KR100567120B1 - Microbiological preparation of materials derived from aromatic metabolism / III - Google Patents

Abstract

The present invention relates to methods and gene constructs and transformed cells by microorganisms for producing substances in aromatic metabolism, in particular aromatic amino acid metabolism,

According to the invention, an increase in substance production, in particular an increase in aromatic amino acids, is observed by introducing or increasing the activity of glucose dehydrogenase, and by increasing the activity of glucose-oxidase, in the glucose-containing substrate gluconolactone And inducing intracellular formation of gluconic acid, in a preferred embodiment, glucose dehydrogenase is derived from Bacillus megaterium and the method according to the invention is characterized in that it is used to provide a broader spectrum of substances.

Description

Method for microbiological preparation of substance derived from aromatic metabolism / III {MICROBIAL PREPARATION OF SUBSTANCES FROM AROMATIC METABOLISM / Ⅲ}

The present invention relates to a method for producing by a microorganism a substance according to claims 1 to 19 and 29, in particular an aromatic amino acid, a genetic construct according to claims 20 to 22 and a transformed cell according to claims 23 to 28.

Microbiologically produced materials, especially aromatic amino acids, are of economic interest, for example, because the demand for amino acids continues to increase.

Thus, for example, L-phenylalanine is used for the preparation of medicaments, in particular for the preparation of the sweetening agent aspartame (α-L-aspartyl-L-phenylalanine methyl ester). L-tryptophan is required as an additive for pharmaceuticals and feed; L-tyrosine is likewise required as a raw material in the pharmaceutical and pharmaceutical industries. In addition to being isolated from natural substances, biotechnological preparations which yield amino acids in the desired optically active form under economically beneficial conditions are very useful. Biotechnological preparation is carried out enzymatically or using microorganisms.

The manufacturing method using the latter microorganism has the advantage that a simple and inexpensive raw material is used. However, since biosynthesis of amino acids is controlled in a wide variety of ways in cells, many other attempts have already been made to increase the formation of products. Thus, for example, amino acid homologues are used to switch off biosynthesis regulation. By selecting resistance to, for example, phenylalanine homologues, variants of Escherichia coli can be obtained and the production of L-phenylalanine can be increased (GB-2,053,906). Induces Corynebacterium (Corynebacterium, JP-19037/1976 and JP-39517/1978) and Bacillus (Bacillus, EP-0,138,526) over-production of the strains in a similar manner.

In addition, microorganisms produced by recombinant DNA techniques are known and control of biosynthesis is likewise destroyed by expression and cloning of genes encoding important enzymes that are no longer feedback inhibited. For example, in EP-0,077,196, 3-deoxy-D-arabinoheptulsonate-7-phosphate synthase, which is no longer feedback inhibited, is overexpressed in Escherichia coli to produce aromatic amino acids. This is described. EP-0,145,156 describes Escherichia coli strains in which over-expression of corismate mutase / prephenate dehydratase is further overexpressed to produce L-phenylalanine.

The mentioned method shares a common feature that adjustments to improve productivity are limited to biosynthetic pathways specific for aromatic amino acids. However, to further increase productivity, efforts are needed to improve the supply of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (Ery4P), the primary metabolites required to prepare aromatic amino acids. .

PEP is the activating precursor of pyruvate (pyruvic acid), a product from the glycolysis; Ery4P is an intermediate of the pentose phosphate pathway.

Many other attempts to increase productivity are of interest in overcoming limitations to intracellular synthesis of amino acids. In the preparation of aromatic amino acids, the primary metabolites, phosphoenolpyruvate (PEP) and Ery4P, are condensed to form 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP).

Several methods for increasing the availability of Ery4P have been described in the literature, for example, an increase in Ery4P supply to enhance the formation of L-tryptophan, L-tyrosine or L-phenylalanine products is made possible by overexpression of transketolase. (EP Patent Application 0 600 463; Frost & Draths, Ann. Rev. Microbiol. 49 (1995) 557-579). It has recently been suggested that spontaneous glucose-positive return variants of PTS-negative variants of Escherichia coli can grow on glucose by concentrating glucose in cells by the GalP system (Flores et al., Nature Biotechnology 14 (1996) 620). -623). Further expression of the transketolase gene tktA was observed to increase the formation of intermediate DAHP (Flores et al., Nature Biotechnology 14 (1996) 620-623).

Although two German patent applications of the reference numbers 196 44 566.3 and 196 44 567.1 have not been published, the applicants have for example enzymatic activities of transaldolase or transaldolase and transketolase in Escherichia coli. By increasing the activity or by increasing the activity of the PEP-independent delivery system for glucokinase and sugars in Escherichia coli and glucose in Escherichia coli or by combining the cited enzymes and delivery system. It has been demonstrated that it can increase the amount of phenylalanine.

It is an object of the present invention to utilize additional processes to improve the synthesis of substances, in particular aromatic amino acids, using microorganisms in aromatic compound metabolism.

Microorganisms can be produced that increase the formation of substances in aromatic compound metabolism.

Surprisingly, this object can be achieved by the transformation of glucose or glucose-containing substances in microorganisms that produce aromatic metabolites by selective metabolic pathways that increase the activity of glucose-oxidase. The pathway consists of oxidizing free glucose to gluconolactone / gluconate and forming 6-phosphogluconate by phosphorylating the gluconate.

The results are surprising because they play an important role in producing substances in aromatic metabolism by simply increasing the activity of glucose-oxidase.

In the sense of the present invention, substances are fine chemicals such as aromatic amino acids, indigo, indoleacetic acid, adipic acid, melamine, quinone and benzoic acid, and possible derivatives and secondary products thereof. I understand. In the context of the present invention, all such substances are also considered to be substances derived from the metabolism of aromatic compounds. This includes all compounds whose biochemical synthesis increases the supply of Ery4P or Ery4P and PEP.

In the above, the genetic selectivity for microorganisms producing substances as well as the control according to the invention is required to prepare indigo, adipic acid and other non-natural secondary products (Frost & Draths, Ann. Rev. Microbiol. 49 (1995) 557-579).
The novel method described above is suitable for preparing a material comprising its produced Ery4P.

Microorganisms that produce substances derived from aromatic compound metabolism can metabolize glucose or glucose-containing substances such as glucose-containing disaccharides or oligosaccharides in various ways: Glucose may be ATP-dependent kinases (hexokinase and glucose). Kinase) and phosphorylation. In addition, many bacteria can use PEP-dependent systems to obtain glucose and phosphorylate it.

