EP1090126A1 - Microbial preparation of substances from aromatic metabolism/iii - Google Patents

Abstract

The invention relates to a process for microbially preparing substances from aromatic metabolism, in particular aromatic amino acids. In addition, the invention relates to gene structures and transformed cells. According to the invention, an increase in the production of substances, in particular aromatic amino acids, is observed by introducing or increasing the activity of a glucose dehydrogenase. Increasing the activity of a glucose-oxidizing enzyme leads to the intracellular formation of gluconolactone and gluconic acid from glucose-containing substrates. In a preferred embodiment, the glucose dehydrogenase is derived from Bacillus megaterium. The process according to the invention can be used to provide a wider spectrum of substances.

Description

MICROBIAL PREPARATION OF SUBSTANCES FROM AROMATIC METABOLISM/III

The invention relates to a process for the microbial preparation of substances from aromatic metabolism according to Claims 1 to 19 and 29, to gene structures according to Claims 20 to 22, and to transformed cells according to Claims 23 to 28.

Microbially prepared substances from aromatic metabolism, in particular aromatic amino acids, are of great economic interest, with the requirement for amino acids, for example, continuing to increase .

Thus, L-phenylalanine, for example, is used for preparing medicaments and, in particular, also in the preparation of the sweetener aspartame (α-L- aspartyl-L-phenylalanine methyl ester) . L-Tryptophan is required as a medicament and as an additive for feedstuffs; there is likewise a need for L-tyrosine as a medicament and as a raw material in the pharmaceutical industry. Besides isolation from natural materials, biotechnological preparation is a very important method for obtaining amino acids, in the desired optically active form, under economically justifiable conditions. The biotechnological preparation is effected either enzymically or using microorganisms.

The latter, microbial preparation has the advantage that simple and inexpensive raw materials can be employed. Since, however, the biosynthesis of the amino acids is controlled in the cell in a great variety of ways, many different attempts have already - 2 -

been made to increase product formation. Thus, amino acid analogues, for example, have been employed for switching off the regulation of biosynthesis. For example, by selecting for resistance to phenylalanine analogues, mutants of Escherichia coli were obtained which made it possible to achieve an increased production of L-phenylalanine (GB-2 , 053 , 906) . A similar strategy also led to overproducing strains of Corynebacterium (JP-19037/1976 and JP-39517/1978) and Bacillus (EP-0 , 138 , 526) .

In addition, microorganisms which have been constructed by recombinant DNA techniques are known in which the regulation of biosynthesis is likewise abolished by the cloning and expression of the genes which encode key enzymes which are no longer feedback- inhibited. As a model, EP-0,077,196 describes a process for producing aromatic amino acids in which a 3-deoxy- D-arabinoheptulosonate- 7 -phosphate synthase (DAHP synthase) which is no longer feedback- inhibited is overexpressed in E. coli . EP-0,145,156 describes an

E. coli strain in which, for producing L-phenylalanine, chorismate mutase/prephenate dehydratase is additionally overexpressed.

The strategies mentioned share the feature in common that the intervention for improving production is confined to the biosynthetic pathway which is specific for the aromatic amino acids. However, in order to increase production further, it is necessary to endeavour to improve the provision of the primary metabolites, phosphoenolpyruvate (PEP) and erythrose-4 -phosphate (Ery4P) , which are required for producing aromatic amino acids.

PEP is an activated precursor of the glycolysis product pyruvate (pyruvic acid) ; Ery4P is an intermediate of the pentose phosphate pathway. - 3 -

These many different attempts to increase productivity are all directed towards overcoming the limitation to the cytosolic synthesis of the amino acids. In the production of aromatic amino acids, the primary metabolites phosphoenolpyruvate (PEP) and Ery4P are required for the condensation to form 3-deoxy-D- arabinoheptulosonate-7-phosphate (DAHP) .

The literature describes several strategies for increasing the availability of Ery4P, for example an increased provision of Ery4P, and as a consequence an improved formation of L-tryptophan, L-tyrosine or L-phenylalanine product, is made possible by overexpressing transketolase (EP Patent Application No. 0 600 463; Frost & Draths , Ann. Rev. Microbiol . 49 (1995) 557-579) . It was recently demonstrated (Flores et al . , Nature Biotechnology 14 (1996) 620-623) that a spontaneous glucose-positive revertant of a PTS- negative mutant of Escherichia coli funnelled glucose into the cells by way of the GalP system and was able to grow on glucose. Additional expression of the transketolase gene tktA resulted in an increased formation of the intermediate DAHP being observed (Flores et al . , nature Biotechnology 14 (1996) 620- 623) . In 2 German patent applications, which have the reference numbers DE 196 44 566.3 and DE 196 44 567.1 and which have not yet been published, the applicants demonstrated that it was possible to produce phenylalanine, for example, in increased quantity by increasing the enzyme activities of transaldolase or transaldolase and transketolase in Escherichia coli or by increasing the activity of a glucokinase in Escherichia coli or of a glucokinase and a PEP- independent transport system for sugars in Escherichia coli or by combining the cited enzymes and the - 4 -

transport system.

