25113 ΔptsG (No Collection, Japan) were transformed with pUC-GAP-VvSTS-At4Cl1. The preparation and transformation of the competent cells was carried out in a manner known to the person skilled in the art.

Example 2: Cultivation of the temperature-sensitive recombinant E. coli strains JP1111 and L48-pUC-GAP-VvSTS-At4Cl1 to produce resveratrol

Strain Background Table 1: E. coli strains JP1111 and L48 used, available from E. coli Genetic Resources at Yale CGSC, The Coli Genetic Stock Center as CGSC # Tribe name CGSC # / Source Chromosomal marker JP1111 6700 / Egan & Russell galE45 (GalS), LAM, fabI1392 (ts), relA1, spoT1, thi-1 L48 5638 / DF Silbert araC14, lacY1, tsx-57, glnV44 (AS), gltA5, galK2 (Oc), fabD89 (ts), rpsL20 (strR), xylA5, mtl-1, IIdD1, thi-1, tfr-5

The genes fabI1392 (ts) and fabD89 (ts) encode a temperature-sensitive enoyl [acyl carrier protein] reductase (enoyl-ACP reductase) or malonyl-CoA-ACP transacylase. At temperatures> 30 ° C inactivation of these enzymes,

The production of electrochemically and / or chemically competent E. coli JP1111 / L48 cells and their transformation with pUC-GAP-VvSTS-At4Cl1 were carried out in a manner known to the person skilled in the art.

1 ml of modified M9 medium (M9 with 10 g / l yeast extract, 1% glycerol as carbon source, 200 mM MOPS as buffer, base medium M9: 6.8 g / l disodium hydrogen phosphate dihydrate, 3 g / l potassium dihydrogen phosphate, 0.5 g / 1 sodium chloride, 1 g / l ammonium chloride, 15 mg / l calcium chloride dihydrate, 250 mg / l magnesium sulfate heptahydrate, pH 7.0) containing 100 μg / ml ampicillin was incubated in the morning with E. coli JP1111 / L48 pUC -GAP-VvSTS-At4Cl1 of freshly streaked and incubated overnight at 37 ° C LB agar plates (LB agar to MILLER, 37 g / l) with 100 ug / ml ampicillin inoculated, and for 6 h at 37 ° C and 250 rpm in 10 ml tubes. In the afternoon, 50 ml of modified M9 medium (including 10 g / l yeast extract) was inoculated into a 250 ml shake flask with baffled whole cell suspension from the preculture. Incubation of the shake flasks was overnight for 16-18 h at 37 ° C and 250 rpm. The next morning, the cell broth was transferred to 50 ml Falcon tubes and spun down at 4000 rpm for 10 minutes at room temperature. The cell pellet was resuspended in 50 ml of fresh modified M9 medium supplemented with 4 mM 4-hydroxycinnamic acid and 100 g / l PEG 3350 (Sigma Aldrich) and each baffled into a 250 ml shake flask. This was incubated for 7 hours to determine product formation rates at 30 ° C and 37 ° C at 250 rpm. The growth of the E. coli cells was monitored by regular measurement of the optical density at 600 nm (OD ₆₀₀ ). As a reference, strain E. coli BW27784 available from E. coli Genetic Resources at Yale, CGSC, The Coli Genetic Stock Center as CGSC # 7881 transformed with pUC-GAP-VvSTS-At4Cl1 was described in the same manner as for JP1111 and L48 used. The results of resveratrol formation are in 1 - 5 shown.

Example 3: Quantification of resveratrol and 4-hydroxycinnamic acid in the cell suspension

For extraction, 1 ml of the cell suspension was used. The cell suspension was transferred to 2 ml tubes and acidified by the addition of 50 μl of 3 M HCl. After addition of 950 .mu.l of ethyl acetate, the samples were extracted in a recycle mill MM100 (REISCH, Hahn) for 30 sec at 30 Hz, and then centrifuged for 1 min at 13,200 rpm. The ethyl acetate phase was transferred to a 1.5 ml HPLC vessel (Agilent Technologies, Waldbronn) and sealed.