Glucose can be oxidized by various soluble or membrane-binding enzymes (gluconolactone to gluconic acid). Such enzymes include glucose oxidase or glucose dehydrogenase. Glucose oxidase oxidizes glucose to gluconolactone by reducing molecular oxygen. While glucose dehydrogenase oxidizes glucose to gluconolactone, they use other electron acceptors such as pyrroloquinoline quinone (PQQ) or other cofactors such as nicotine adenine dinucleotide (NAD) or NADP. Membrane-bound glucose dehydrogenase oxidizes glucose using a cofactor, pyrroloquinolone quinone (PQQ), and the reaction is known to occur on the outer side of the membrane. In order to collect the product (gluconolactone or gluconic acid) into cells, a specific delivery system is required, which is described in van Schie et al., Journal of Bacteriology 163 (1985) 493-499; Journal of General Microbiology 132 (1986) 3209-3219; Isturiz et al .; Izu et al., Journal of Molecular Biology 267 (1997) 778-793, whose expression suppressed by the presence of glucose is largely (eg, Escherichia coli). Journal of Molecular Biology 267 (1997) 778-793 And Conway. FEMS Microbiology Reviews 103 (1992) 1-27.

Glucose dehydrogenase using cofactor NAD or NADP is a soluble enzyme found in cells. Known manufacturers include several isoenzymes of Bacillus strains, glucose dehydrogenases (eg, glucose dehydrogenases I to IV in the case of Bacillus megaterium ; 1990, Journal of Fermentation and Bioengineering 70, 363). -369). Expression of glucose dehydrogenase is strictly regulated and is known to occur only at the progenitor stage during endogenous spore formation, eg by Bacillus spp .; The physiological role of glucose dehydrogenase in relation to growth on glucose is unclear, and Lampel et al. Journal of Bacteriology 166 (1986) 238-243 and recently Steinmetz or Fortnagel (" Bacillus subtilis " and other Gram-positives) Bacteria "(Sonenshein, Hoch and Losick, eds), M. Steinmetz pp. 157-170 and P. Fortnagel pp. 171-180; ISBN 1-55581-053-5; ASM Press, Washington, DC, 1993 It is.

NAD (P) -dependent glucose dehydrogenases are not described in particular in Escherichia, Corynebacteria or Brevibacteria . Bacillus strain-derived genes for glucose dehydrogenase are cloned into Escherichia and expressed in the bacteria (Hilt et al. Biochimica et Biophysica Acta 1076 (1991) 298-304). In particular, it is an object of the present invention to obtain recombinant glucose dehydrogenase, which can be used as a detection system for regenerating glucose or cofactors (Bilchi et al. Biochimica et Biophysica Acta 1076 (1991) 298-304; DE Patent Application No. 3 711 881). In contrast, the gene is not described using glucose as a substrate for obtaining aromatic amino acids as substances in aromatic compound metabolism.

According to the present invention, it has been found that substances are formed by introducing and increasing the activity of glucose dehydrogenase. Increasing the activity of glucose-oxidase leads to the intracellular formation of gluconolactone and gluconic acid in the glucose-containing substrate. In a preferred embodiment, glucose dehydrogenase is derived from Bacillus megaterium, in particular Bacillus megaterium glucose dehydrogenase IV, according to the Journal of Fermentation and Bioengineering 70 (1990) 363-369 by Mitamura et al., Journal of Bacteriology 15 by Nagao et al. (1992) 5013-5020.

Gluconic acid relies on gluconic acid-phosphatase in its activity. For example, it is known that the enzyme may be phosphoenol-pyruvate-dependent enzyme II having specificity to gluconic acid or ATP-dependent kinase having specificity to gluconic acid. See, eg, FEBS Letters 394 (1996) 14-16 by Izu et al .; Escherichia coli ATP-dependent gluconate kinase GntK described in Journal of Molecular Biology 267 (1997) 778-793 in Izu et al. And Journal of Bacteriology 178 (1996) 3260-3269 in Tong et al. Are known.

In Escherichia coli, the product 6-phosphogluconate is an intermediate of oxidation of the pentose phosphate pathway and the Entner-Douderoff pathway, which is described in Fraenkel, pp 189-198, "Escherichia coli". and Salmonella ". 2nd edition (Neidhardt et al., Eds.), ASM Press, Washington, USA, ISBN-1-55581-084-5, 1996.

The production of the substance can be enhanced by further increasing the activity of gluconic acid (gluconate) -phosphatase. When gluconic acid-phosphatase is used, for example, gluconic acid-phosphatase is suitable in various microorganisms, which should be able to functionally express in microorganisms the substances produced in metabolism of aromatic compounds. ATP-dependent gluconate kinase, preferably Escherichia coli gluconate kinase, in particular gluconate kinase (GntK) in Escherichia coli K-12 is used. Other genes for the gluconic acid-phosphatase in its gene product phosphorylated gluconic acid are suitable for the method according to the invention. Other enterobacteria, Zymomonas mobilis , Bacillus subtilis and Corynebacterium glutamicum are mentioned in the examples.

The effect of gluconate kinase is limited to activating gluconic acid / gluconate, but is not metabolized by Escherichia coli or other bacteria, for example in the presence of glucose. For example in Escherichia coli, the gluconate kinase GntK is important when the organism grows in gluconic acid and does not contribute to the metabolism of glucose; Indeed its formation is actually inhibited in the presence of glucose and is described in the Journal of Molecular Biology 267 (1997) 778-793 in Izu et al. And the Journal of Bacteriology 178 (1996) 3326-3269 in Tong et al. Do not.

Therefore, in this particular embodiment, the activity of gluconate kinase GntK is increased by increasing the activity of gluconate-phosphorylation enzymes, in particular gluconate kinase, in particular all eschericia coli gluconate kinase and in particular escheria coli K-12, Provided are enzymes that are gluconic acid-phosphorylating enzymes for use in substance-producing microorganisms in the absence of gluconate or in the presence of glucose. Selective metabolic pathways have the advantage of increasing material flow. This can convert gluconic acid to 6-phosphogluconate to increase even in the presence of glucose. This increases the intracellular ratio of 6-phosphogluconate and can be converted to the substance by known metabolic sequences.