The object of the invention is to make available a further process by which an improved microbial synthesis of substances from aromatic metabolism is achieved.

A microorganism in which the formation of substances from aromatic metabolism is increased is also to be constructed.

Surprisingly, the object is achieved by glucose or glucose-containing substrates being transformed, in a microorganism producing substances from aromatic metabolism, by way of increasing the activity of a glucose-oxidizing enzyme. In this way an alternative metabolic pathway is provided. This pathway comprises oxidation of the free glucose to gluconolactone/gluconate, and the phosphorylation of the gluconate to form 6-phosphogluconate .

This result is particularly surprising since it is in no way self-evident that simply increasing the activity of a glucose-oxidizing enzyme plays an important role in producing substances from aromatic metabolism.

Within the meaning of the invention, substances from aromatic metabolism are understood as being all compounds whose biochemical synthesis is favoured by the increased provision of Ery4P or Ery4P and PEP. For example, aromatic amino acids, indigo, indoleacetic acid, adipic acid, melanin, shikimic acid, chorismic acid, quinone and benzoic acid, and also their potential derivatives and secondary products.

In this context, it is to be noted that further genetic alterations to the substance-producing microorganisms, in addition to the interventions according to the invention, are required for preparing indigo, adipic acid and other non-natural secondary products. (Frost & Draths, Ann. Rev. Microbiol . 49 (1995) 557-579) .

Microorganisms producing substances from aromatic metabolism can metabolize glucose or glucose- containing substrates, i.e. glucose-containing disaccharides or oligosaccharides , in a variety of ways: thus, it is known that glucose is phosphorylated by ATP-dependent kinases (hexokinase and glucokinases) and thereby funnelled into glycolysis. In addition, many bacteria have available a PEP-dependent system for taking up glucose and phosphorylating it.

Glucose can also be oxidized by various soluble or membrane-bound enzymes (to gluconic acid by way of gluconolactone) . These enzymes include glucose oxidases or glucose dehydrogenases . Glucose oxidases oxidize glucose to gluconolactone with the reduction of molecular oxygen. While glucose dehydrogenases also oxidize glucose to gluconolactone, they use other electron acceptors such as pyrroloquinoline quinone (PQQ) or other cofactors such as nicotine adenine dinucleotide (NAD) or NADP . It is known that membrane- bound glucose dehydrogenases can oxidize glucose using the cofactor pyrroloquinolone quinone (PQQ) , with the reaction taking place on the outer side of the membrane. In order to take up the product

(gluconolactone or gluconic acid) into the cell, there is then a need for a specific transport system, as has been described by van Schie et al . , Journal of Bacteriology 163 (1985) 493-499; Isturiz et al . , Journal of General Microbiology 132 (1986) 3209-3219; Izu et al . , Journal of Molecular Biology 267 (1997) 778-793, whose expression is, as a rule (e.g. in Escherichia coli ) , repressed by the presence of glucose, as described by Izu et al . , Journal of Molecular Biology 267 (1997) 778-793, and Conway. FEMS Microbiology Reviews 103 (1992) 1-27.

Glucose dehydrogenases using the cofactor NAD or NADP are soluble enzymes which are found within the cell. Known producers include Bacillus strains, some of which possess several isoenzymes of glucose dehydrogenase (e.g. glucose dehydrogenases I to IV in the case of Bacillus megaterium; Mitamura et al . , 1990, Journal of Fermentation and Bioengineering 70, 363- 369) . Expression of the glucose dehydrogenases is strictly regulated and is known to occur, for example, only in prespore stages during the formation of endospores by Bacillus species; the physiological role of glucose dehydrogenase in association with growth on glucose is unclear, as has been reported by Lampel et al . , Journal of Bacteriology 166 (1986) 238-243, and recently by Steinmetz or Fortnagel { "Bacillus subtilis and other Gram-positive 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, D.C., 1993).