2 μl of the extracts were injected onto a Zorbax SB-C18 column (4.6 x 150 mm, 3.5 μm, Agilent Technologies, Waldbronn) using the Agilent 1200 HPLC system equipped with a photodiode array detector and a binary gradient mixer. Resveratrol was mixed with a mobile phase consisting of 580 g acetonitrile, 265 g water and 0.5 ml trifluoroacetic acid per liter (eluent A) and water (eluent B) mixed in a 48:52 (A: B) ratio Flow rate of 1.0 ml / min and a column temperature of 25 ° C eluted. Resveratrol as well as 4-hydroxycinnamic acid was identified by comparison with the residence time and the UV / VIS spectrum of a reference substance (SIGMA-ALDRICH, Taufkirchen and Merck, Darmstadt, respectively). To quantify the educt 4-hydroxycinnamic acid or product resveratrol in the extracts, standard curves were prepared by applying the peak areas of reference samples of known concentration to the corresponding concentration in these reference samples. Resveratrol and 4-hydroxycinnamic acid were detected and quantified at a wavelength of 308 nm.

In 6 is the percentage increase in resveratrol production in the temperature-sensitive E. coli mutants E. coli L48 fabD (ts) and E. coli JP1111 fabI (ts), respectively, at different temperatures (30 ° C and 37 ° C) relative to the non-temperature sensitive reference strain BW27784 shown. This means that if the biotransformation is carried out at 30 ° C. and parallel at 37 ° C., an increase in the production of resveratrol at 37 ° C. is due to the inactivation of FabD in E. coli L48 fabD (ts) or of FabI in E. coli JP1111 fabI (ts) of up to 300% (for E. coli L48 fabD (ts)) and of 100% (for E. coli JP1111 fabI (ts)). While the enzymes FabD / FabI are also active at 37 ° C. for the reference strain BW27784, the temperature-sensitive E. coli mutants at temperatures> 30 ° C. lead to inactivation of FabD or FabI.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6552213 [0007]
• WO 2005023740 [0007]
• US 7235324 [0007]
• WO 06089898 [0007, 0011]
• WO 06124999 [0007, 0012]
• WO 05084305 [0011]
• WO 2006/089898 A [0067, 0069]
• WO 2005/084305 A [0067, 0069]
• DE 102009054984 [0078, 0081]
• DE 102010028874 [0079]
• WO 2009/016108 [0080]