Gluconolactone is the reaction product of glucose-oxidase. Gluconolactone can be spontaneously converted to gluconic acid, and enzymes have been described that catalyze the conversion catalytically ( Zymomonas mobilis gluconolactonases such as Kanagasundaram and Scopes, Biochimica et Biophysica Acta) 1171 (1992) 198-200). Therefore, in another embodiment, the gene for gluconolactonase (such as Zimomonas mobilis) is expressed, and further glucose-oxidase or glucose-oxidase and gluconic acid-phosphatase are expressed to convert gluconolactone to gluconic acid. (Or 6-P-gluconate, respectively).

Genes for glucose dehydrogenase and gluconate kinase are known to occur naturally in several Bacillus species; However, the genes for these enzymes are arranged in different operons and are not used together to metabolize glucose, which is Steinmetz or Fortnagel ("Bacillus subtilis and other Gram-positive bacteria" (Sonenshein, Hoch and Losick, Eds) , Steinmetz pp. 157-170 and Fortnagel pp. 171-180; ISBN 1-55581-053-5; ASM Press, Washington, DC, 1993). Also, due to the specific induction of glucose dehydrogenase under sporulation conditions, the two enzymes are not expected to contribute together to the formation of the substance.

As a result, glucose-oxidases, or glucose-oxidases and gluconic acid-phosphatases, to produce substances in metabolism of aromatic compounds, in particular aromatic amino acids, in combination with growth in the glucose or glucose-containing substrates described above in the present invention. The effect of each increased activity of is not entirely expected.

In another preferred embodiment of the invention, the activity of the carrier protein for PEP-independent absorption of sugars is increased, and also glucose-oxidase or glucose-oxidase and gluconic acid-phosphatase are increased.

This embodiment also increases the activity of a glucose-containing substance or carrier protein for PEP-independent absorption of glucose in a microorganism that produces a substance derived from metabolism of an aromatic compound capable of absorbing sugar by a PEP-dependent delivery system. It involves making. Further integration of the PEP-independent delivery system may increase the supply of sugars in the microorganisms producing the substance. According to the present invention, the sugar is converted to gluconolactone by intracellular glucose-oxidase, which subsequently becomes gluconic acid. And gluconic acid is a substance for gluconic acid-phosphatase. Usually PEP is not required as an energy donor in the reaction, and therefore can be used based on a constant flow of material in glycolysis, for the pentose phosphate pathway, for the condensation of large amounts of Ery4P, and for general aromatic compounds. 3-deoxy-D-arabino-heptulsonate-7-phosphate (DAHP), which is the primary metabolite of the biosynthetic pathway, can be formed and produce substances in aromatic compound metabolism.

In the case of carrier proteins, activity means protein-mediated uptake.

For carrier proteins for PEP-independent absorption of glucose or glucose-containing substrates, it is advantageous to use carrier proteins, particularly promoters, which are carrier proteins that act on the principle of protein-mediated dispersion. Particularly suitable is the use of glucose promoter protein (Glf) in Zimomonas mobilis. In the latter case, a gene encoding a protein, such as glf, is derived from Zimomonas mobilis, in particular the promoter gene glf is isolated from Zimomonas mobilis ATCC 31821, Parker et al. (1995) Molecular Microbiology 15 (1995). 795-802 and Weisser et al. (1995) Journal of Bacteriology 177 (1995) 3351-3354. However, sugar transport genes derived from other bacteria, gene products thereof, carry glucose, with no specific PEP, such as the Escherichia coli GalP system being suitable in the method according to the invention. In addition, the gene for the sugar transport system derived from eukaryotic microorganisms Saccharomyces cerevisiae , Pichia stipitis and Kluyveromyces lactis , such as HXT1 Sugar transport genes derived from HXT7, or other microorganisms that can be functionally expressed in microorganisms, can be used, while the gene product can operate without PEP to phosphorylate and / or transport glucose. In particular, it is advantageous that the sugar transport gene can be expressed in amino acid producers.

In the sense of the present invention, a method of increasing activity increases the activity of glucose-oxidase or glucose-oxidase activity and also a carrier protein for PEP-independent absorption of gluconate phosphatase, gluconolactonase and sugars. Means any suitable way to increase at least one activity. The following are particularly suitable for this purpose:

Introducing the gene using, for example, a vector or a source phage;

Increase the number of gene copies to introduce the gene according to the invention into the microorganism with an increased number of copies, such as a slightly increased number (e.g. 2 to 5 times) and a significantly increased number of copies (e.g. 15 to 50 times) To;

Gene expression by, for example, by using a facilitator such as Ptac, Ptet or other regulatory nucleotide sequence, for example by increasing the rate of transcription and / or by increasing the rate of translation by eg using consensus ribosomal binding sites. Increases;

For example by mutagenesis, eg by UV irradiation or by mutation-producing chemicals, or by recombinant DNA techniques such as deletion, insertion and / or nucleotide exchange, which are produced in a non-aromatic manner according to conventional methods. Increase the endogenous activity of the enzymes present by the mutations that occur;

Altering the structure of the enzyme to increase the activity of the enzyme, for example by mutagenesis using physical, chemical, molecular biological or other microbiological methods;

Use a deregulated enzyme, eg, an enzyme that no longer feedback-suppression occurs;

Introduce the corresponding gene encoding a deregulated enzyme.

It is also possible to use a combination of the cited methods and other similar methods to increase activity. In the case of carrier proteins, for example, endogenous activity can be increased by cloning the gene using the above methods or by selecting variants that exhibit an increase in substrate transport.

Preferably, the increase in activity is effected by the gene or gene construct or by a gene integrated into several gene constructs, the gene or genes being introduced into the gene construct in a single copy or increased copy number.

In the sense of the present invention, a genetic construct is understood to be a gene or a specific nucleotide sequence carrying a gene according to the invention. For example, a suitable nucleotide sequence can be a plasmid, vector, chromosome, phage or other nucleotide sequence that is not closed by the circular method.

The availability of PEP to prepare the first intermediate of aromatic amino acid metabolism is limited to microorganisms with increased flow of material into Ery4P. In this case, other PEP-consuming responses in metabolism, if present, are beneficial for reducing or blocking the response of the PEP: sugar phosphotransferase system (PTS), and activate the PEP-dependent sugar uptake rate.

According to the present invention, organisms exhibiting natural levels of PTS activity are used; However, as a method for further improving the method, PTS variants having reduced activity of PTS are used. Reduction of this property can be effected at the level of enzymes or by genetic methods, such as by using a selectively strong inhibitory promoter that expresses the pts gene, or by inserting the glf gene into the chromosome and especially into the locus of the ptsI gene. And at the same time stabilization (separation stability) of the recombinant DNA on the chromosome and consequently no vector is used. In addition, in combination with regulatory promoters, the activity of PTS can be influenced by adding the trigger or inhibitor of the relative promoter during culture.