No NAD (P) -dependent glucose dehydrogenases have so far been described in Escherichia coli , Corynebacteria or Brevibacteria, inter alia. Bacillus strain-derived genes for glucose dehydrogenase (s) have been cloned into Escherichia coli and expressed in this bacterium (Hilt et al . , Biochimica et Biophysica Acta 1076 (1991) 298-304) . The aim was, in particular, to obtain recombinant glucose dehydrogenase, which was employed as a detection system for glucose or for cofactor regeneration (Hilt et al . , Biochimica et Biophysica Acta 1076 (1991) 298-304; DE Patent Application 3 711 881) . By contrast, these genes have not so far been described for exploiting glucose, for example, as a substrate for obtaining substances from aromatic metabolism. - 7 -

In accordance with the invention, it has been found that introducing, or increasing the activity of, a glucose dehydrogenase surprisingly leads to the formation of substances. An increase in the activity of a glucose-oxidizing enzyme leads to the intracellular formation of gluconolactone and gluconic acid from glucose-containing substrates. In a preferred embodiment, the glucose dehydrogenase derives from Bacillus megaterium, in particular the Bacillus znecjateriuxπ glucose dehydrogenase IV, as described by Mitamura et al . , Journal of Fermentation and Bioengineering 70 (1990) 363-369, and Nagao et al . , Journal of Bacteriology 15 (1992) 5013-5020.

Gluconic acid relies on a gluconic acid- phosphorylating enzyme for its activation. It is known that such enzymes can, for example, be phosphoenolpyruvate-dependent enzymes II which have a specificity for gluconic acid, or ATP-dependent kinases having a specificity for gluconic acid. The Escherichia coli ATP-dependent gluconate kinase GntK, as described by Izu et al., FEBS Letters 394 (1996) 14-16; Izu et al . Journal of Molecular Biology 267 (1997) 778-793, and Tong et al . , Journal of Bacteriology 178 (1996) 3260- 3269 is known, for example. In Escherichia coli , the product, 6- phosphogluconate, is an intermediate of both the oxidative branch of the pentose phosphate pathway and of the Entner-Douderoff pathway, as described by Fraenkel , pp. 189-198 in "Escherichia coli and Salmonella" , 2nd Edition (Neidhardt et al . , Eds.), ASM

Press, Washington, USA, ISBN-1-55581-084-5 , 1996.

The production of substances can be improved by additionally increasing the activity of a gluconic acid (gluconate) -phosphorylating enzyme. When a gluconic acid-phosphorylating enzyme is being used, gluconic acid-phosphorylating enzymes, for example, from a variety of microorganisms are suitable, provided that they can be expressed functionally in the microorganisms producing substances from aromatic metabolism. The use of an ATP-dependent gluconate kinase, preferably an Escherichia coli gluconate kinase, in particular the gluconate kinase (GntK) from Escherichia coli K-12, is particularly advisable. Other genes for gluconic acid-phosphorylating enzymes, whose gene products phosphorylate gluconic acid, are just as suitable for the process according to the invention. Genes from other enterobacteria, Zymomonas mobilis , Bacillus subtilis and Corynebacterium glutamicum may be mentioned by way of example. The effect of gluconate kinase is restricted to activating gluconic acid/gluconate, which is not, however, metabolized by Escherichia coli or other bacteria in the presence of glucose, for example. For example, in Escherichia coli , the gluconate kinase GntK is only of importance when the organism is growing on gluconic acid and does not contribute to the metabolism of glucose; indeed its formation is actually repressed in the presence of glucose, as described by Izu et al . , Journal of Molecular Biology 267 (1997) 778-793, and Tong et al . , Journal of Bacteriology 178 (1996) 3260-3269, and does not take place in the presence of glucose.

In this particular embodiment, therefore, the additional, increased activity of a gluconic acid- phosphorylating enzyme, in particular a gluconate kinase, best of all an Escherichia coli gluconate kinase, and, in particular, the gluconate kinase GntK from Escherichia coli K-12, provides an enzyme which enables a gluconic acid-phosphorylating enzyme to be made available in microorganisms in the absence of 9 -

extracellular gluconate or in the presence of glucose. The advantage is an increased flow of material by way of the metabolic pathway according to the invention. This enables the conversion of gluconic acid to 6-phosphogluconate to be increased even in the presence of glucose. This leads to an increase in the intracellular proportion of 6-phosphogluconate present, which can be converted into the said substances by way of known metabolic sequences. Gluconolactone is a reaction product of the glucose-oxidizing enzyme. While gluconolactone can convert spontaneously into gluconic acid, enzymes have also been described which catalytically accelerate this conversion {Zymomonas mobilis gluconolactonase, for example, as described by Kanagasundaram and Scopes,

Biochimica et Biophysica Acta 1171 (1992) 198-200) . In another embodiment, therefore, the gene for a gluconolactonase (e.g. from Zymomonas mobilis) is expressed, in addition to the glucose-oxidizing enzyme or in addition to the glucose-oxidizing enzyme and the gluconic acid-phosphorylating enzyme, in order to accelerate the conversion of gluconolactone to gluconic acid (or to 6-P-gluconate, respectively) .