Cited non-patent literature

• Hart, JH (1981) Annu. Rev. Phytopathology 19, 437-458 [0003]
• Hart, JH, Shrimpton, DM (1979) Phytopathology 69, 1138-1143 [0003]
• Beekwilder et al., 2006 [0011]
• Watts et al., 2006 [0011]
• Katsuyama et al., 2007 [0011]
• Becker et al., 2003 [0011]
• Beekwilder et al., 2006 [0011]
• Zhang et. al. (2006) Inhibiting bacterial fatty acid synthesis JBC 281 (26) pp. 17541-17544 [0042]
• Wright, H.T., and Reynolds, KA Antibacterial Targets in Fatty Acid Biosynthesis Current Op. Microbiol., 10, 447-453, 2007 [0042]
• Georgopapadakou et al. (1986) Effect of Antifungal Agents on Lipid Biosynthesis and Membrane Integrity in Candida albicans ACC 31 (1) pp. 46-51 [0043]
• Kizer et. al. (2008) Application of functional genomics to pathway optimization for increased isoprenoid production. Applied and Environmental Microbiology 74 (10) pp. 3229-3241 [0044]
• Wintrode et al. (2000) Cold Adaptation of a mesophilic subtilisin-like protease by laboratory evolution, J. Bio. Chem. 275 (41), pp. 31635-31640 [0050]
• Merz et al. (2000) Improving the catalytic activity of a thermophilic enzyme at low temperatures, Biochemistry 39, pp. 880-889 [0051]
• Lebbink et al. (2000) Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus β-glucosidase CelB by directed evolution, Biochemistry 39 pp. 3656-3665 [0052]
• Reetz et al. (2008) Knowledge-guided laboratory evolution of protein thermolability, Biotechnology & Bioengineering 102 (6), pp. 1712-1717 [0053]
• Knobling et al. (1975) Temperature-Sensitive Mutants of the Yeast Fatty Acid Synthetase Complex Eur. J. Biochem. 59 pp 415-421 [0055]
• C. Rieck (2006) Genetic and biochemical characterization of the fatty acid elongase in the yeast Saccharomyces cerevisiae (dissertation) [0056]
• Egan et al. (1973) Conditional mutations affecting the cell envelope of Escherichia coli K-12. Genet. Res. 21: 139-152 [0057]
• Harder et al. (1972) Temperature-sensitive mutants of Escherichia coli required saturated and unsaturated fatty acids for growth: isolation and properties. Proc. Natl. Acad. Sci. USA 69: 310 [0057]
• Semple et al. (1975) Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 121 (3): 1036-46 [0057]
• Verwoert et al. (1994) Molecular characterization of Escherichia coli mutant with a temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase. FEBS Letters, 348 (3), 311-16 [0057]
• Flaks et al. (1966) Mutations and genetics concerned with the ribosomes. Cold Spr. Harb. Symp. Quant. Biol. 31: 623-631 [0057]
• Song et al. (1992) coaA and rts are allelic and located at kilobase 3532 on the Escherichia coli physical map. J. Bacteriol. 174: 1705-1706 [0057]
• Vallari at al. (1987) Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli. J. Bacteriol. 169: 5795-5800 [0057]
• Vallari at al. (1988) Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli. J. Bacteriol. 170: 3961-3966 [0057]
• Broekman et al. (1974) Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J. Bacteriol. 117 (3): 971-7. [0057]
• Broekman et al. (1973) Growth in high osmotic medium of an unsaturated fatty acid auxotroph of Escherichia coli K-12. J. Bacteriol. 116 (1): 285-9 [0057]
• Broekman et al. (1974) Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J. Bacteriol. 117 (3): 971-7 [0057]
• Johnson et al. (1975) Mapping of sul, the suppressor of ion in Escherichia coli. J. Bacteriol. 122 (2): 570-4 [0057]
• Cronan et al. (1972) Mapping of the fabA locus for fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 112: 206-211 [0057]
• Lai et al. (2004) Isolation and characterization of .beta.-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar typhimurium. Journal of Bacteriology, 186 (6), 1869-1878 [0057]
• Watts et al., "Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli" (BMC Biotechnology 2006, 6:22 ) [0093]

Claims (12)