In the process according to the invention for preparing aromatic metabolites, it is preferred to use microorganisms which deregulate and / or deregulate one or more enzymes further included in the synthesis of said substances.

In particular the enzymes are aromatic amino acid metabolism and in particular DAHP synthase, schimate kinases and corismate mutases / prephenate dehydrogenases and also all other enzymes, in particular transaldolases, transketolases and glucokinases, aromatic Metabolic intermediates and secondary products are included in compound synthesis.

Unlike the enzymes according to the invention, it deregulates and overexpresses DAHP synthase, which is particularly important for the preparation of substances such as adipic acid, bilic acid and quinone compounds and their derivatives. In addition, sikimate kinases must be deregulated, such as to increase their activity to oversynthesize the derivatives of L-tryptophan, L-tyrosine, indigo and hydroxy- and aminobenzoic acids and naphtho- and anthraquinones and their secondary products. Let's do it. Deregulated and overexpressed corismate mutase / prephenate dehydratase is particularly important for the efficient preparation of phenylalanine and phenylpyruvic acid and derivatives thereof. However, this includes Ery4P or all other enzymes whose activity is contributed to the biochemical synthesis of the substance by the activity of all other enzymes, compounds which are promoted by the supply of Ery4P and PEP.

Contrary to the inventive adjustments, additional genetic choices for the microorganisms producing the material are required to produce indigo, adipic acid and other non-natural second products. Such methods are known to those of ordinary skill in the art (Frost & Draths, Ann. Rev. Microbiol. 49 (1995) 557-579).

The process according to the invention is suitable for preparing aromatic amino acids, in particular L-phenylalanine. In the case of L-phenylalanine, gene expression and / or enzymes of deregulated DAHP synthase (such as Escherichia coli AroF or AroH) and / or likewise deregulated corismate mutase / prefenate dehydratase (PheA) Increase activity.

Suitable preparation organisms include Escherichia coli and Serratia , Bacillus, Corynebacterium or Brevibacterium and other strains known from conventional amino acid methods. In addition, the bacteria are derived from Nocardiaceae and Actinomycetales. Escherichia coli is particularly suitable.

The present invention also relates to the supply of suitable genetic constructs and transformed cells which can carry such gene constructs and in particular can successfully carry out the invention.

In the context of the present invention, new genetic constructs can be used, and recombinant forms of the gene constructs encode carrier proteins for (a) a gene encoding gluconic acid-phosphatase or (b) for PEP-independent uptake of sugars. Or (c) glucose-oxidation together with at least two of three genes encoding a gluconic acid-phosphatase, a gene encoding gluconolactonase, or a carrier protein for PEP-independent uptake of sugars. One containing a gene encoding an enzyme is used.

In particular, the gene of glucose-oxidase encodes glucose dehydrogenase and the gene of gluconate-phosphatase encodes gluconate kinase.

Genes for glucose dehydrogenase are preferably derived from Bacillus megaterium, genes for gluconate kinase are preferably derived from Escherichia coli, and genes and carrier proteins for gluconolactonase are preferably Zimomonas mobil. It is preferred to derive from the lease. The gene for glucose dehydrogenase is glucose dehydrogenase IV ( gdhIV ) in Bacillus megaterium , the gntK gene for gluconate kinase is GntK derived from Escherichia coli, and the genes for the carrier protein and the gluconanolactonase are Particularly beneficial are glf and gnl derived from Zimomonas mobilis. Isolation of related genes and cell transformation are carried out according to the current method; For example, when cloning the Escherichia coli gluconate kinase gene gntK , the Bacillus megaterium glucose dehydrogenase IV ( gdh IV ) gene, or the Zimomonas mobilis gluconolactonase (gnl) gene or the carrier gene ( glf ) In order to specifically amplify genes, polymerase chain reaction (PCR) methods were used for E. coli K-12 (gntK), Bacillus megaterium (gdhIV), and Jimonas mobilis strain ATCC 29191 or ATCC 31821 ( gnl). It is appropriate to use chromosomal DNA derived from glf ).

After amplifying the DNA and recombining it with known vectors (pGEM7, pUCBM20, pUC19 or others) in vitro, the host cells are transformed by chemical methods, electrophoresis, transduction or conjugation.

The complete nucleotide sequence of the gntK , gdhIV , gnl and glf genes in three donor organisms is known and is known as Heidelberg's Accession Numbers D 84362 ( gntK ), D 10626 ( gdhIV ), X 67189 ( gnl ) and M 60615 ( glf ). It can be obtained from those deposited in a database such as EMBL / HUSAR. Genes using chromosomal DNA from Escherichia coli K-12 strains and the gene sequences described in Journal of Molecular Biology 267 (1997) 778-793 and Iong et al. Journal of Bacteriology 178 (1996) 3260-3269 PCR for amplifying the target is suitable for cloning the Escherichia coli gntK gene. Chromosomal DNA derived from, for example, Bacillus megaterium , is suitable for cloning the Bacillus megaterium gdhIV gene (Nagao et al. Journal of Bacteriology 174 (1992) 5013-5020).

An isolated glucose dehydrogenase IV gene may be integrated into a gene construct or several gene constructs in certain combinations with one or more genes described herein. Regardless of whether it was correctly distributed into the gene constructs, these include gdhIV + gntK , gdhIV + glf , gdhIV + gntK + glf ; gdhIV + gntK + gnl ; gdhIV + gnl + glf ; This is a combination of gdhIV + gntK + gnl + glf .

In distributing genes, glf should preferably be introduced into the gene construct or gene constructs at low copy numbers (X) (eg X = 1 to 10) to avoid possible adverse consequences of overexpressing the membrane protein.

Genetic constructs comprising at least one regulatory gene sequence that can be identified as one of the genes are advantageous.

As such, the modulator may preferably be enhanced in the transfer step, in particular to enhance the transfer signal. This can be done, for example, by increasing the activity of the promoter or promoters by altering the promoter sequence located upstream of the structural gene or by completely replacing the promoter with many active promoters. Transcription can also be enhanced by appropriately affecting regulatory genes identified as genes; However, it can also enhance translation by improving the stability of messenger RNA (mRNA).