It may be noted that genes for glucose dehydrogenase and gluconate kinase can occur naturally in some Bacillus species; however, the genes for the enzymes are arranged in different operons and are evidently not used jointly for metabolizing glucose, as described by 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, D.C., 1993). In addition, because of the specific induction of the glucose dehydrogenases under sporulation conditions, it is not to be expected that 10 -

the two enzymes would contribute together to the formation of substances.

Consequently, the effect, which is described within the context of this invention, of the increased activity of a glucose-oxidizing enzyme, or of a glucose-oxidizing enz me and a gluconic acid- phosphorylating enzyme, respectively, for the production of substances from aromatic metabolism, in association with growth on glucose or glucose- containing substrates, is completely unexpected.

In another preferred embodiment of the invention, the activity of a transport protein for the PEP-independent uptake of a sugar is increased in addition to increasing the glucose-oxidizing enzyme or the glucose-oxidizing enzyme and the gluconic acid- phosphorylating enzyme.

This embodiment also includes increasing the activity of a transport protein for the PEP- independent uptake of glucose or of glucose-containing substrates in a microorganism producing substances from aromatic metabolism which is able to take up the sugar by means of a PEP-dependent transport system. The additional integration of a PEP-independent transport system makes it possible to increase the provision of the sugar in the microorganism producing said substances. According to the invention, this sugar can be converted by an intracellular glucose-oxidizing enzyme into gluconolactone and subsequently gluconic acid. Gluconic acid is then the substrate for a gluconic acid-phosphorylating enzyme. Usually, PEP is not required as an energy donor for these reactions and is therefore available, on the basis of a constant flow of material in glycolysis and the pentose phosphate pathway, in greater quantity for the condensation with Ery4P to form the primary metabolite of the common - 11 -

biosynthetic pathway for aromatic compounds, i.e. 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), and subsequently for producing substances from aromatic metabolism. In the case of the transport protein, activity is understood as being the protein-mediated uptake rate.

With regard to the transport protein for the PEP-independent uptake of glucose or glucose- containing substrates, it is advisable to use transport proteins, in particular a facilitator, that is a transport protein which acts in accordance with the principle of protein-mediated facilitated diffusion. The use of the glucose facilitator protein (Glf) from Zymomonas mobilis is particularly suitable. When the latter is used, the gene, i.e. glf, encoding the protein is derived, for example, from Z . mobilis, in particular the facilitator gene glf isolated from Z. mobilis ATCC 31821, as described by Parker et al . , 1995, Molecular Microbiology 15 (1995) 795-802, and Weisser et al . , 1995, Journal of Bacteriology 177 (1995) 3351-3354. However, other bacterium-derived sugar transport genes, whose gene products transport glucose and do not use any PEP in doing so, for example the Escherichia coli GalP system, are just as suitable for the process according to the invention. In addition, use can be made of genes for sugar transport systems, such as HXT1 to HXT7 , which are derived from eukaryotic microorganisms such as Saccharomyces cerevisiae, Pichia stipi tis or Kluyveromyces lactis , or, quite generally, of sugar transport genes derived from other organisms provided that they can be expressed functionally in the microorganisms and that, at the same time, the gene products can operate without PEP for phosphorylating and/or transporting the - 12 -

glucose. It is particularly advisable that the sugar transport genes can be expressed in amino acid producers .