A cell which is capable of forming resveratrol, the cell having at least one reduced activity of an enzyme E ₁ , which is involved in malonyl-coenzyme A cataplerotic pathway, as compared to its wild-type. A cell according to claim 1, characterized in that the enzyme E _{1 is} selected from the group comprising: an enzyme E _1a , which catalyzes the conversion of malonyl-CoA to malonic acid semialdehyde; an enzyme E _1b , which catalyzes the conversion of malonyl-CoA to acetyl-CoA; an enzyme E _1c , which catalyzes the conversion of holo- [ACP] + malonyl-CoA to malonyl- [ACP] + coenzyme A; an enzyme E _{1d having} polyketide synthase activity; an enzyme E _1e , which catalyzes the conversion of acetyl-CoA + malonyl- [ACP] to acetoacetyl- [ACP] and / or of holo- [ACP] + acetyl-CoA to acetyl- [ACP]; an enzyme E _1f , which is the reaction of trans-Δ ³ -cis-Δ ⁵ -dodecenoyl- [ACP] to cis-Δ ⁵ -dodecenoyl- [ACP] and / or of 2,3,4-saturated acyl- [ACP ] catalyses trans-Δ ² -enoyl-acyl- [ACP]; an enzyme E _1g , which is the reaction of octanoyl- [ACP] + malonyl- [ACP] to 3-oxo-decanoyl- [ACP] + holo- [ACP] and / or decanoyl- [ACP] + malonyl- [ACP ] to 3-oxo-dodecanoyl- [ACP] + holo- [ACP] and / or from dodecanoyl- [ACP] + malonyl- [ACP] to 3-oxo-meristoyl- [ACP] + holo- [ACP] and / or from myristoyl- [ACP] + malonyl- [ACP] to 3-oxo-palmitoyl- [ACP] + holo- [ACP] and / or from butyryl- [ACP] + malonyl- [ACP] to 3-oxo-hexanoyl - [ACP] + holo- [ACP] and / or of hexanoyl- [ACP] + malonyl- [ACP] to 3-oxo-octanoyl- [ACP] + holo- [ACP] and / or of cis-Δ ³ - Decenoyl- [ACP] + malonyl- [ACP] to holo-keto-cis-Δ ⁵ -dodecenoyl- [ACP] + holo- [ACP] and / or malonyl- [ACP] + acetyl- [ACP] to holo- [ACP] + acetoacetyl- [ACP] and / or malonyl- [ACP] + 2,3,4-saturated acyl- [ACP] to holo- [ACP] + β-ketoacyl- [ACP] and / or malonyl Catalysed [ACP] to acetyl- [ACP] and / or palmitoleoyl- [ACP] + malonyl- [ACP] to 3-oxo-cis-vaccenoyl- [ACP] + holo- [ACP] and / or malonyl- [ACP] + acetyl- [ACP] to holo- [ACP] + A cetoacetyl- [ACP] and / or malonyl- [ACP] + 2,3,4-saturated acyl- [ACP] to holo- [ACP] + β-ketoacyl- [ACP] catalysis; an enzyme E _1h , which is the reaction of β-keto-cis-Δ ⁵ -dodecenoyl- [ACP] to β-hydroxy cis Δ ⁵ -dodecenoyl- [ACP] and / or of (3R) -3-hydroxyacyl- [ACP ] catalyses β-ketoacyl [ACP]; an enzyme E _1i , which is the reaction of β-hydroxy cis Δ ⁵ -dodecenoyl- [ACP] to trans-Δ ³ -cis-Δ ⁵ -dodecenoyl- [ACP] and / or of (R) -3-hydroxydecanoyl- [ ACP] to trans-Δ ² -deenoyl- [ACP] and / or from (3R) -3-hydroxyacyl- [ACP] to trans-Δ ² -enoyl-acyl- [ACP] and / or from trans-Δ ² - Decenoyl [ACP] catalyses cis-Δ ³ -deenoyl- [ACP]; an enzyme E _1j which catalyzes the conversion of apo [ACP] to adenosine 3 ', 5'-bis-bisphosphate + holo- [ACP]; an enzyme E _{1k having} fatty acid synthase activity; an enzyme _E1l which catalyzes the reaction of pantothenate + ATP into D-4'-phosphopantothenate + ADP; an enzyme E _1m , which catalyzes the conversion of 4'-phosphopantetheine + ATP to dephospho-CoA + diphosphate; an enzyme E _1n which catalyzes the conversion of dephospho-CoA + ATP into coenzyme A + ADP and an enzyme E _1o , which catalyzes the conversion of D-4'-phosphopantothenate + L-cysteine + CTP to diphosphate + CMP + R-4 ' Phosphopantothenoyl-L-cysteine and / or R-4'-phosphopantothenoyl-L-cysteine catalyzed to 4'-phosphopantetheine. A cell according to claim 1 or 2, characterized in that E _{1a is} a malonate semialdehyde dehydrogenase (EC: 1.2.1.15 or EC: 1.2.1.18), E _{1b is} a malonyl-CoA decarboxylase (EC: 4.1.1.9), E _1c an [acyl carrier protein] S malonyltransferase (EC: 2.3.1.39), E _1e an [acyl carrier protein] S acetyltransferase (EC: 2.3.1.38) or a [myelin proteolipid] -O-palmitoyltransferase (EC: 2.3.1.100), E _{1f is} an enoyl [acyl carrier protein] reductase (EC: 1.3.1.9), E _{1g is} a beta-ketoacyl-acyl-carrier-protein synthase I or II (EC: 2.3.1.41/179), E _1h a 3-oxoacyl [acyl carrier protein] reductase (EC: 1.1.1.100), E _1i a 3-hydroxydecanoyl [acyl carrier protein] - Dehydratase or 3-hydroxyoctanoyl [acyl carrier protein] dehydratase (EC: 4.2.1.60 or EC: 4.2.1.59) or a crotonoyl [acyl carrier protein] hydratase (EC: 4.2.1.58) or a trans-2-decenoyl [acyl carrier protein] isomerase (EC: 5.3.3.14), E _{1j is} a holo [acyl carrier protein] synthase (EC: 2.7.8.7), E _{1k is} a fatty acid Synthase or fatty acid-acyl-CoA synthase (EC: 2.3.1.85 or EC: 2.3.1.86), E _1l a pantothenate kinase (EC: 2.7.1.33), E _1m a pantetheine phosphate adenylyltransferase (EC: 2.7. 