In addition, in the constitution of the present invention, transformed cells comprising the gene construct according to the present invention in replicable form may also be used. In the sense of the present invention, a transformed cell is understood as a particular microorganism carrying the genetic construct according to the invention which can increase the intracellular formation of a substance in aromatic compound metabolism. The host cell can be transformed by chemical methods (Hanahan J. Mol. Biol. 166 (1983) 557-580) and also by electrophoresis, conjugation or transduction.

In the transformation, it is advantageous to use a host cell with deregulated and / or deregulated one or more enzymes further included in the synthesis of the substance, or with their increased activity. A microbial strain according to the invention, in particular producing an aromatic amino acid or other substance in aromatic compound metabolism, is transformed into a gene construct comprising an E. coli related gene.

In transformation with the gene construct, it is also advantageous to use host cells in which the PEP-dependent sugar uptake system, if present, has reduced or blocked its activity.

In particular, it is used that the transformed cell can produce an aromatic amino acid, and the aromatic amino acid is preferably L-phenylalanine.

Methods for producing substances with microorganisms in aromatic metabolism can be used conclusively, using the transformed cells described above having the genetic construct as described above.

In a particularly preferred embodiment of the method according to the invention, transformed cells comprising other metabolites of central metabolism can be used with increased availability other than Ery4P. Examples of such metabolites are α-oxoglutarate or oxaloacetate resulting from the intracellular synthesis method, or as a metabolite of the citric acid cycle, or corresponding precursors thereof, or their precursors such as by feeding from fumarate or malate Grow the cells.

Escherichia coli strain AT2471 / pGEM7gntKgdhIV was deposited in the German Collection of Microorganisms and Cell Cultures (SMZ) under accession number DSM 12118 on April 15, 1998 under the Budapest Treaty.

As host organisms used, for example AT2471 has been deposited with CGSC as heading 4510 by Taylor and Trotter (Bacteriol. Rev. 13 (1967) 332-53) and can be obtained free of charge.

The materials and methods used will be described below and the invention will be supported by experimental and comparative examples:

Common way

In genetic studies, Escherichia coli strains were identified in LB medium consisting of Difco Bacto tryptone (10 g · l ⁻¹ ), Difco yeast extract (5 g · l ⁻¹ ) and NaCl (10 g · l ⁻¹ ) Incubated. Therefore, the resistance of the strain to be used, ampicillin (100㎎ · ℓ ^-1) and chloramphenicol (17-34㎎ · ℓ ^-1), if the need is added to the culture medium. In the above, ampicillin was previously dissolved in water, chloramphenicol was previously dissolved in ethanol, and then sterilized by filtration and then added to a pre-autoclaved medium. Difco Bacto agar (1.5%) is added to LB medium to prepare agar plates.

Plasmid DNA in Escherichia coli is isolated by alkaline digestion using a commercially available system (Qiagen, Hilden). Chromosome DNA is isolated from Escherichia coli and Bacillus megaterium DSM 319 using the method of Chen and Kuo (Nucl. Acid Res. 21 (1993) 2260). Restriction enzymes, Taq DNA polymerase, DNA polymerase I, alkaline kinase, RNase and T4 DNA ligase are used according to the manufacturer's instructions (Boehringer, Mannheim, Germany or Promega, Heidelberg, Germany). In restriction analysis, DNA fragments are fractionated on agarose gel (0.8%) and separated from agarose by extraction using a commercially available system (QuiaExII, Hilden, Germany).

Prior to transformation, the cells are incubated at 200 rpm and 37 ° C. for 2.5-3 hours in LB medium (5 ml tubes). At an optical density of approximately 0.4 (620 nm), the cells are centrifuged and 1 volume of TSS (LB medium containing 10% (w / v) PEG 8000, 5% (v / v) DMSO and 50 mM MgCl ₂ ) / 10 is obtained. After incubating 0.1-100 ng of DNA for 30 min at 4 ° C., followed by continuous incubation at 37 ° C. for 1 hour, the cells are plated in LB medium containing the appropriate antibiotic.

Example 1

Preparation of pGEM7gntKgdhIV as a Model of Plasmid-Based Gene Constructs According to the Present Invention

After cloning the Bacillus megaterium DSM 319 gdhIV gene encoding glucose dehydrogenase IV, the polymerase was based on the known DNA sequence of the gene described in Nagao et al., Journal Bacteriology 15 (1992) 5013-5020. Amplification of the chromosomal DNA of Bacillus megaterium DSM 319 is specifically amplified by chain reaction (PCR). PCR oligonucleotide primers were provided with cleavage sites for restriction enzymes BamHI (5 'terminus) and SacI (3' terminus). Primer 1 (BamHI) consists of 5 'ATG GAT CCA TGA AAA CAC TAG GAG GAT TTT 3'. Primer 2 (SacI) consists of 5 ′ GCC AGA GCT CTT TTT TCC ACA TCG ATT AAA AAC TAT 3 ′ and is complementary to the 3 ′ end of the gdhIV gene. The resulting DNA amplification product of approximately 800 base pairs is limited to BamHI + SacI and linked to the vector pGEM7 and treated in the same way (see Table 1). The transformation is selective for LB agar plates containing X-Gal and ampicillin. It is carried out with JM109DE3 strain. Test for cloning is successful by measuring the DNA sequence of the cloned gdhIV gene. The vector (pGEM7gdhIV) activates glucose dehydrogenase IV expressed without the T7 polymerase system (strain JM109DE3). The gntK gene for Escherichia coli K-12 gluconate kinase is cloned by specifically amplifying DNA using Escherichia coli K-12 W3110 as a chromosome template. The sequence of the gntK gene is described in Tong et al. Journal of Bacteriology 178 (1966) 3260-3269. For amplification by PCR, it is selected that the oligonucleotide primer is further provided as a restriction cleavage site for EcoRI (5 ') and BamHI (3'). Primer 1 consists of 5 ′ CCG AAT TCT TGT ATT GTG GGG GCA C 3 ′ and binds 5 ′ upstream of the gntK gene; Primer 2 consists of 5 ′ CCG GAT CCG TTA ATG TAG TCA CTA CTT A 3 ′ and is complementary to the 3 ′ end of the gntK gene. Approximately 600 base pairs of amplification products are purified, digested with EcoRI + BamHI and ligated into vector pGEM7, which is also open. Transformation is carried out in JM109DE3 strain that is selective for LB agar plates containing X-Gal and ampicillin. Test for cloning is successful by measuring the DNA sequence of the cloned gntK gene. The gluconate kinase in which the vector (pGEM7gntK) is expressed without the T7 polymerase system (JM109DE3) is activated (see Table 2).

The gntK and gdhIV genes are combined by opening the vector pGEM7gntK by double cleavage with BamHI + SacI. Obtained after restriction with the vector pGEM7gdhIV, an 800 base pair fragment comprising the gdhIV gene is ligated into the vector which has been ring-opened by this method. Transformation is again carried out with selectivity for ampicillin. The new gene construct pGEM7gntKgdhIV according to the present invention mediates T7 polymerase-independent expression of enzyme activity against glucose dehydrogenase IV and gluconate kinase GntK (see Table 2).

The resulting transformants are stored in LB medium in the form of glycerol culture (30%) at -80 ° C. If necessary, the glycerol culture is thawed immediately before use.

Example 2

Determination of Enzyme Activity of Glucose Dehydrogenase and Gluconate Kinase

To measure enzyme activity in crude bacterial extracts, cells of E. coli cells and variants with plasmids are cultured in mineral medium. The medium was sodium citrate.3H ₂ O (1.0 g.l ^-1 ), MgSO ₄ .7H ₂ O (0.3 g.l ^-1 ), KH ₂ PO ₄ (3.0 g.l ^-1 ), K ₂ HPO ₄ (12.0 g · l ⁻¹ ), NaCl (0.1 g · l ⁻¹ ), (NH ₄ ) ₂ SO ₄ (5.0 g · l ⁻¹ ), CaCl ₂ · 2H ₂ O (15.0 mg · L ⁻¹ ), FeSO ₄ .7H ₂ O (0.075 g · l ⁻¹ ) and L-tyrosine (0.04 g · l ⁻¹ ). In addition, minerals _{Al 2 (SO 4) 3 ·} 18H 2 O (2.0g · ℓ -1), CoSO 4 · 6H 2 O (0.7g · ℓ -1), CuSO 4 · 5H 2 O (2.5g · ℓ - ¹ ), H ₃ BO ₃ (0.5 mg · L ⁻¹ ), MnCl ₂ · 4H ₂ O (20.0 g · L ⁻¹ ), Na ₂ MoO ₄ · 2H ₂ O (3.0 g · L ⁻¹ ), NiSO ₄ It is added in the form of a trace element solution (1 ml·l ⁻¹ ) consisting of 3H ₂ O (2.0 g · l ⁻¹ ) and ZnSO ₄ · 7H ₂ O (15.0 g · l ⁻¹ ). Vitamin B1 (5.0 mg · L ⁻¹ ) is dissolved in water, sterilized by filtration and then added to the autoclaved medium, and ampicillin and / or ampicillin and chloramphenicol are added if necessary. Glucose (30 g · L ⁻¹ ) is autoclaved, respectively, and is added to the autoclaved medium as well.

The cells obtained are washed in 100 mM Tris / HCl buffer, pH 8.0. The precipitated cells are degraded by ultrasound in a 25% ultrasonic cycle of 40 watts intensity for 4 minutes per ml of cell suspension (Branson sonifier 250 with microtips). After centrifugation at 18,000 g and 4 ° C. for 30 minutes, the supernatant (crude extract) is used to measure the activity of glucose dehydrogenase and / or gluconate kinase.

Glucose dehydrogenase activity is measured according to Harwood & Cutting, Molecular Biological Methods for Bacillus, John Wiley & Sons. Glucose dehydrogenase activates the oxidation of glucose to gluconolactone. Enzyme activity is measured optically at a wavelength of 340 nm by increasing the concentration of reduced cofactor NADH + H ⁺ . The measurement is carried out in a quartz cuvette with a total volume of 1 ml. The reaction mixture consists of Tris HCl (final concentration 250 mM, pH 8.0), 2.5 mM EDTA sodium, 100 mM KCl and 2 mM NAD. The crude extract is preincubated at 25 ° C. for 5 minutes in buffer. Glucose (final concentration, 100 mM) is added to initiate the detection reaction. The increase in absorbance is monitored at 340 nm. A mixture that does not contain glucose serves as a control in each case. Specific glucose dehydrogenase activity is expressed in U / mg and is defined as the formation of 1 μmol of NADH per minute and per mg of protein and is set to correspond to a conversion of 1 μmol of glucose per minute and per mg of protein.

Gluconate kinase is measured in crude extracts such as those described in FEBS Letters 394 (1996) 14-16 by Izu et al.

Gluconate kinase activates ATP-dependent phosphorylation of gluconate with 6-phosphogluconate. In enzymatic testing, the 6-phosphogluconate formed is optically measured at a wavelength of 340 nm by increasing the NADPH concentration when using NADP-dependent coenzyme 6-phosphogluconate dehydrogenase (Boehringer Mannheim, No. 108 405). In the above, the formation of 1 μmol of NADPH corresponds to the phosphorylation of 1 μmol of gluconate. Detection of the enzyme is carried out at 25 ° C. of quartz cuvette with a total volume of 1 ml. The reaction mixture contains 50 mM Tris HCl, pH 8.0, 100 mM ATP, 0.25 mM NADP, 1.2 units of coenzyme 6-phosphogluconate dehydrogenase and various amounts of crude extract. The mixture is preincubated at 25 ° C. for 5 minutes and the reaction is initiated by adding gluconic acid (pH 6.8; final concentration in the mixture, 10 mM). The mixture without addition of gluconic acid is used as a control.

Protein concentrations in crude extracts are measured according to Bradford M.M. by using commercially available colored reagents (Anal. Biochem. 72 (1976) 248-254). Bovine serum albumin is used as standard.

Table 2 shows the results of enzyme measurements when using the host strain Escherichia coli W3110 and its variants with plasmids pGEM7gdhIV, pGEM7gntK or pGEM7gntKgdhIV. When the gene construct according to the invention is used, it has been found that the enzyme can be expressed in a functional way in the cells according to the invention.

Example 3

Preparation of Substances Using Strains Showing Increased Glucose Dehydrogenase Activity

The synthetic efficiency of Escherichia coli AT2471 and Escherichia coli AT2471 / pGEM7ghdIV was measured in the mineral medium described in Example 2. In the above, 2 ml of glycerol culture was inoculated while shaking the flask (1000 ml containing 100 ml of medium) and incubated in an orbital shaker at 150 rpm and 37 ° C. for 72 hours. The pH of the culture is measured at approximately 12 hour intervals and stored at a starting value of 7.2 by adding KOH (45%) if necessary. Samples are also taken after 24 and 48 hours to determine the optical density and concentration of glucose and L-phenylalanine.