7.3), E _1n a dephospho -CoA kinase (EC: 2.7.1.24) and E _{1o is} a phosphopantothenoylcysteine decarboxylase (EC: 4.1.1.36) or a phosphopantothenoylcysteine ligase (6.3.2.5). Cell according to at least one of the preceding claims, characterized in that the activity of the enzyme E _{1 is} reduced by an inhibitor selected from the group consisting of: Sch 538415, 4H-oxazol-5-one, anthranilic acid, corytuberin, HR12, RWJ-3981, Indole analogs, SB418001, 1,2-dithiole-3-one, benzoylaminobenzoic acids, platensimycin, platencin phomallic acid, cerulenin, thiolactomycin, BABX, phomallic acids, polyphenols, alkenoic acids, 3-decynoyl-NAC, NAS-91, NAS-21, isoniazid, triclosan , Diazoborin, aminopyridines, bischloroanthrabenzoxocinone, BABX, aminopyridine, pantothenamides, clotrimazole, econazole, ketoconazole, miconazole, naftifine, tolnaftate, azaterol, cerulenin, 3-HMG-CoA, Cell according to at least one of the preceding claims, characterized in that the activity of the enzyme E ₁ conditioned, in particular by a change in temperature, is reduced. Cell according to claim 5, characterized in that it is selected from the group consisting of: E. coli JP1111 containing temperature-sensitive FabI; E. coli L48 and E. coli LA2-89 containing temperature-sensitive FabD; E. coli DV62, E. coli DV79 and E. coli ts9 containing temperature-sensitive CoaA; E. coli M5 containing a temperature-sensitive FabB; E. coli M8, E. coli PAM155, E. coli CY53, E. coli CY55 and E. coli CY50, containing temperature sensitive FabA and E. coli CL37, E. coli CL48 and E. coli CL104 containing temperature-sensitive FabG. Cell according to at least one of the preceding claims, characterized in that the cell is genetically engineered such that it is able to form more resveratrol compared to its wild type without the reduced activity of an enzyme E ₁ already. Cell according to at least one of the preceding claims, characterized in that the cell has a compared to its wild type increased activity of at least one of the following enzymes: an enzyme E ₂ , which is the reaction of tyrosine to 4-coumarate or the reaction of L-phenylalanine catalysed to trans-cinnamate; an enzyme E ₃ , which catalyzes the conversion of trans-cinnamate to 4-coumarate; an enzyme E ₄ which catalyzes the conversion of 4-coumarate to 4-coumaryl coenzyme A; an enzyme E ₅ , which catalyzes the conversion of 4-coumaryl coenzyme A to resveratrol. A cell according to claim 8, characterized in that in the cell, the activity of the following enzyme or the following enzymes is increased: E ₂ , E ₃ , E ₄ , E ₅ , E ₂ + E ₄ , E ₂ + E ₅ , E ₄ + E ₅ , E ₂ + E ₄ + E ₅ , E ₃ + E ₄ , E ₃ + E ₅ , E ₃ + E ₄ + E ₅ , E ₂ + E ₃ + E ₄ + E ₅ , where E ₄ + E _{5 is} particularly preferred. A process for the preparation of resveratrol comprising the steps of: i) contacting the cell according to any one of claims 1 to 6 with a nutrient medium including at least one carbon source II) cultivating the cell under conditions permitting the cell to recover from the carbon source resveratrol to form and if necessary III) Isolation of Resveratrol formed. A method according to claim 10, characterized in that method step II) comprises the steps A) Extracting the resveratrol from the cells and / or the nutrient medium with at least one water-immiscible solvent B) contacting the at least one water-immiscible solvent with an aqueous phase, C) separating the aqueous phase from the water-immiscible solvent and optionally D) Isolation of stilbenoid from the aqueous phase. A method according to claim 10 or 11, characterized in that method step II) comprises the steps: IIa) attracting cells according to at least one of claims 1 to 6 to high cell densities, without the activity of an enzyme E _{1 is} reduced and IIb) induction of Reduction of the activity of the enzyme E ₁ .

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