The concentration of phenylalanine was investigated by high pressure liquid chromatography (HPLC, Hewlett Packard, Munich, Germany) in combination with that detected by fluorescence (absorption 335 nm, emission 570 nm), and nucleosyl-120-8 C18 column (250. 4.6 mm) is used as the solid phase; Gradient (eluent A: 90% 50mM phosphoric acid, 10% methanol, pH 2.5; eluent B: 20% 50mM phosphoric acid, 80% methanol, pH 2.5; gradient: 0-8 minutes, 100% A, 8-13 minutes, 0% Elution is carried out using A, 13-19 min, 100% A). The elution rate is set at a column temperature of 1.0 ml · min ⁻¹ and 40 ° C. Post-column derivatization is carried out using o-phthaldialdehyde in a reaction capillary (14 m · 0.35 mm) at room temperature. Under the conditions described, it was found that L-phenylalanine had a retention time of 6.7 minutes.

By measuring the glucose concentration using an enzymatic test strip (Diabur, Boehringer Mannheim, Germany) and 2 ml of concentrated glucose solution (500 g · l ⁻¹ ), it was confirmed that glucose was not limited in the experimental mixture. It became.

The plasmid pGEM7gdhIV is briefly introduced and the index value describing the phenylalanine concentration after 48 hours of incubation is 145 compared to the index value of 100 (phenylalanine) for the host strain Escherichia coli AT2471. The results demonstrated the aromatic compound synthesis-increasing effect according to the present invention to increase the activity of glucose dehydrogenase in continuously produced microorganisms.

Example 4

Preparation of Substances Using Strains That Not Only Increase Glucose Dehydrogenase Activity, but Express a PEP-Independent Sugar Absorption System

The Zimomonas mobilis glf gene is amplified using plasmid pZY600 as a template (J. Bacteriol 177 (1995) 3351-3345 by Weisser et al.). At the same time, the selection of primers introduces the BamHI cleavage site and the KpnI cleavage site. Using this unique cleavage site, the gene is inserted into the vector pUCBM20 (Boehringer Mannheim) and opened to BamHI and KpnI. A 1.5 kb DNA fragment was cut with BamHI and HindIII and isolated from the vector (pBM20glf), ligated with vector plasmid pZY507 (J. Bacteriol 177 (1995) 3351-3345, Weisser et al.) And restriction enzymes BamHI and HindIII Open. The recombinant plasmid pZY507glf is obtained by transforming Escherichia coli and cloning the transformant. The vector is resistant to chloramphenicol, comprises a lacI ^q -tac promoter system and has a low copy number.

The vector pZY507glf is transformed into a host AT2471 strain with the gene construct of the invention obtained as described in Example 1.

According to the experimental conditions described in Example 3, a mixture of two parallel variants, Escherichia coli AT2471glf, Escherichia coli AT2471glf / pGEM7, Escherichia coli AT2471glf / pGEM7gntK and Escherichia coli AT2471glf / pGEM7gntKgdhIV in each case Are incubated. After 48 hours, the concentration of L-phenylalanine in the medium is measured.

In order to compare with the starting strain Escherichia coli AT2471glf such that there is an L-phenylalanine concentration corresponding to an index value of 100, the presence of the vector pGEM7 is an index value of 96 and consequently in fact the same concentration is obtained. On the other hand, the use of Escherichia coli AT247glf / pGEM7gntK yields an L-phenylalanine concentration corresponding to an index value of 179 compared to previously known strains. Expression of selective metabolic genes, such as glucose dehydrogenase and gluconate kinase, in Escherichia coli AT2471glf / pGEMgntKgdhIV can be further increased with an index value indicating a phenylalanine concentration of 195.

The results suggest that expression of selective metabolic pathways by introducing glucose dehydrogenase activity and increasing gluconate kinase activity not only positively affects L-phenylalanine synthesis in microorganisms, but at the same time PEP-independent sugar uptake system. Is transformed and expressed.

Example 5

Preparation of substances using PTS ^- variants that not only increase glucose dehydrogenase activity but also express a PEP-independent sugar uptake system

To integrate the glf gene into a gene encoding components of the Escherichia coli PTS system, the plasmid pPTS1 is digested with BglII and treated with a Klenow fragment. The only cleavage site is in the ptsI gene. The glf gene is isolated from the plasmid pBM20glfglk as a BamHI / KpnI fragment, and likewise treated with cleno fragments. Clones carrying the glf gene in the same orientation as the ptsHI gene are obtained by blunt end ligation. The ptsH gene with integrated glf and crr and 4.6 kb of PstI fragment carrying the 3 'region of ptsI are obtained in plasmid pPTSglf. The fragment is ligated to the EcoRV cleavage site of the vector pGP704. Since the vector can be replicated in a λpir strain, the vector is incorporated into the chromosome if the transformant without the phage can grow in carbenicillin. This integration is confirmed by Southern analysis (J. Bacteriol. 170 (1988) 2575-83 by Miller V.L. et al.). In addition to the glf gene, the resulting transformants also include the complete PTS gene.

The vector component can be recombined at the second homologous crossover and loses resistance to carbenicillin. The PTS is not functionally expressed in the variant because the pts gene is interrupted by the insertion of the glf gene in this case. The desired PTS ^- variant is selected as follows: after repeated subinoculation of PTS ⁺ transformant on LB medium without antibiotic, LB plate containing 100 μg · l ⁻¹ phosphomycin in fractions of the cell suspension Apply to PTS ^- variants can be grown on the plate. The clones are grown and plated on LB medium containing fosfomycin or 20 μg · l ⁻¹ carbenicillin. Chromosomal DNA is isolated from clones that grow fresh on phosphomycin plates but do not grow on carbenicillin plates. The integration of the glf gene into the gene encoding the PTS system is confirmed by sudden analysis. Corresponding variants are as in phenotypic PTS-deficient.

One clone is selected from the host organism Escherichia coli AT2471glfintPTS ⁻ and used for transformation with plasmid pGEM7gntKgdhIV (see above).

According to the experimental conditions described in Examples 3 and 4, the PTS ^- negative variant Escherichia coli AT2471glfintPTS-/ pGEM7gntKgdhIV and the corresponding host strain AT2471glfintPTS ^- in each case are incubated in a mixture of two parallels. After incubation for 48 hours, integrated biomass-specific productivity is calculated from the results.

Integrated biomass-compared and variants AT2471glfintPTS ^{- -} specific productivity is host strain S. Cherry Escherichia coli AT2471glfintPTS represented by the index value of 100 / pGEM7gntKgdhIV integrates biomass having an exponent value of a 133-form the specific productivity.

The result is a microorganism characterized by a PTS system that introduces glucose dehydrogenase activity and increases the activity of gluconate kinase while simultaneously integrating a PEP-independent sugar uptake system to reduce or completely block enzymatic activity. Has been shown to positively affect phenylalanine synthesis products.

Strain Genotype / Features Sources and References Bacillus Megaterium DSM 319 Donor for gdhIV Gene J. Bacteriol, Nagao et al. 174, 5013-5020 Escherichia coli AT2471 tyr A4, rel A1, spo T1, thi -1 Taylor and Trotter, Bacteriol. Rev. 13 (1967) 332-53 JM109DE3 Δ ( pro-lac ) / F ′ pro ⁺ lacZΔM 15; Carry genes for T7 RNA polymerase Promega Co. Escherichia coli K-12 W3110 F-, prototropic wild type strain, thi- 1; Donor for gnt K Gene Coli Genetic Stock Center, Yale University, New Haven, CT, U.S.A. Plasmid pZY507 Cm ² J. Bacteriol 177 (1995) 3351-4 by Weisser et al. pZY507 glf pZY507 My Zimomonas mobilis glf -gene J. Bacteriol 177 (1995) 3351-4 by Weisser et al. PGEM7 ApR; T7 and SP6 Promoter Promega Co. PGEM7 gnt K PGEM7 containing Escherichia coli gnt K gene This application PGEM7 gdhIV PGEM7 containing the Bacillus megaterium gdh IV gene This application pGEM7 gnt Kg dhIV pGEM7 containing the gnt K and gdh IV genes This application

Determination of Glucose Dehydrogenase and Gluconate Kinase Activity in Crude Extracts of Escherichia coli with Various Genetic Structures Strain Specific Activity of Glucose Dehydrogenase Specific Activity of Gluconate Kinase W3110 / pGEM7 n.d.a. n.d.a. W3110 / pGEM7 gdh IV 0.4U / mg n.d. W3110 / pGEM7 gnt K n.d.a. 0.9U / mg W3110 / pGEM7 gnt K gdh IV 1.0U / mg 0.9U / mg

n.d.a. = undetectable activity; n.d. = not measured

Claims (29)

One or more substances selected from the group consisting of phenylalanine, tryptophan, tyrosine, indigo, indole acetic acid, adipic acid, melanin, shikimic acid, chorismic acid, quinone, benzoic acid and derivatives thereof are microbiological As a method of manufacturing Microorganisms selected from the group consisting of the genus Esccherichia, Serratia, Bacillus, Corynebacterium, and Brevibacterium [microorganisms are Bacillus species Formulated to have an increased activity of glucose dehydrogenase derived from Bacillus species; and a metabolic pathway step of converting the glucose or glucose-containing substrate to gluconolactone or gluconic acid. How to. delete The method of claim 1, Bacillus megaterium Glucose Dehydrogenase Activity is Escherichia, Serratia, Bacillus, Corynebacterium, and Brevibacterium ) Is introduced into and / or increased in the microorganism selected from the group consisting of The method of claim 3, wherein Activity of Bacillus megaterium glucose dehydrogenase IV into Escherichia genus, Serratia genus, Bacillus genus, Corynebacterium genus and Brevibacterium genus Introduced into and / or increased in the microorganism selected from the group consisting of: The method according to any one of claims 1, 3 and 4, A method characterized by an additional increase in the activity of Escherichia coli gluconate kinase. delete delete The method according to any one of claims 1, 3 and 4, A method in which the activity of gluconolactonase is additionally increased. The method according to any one of claims 1, 3 and 4, A method characterized by an additional increase in the activity of Zimomonas mobilis glucose promoter protein (Glf). delete delete The method according to any one of claims 1, 3 and 4, (a) Escherichia coli gluconate kinase, Glunolactonase and Zimomonas mobilis glucose promoters encoding glucose dehydrogenases derived from Bacillus species or together with glucose dehydrogenases derived from Bacillus species Introducing and / or introducing gene (s) encoding one or more enzymes selected from the group consisting of Protein (Glf), (b) increase and / or increase the number of gene copies of said enzyme (s), (c) increase and / or increase gene expression of the gene (s) encoding said enzyme (s), (d) increase / increase the endogenous activity of the enzyme (s), (e) alter the structure of the enzyme (s) and / or (f) use the enzyme (s) as deregulated enzyme (s), and / or (g) introducing a gene encoding said enzyme (s) as deregulated enzyme (s), Escherichia coli gluconate kinase, Glunolactonase and Zimomonas mobilase with increased activity of glucose dehydrogenase derived from Bacillus species or with activity of glucose dehydrogenase derived from Bacillus species A method of increasing activity of at least one enzyme selected from the group consisting of glucose promoter proteins (Glf). The method of claim 12, The increase in activity is achieved by incorporating the gene or genes into a single gene construct or several gene constructs, wherein the gene or genes are introduced into the gene construct as a single copy or the genetic construct of increased copy number. The method of claim 9, Wherein the activity of the PEP-dependent uptake system (if any) on the glucose or glucose-containing substrate is additionally reduced or switched off. The method according to any one of claims 1, 3 and 4, DAHP synthase, shikimate kinase, chorismate mutase / prephenate dehydratase, transaldolase, transketolase and glucose At least one enzyme selected from the group consisting of glucokinase has deregulated and / or increased activity. The method according to any one of claims 1, 3 and 4, Method wherein the material produced is phenylalanine, tryptophan or tyrosine. The method of claim 16, Method wherein the material produced is L-phenylalanine. delete The method according to any one of claims 1, 3 and 4, Escherichia coli is used. delete delete delete delete delete delete delete delete delete The method according to any one of claims 1, 3 and 4, Gene containing a gene encoding glucose dehydrogenase together with a gene encoding Escherichia coli gluconate kinase, or a gene encoding glucose dehydrogenase together with a gene encoding a geomonas mobilis glucose promoter protein (Glf) In a recombinant form, or selected from the group consisting of a gene encoding Escherichia coli gluconate kinase, a gene encoding glucononolactonase, and a gene encoding Jimmonas mobilis glucose promoter protein (Glf). A transgenic cell is used which retains in a replicable form a gene construct containing in recombinant form a gene encoding a glucose dehydrogenase with more than one gene.