Within the meaning of the invention, measures for increasing activity are to be understood as being all measures which are suitable for increasing the activity of a glucose-oxidizing enzyme or the activity of a glucose-oxidizing enzyme and, in addition, at least one activity from gluconic acid- phosphorylating enzyme, gluconolactonase and transport protein for the PEP-independent uptake of sugar. The following are particularly suitable for this purpose: introducing genes, for example using vectors or temperate phages; - increasing the gene copy number, for example using plasmids, with the aim of introducing the genes according to the invention into the microorganism in increased copy number, from slightly (e.g. 2 to 5 times) to greatly increased copy number (e.g. 15 to 50 times) ; increasing gene expression, for example by increasing the transcription rate, for example by using promoter elements such as Ptac, Ptet or other regulatory nucleotide sequences and/or by increasing the translation rate, for example by using a consensus ribosome binding site; increasing the endogenous activity of existing enzymes, for example by mutations which are generated in an undirected manner in accordance with classical methods, for example by UV irradiation or mutation-producing chemicals, or by mutations which are generated specifically by means of recombinant DNA methods such as deletion(s), insertion(s) and/or nucleotide exchange ( s ) ; - 13 -

increasing the activity of enzymes by altering the structure of enzymes, for example by mutagenizing using physical, chemical, molecular biological or other microbiological methods; - using deregulated enzymes, e.g. enzymes which are no longer feedback-inhibited; introducing corresponding genes which encode the deregulated enzymes .

It is also possible to employ combinations of the cited methods and other, analogous methods for increasing activity. In the case of transport proteins, the endogenous activity can be increased, for example, by cloning the gene using abovementioned methods, for example, or by way of selecting mutants which exhibit an increased transport of substrates.

Preferably, the increase in activity is effected by the gene or the genes being integrated into a gene structure or into several gene structures, with the gene or the genes being introduced into the gene structure as single copies or in increased copy number.

Within the meaning of the invention, gene structure is to be understood as being a gene or any nucleotide sequence which carries the genes according to the invention. Appropriate nucleotide sequences can, for example, be plasmids, vectors, chromosomes, phages or other nucleotide sequences which are not closed in a circular manner.

The availability of PEP for producing the first intermediate of aromatic amino acid metabolism can be limited in microorganisms in which the material flow towards Ery4P is elevated. In such cases, it can be advantageous to decrease or switch off other PEP- consuming reactions in metabolism, if present, such as the reaction of the PEP : sugar phosphotransferase system (PTS) , which catalyses a PEP-dependent sugar uptake. 14

According to the invention, use can be made of organisms which exhibit the natural level of PTS activity; however, it is also possible, for the purpose of further improving the process, to use PTS mutants in which the activity of the PTS has been decreased. A decrease of this nature can be effected either at the enzymic level or by means of genetic methods, e.g. by using alternative, strongly repressible promoters for expressing the pts genes, or by inserting a glf gene into the chromosome and in particular into the gene locus of the ptsl gene, which at the same time involves stabilization of the recombinant DNA in the chromosome (segregation stability) and, as a result, the ability to dispense with using a vector. Furthermore, in conjunction with a regulable promoter, the activity of the PTS can also be influenced by adding inducers or inhibitors of the relevant promoter during culture.

In the process according to the invention for producing substances from aromatic metabolism, preference is given to employing microorganisms in which one or more enzymes, which are additionally involved in the synthesis of these substances, are deregulated and/or have had their activity increased. These enzymes are, in particular, the enzymes of aromatic amino acid metabolism and especially DAHP synthase, shikimate kinase and chorismate mutase/prephenate dehydratase, and also all other enzymes, in particular transaldolase, transketolase and glucokinase as well, which are involved in the synthesis of substances from aromatic metabolism.

Apart from the enzymes according to the invention, it is particularly the deregulation and overexpression of DAHP synthase which is of importance for preparing substances such as adipic acid, bile acid - 15 -

and quinone compounds and their derivatives . In addition, shikimate kinase should be deregulated and have its activity increased in order to achieve excess synthesis of, for example, L-tryptophan, L-tyrosine, indigo, and derivatives of hydroxy- and aminobenzoic acid and naphtho- and anthraquinones , and their secondary products. A deregulated and overexpressed chorismate mutase/prephenate dehydratase is additionally of particular importance for efficient production of phenylalanine and phenylpyruvic acid and their derivatives. However, this is also intended to encompass all other enzymes whose activities contribute to the biochemical synthesis of substances whose production is promoted by the provision of Ery4P or Ery4P and PEP.

It may be noted that, apart from the interventions according to the invention, further genetic alterations to the microorganisms are required in order to prepare indigo, adipic acid and other non- natural secondary products. These measures are known to the skilled person (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, preference is given to increasing, at the same time, the gene expression and/or enzyme activity of a deregulated DAHP synthase (e.g. in E. coli AroF or AroH) and/or a likewise deregulated chorismate mutase/prephenate dehydratase (PheA) .

Suitable production organisms are Escherichia species and also microorganisms of the genera Serratia, Bacillus, Corynebacterium or Brevibacterium, and other strains known from classical amino acid methods. Likewise bacteria from the families 16

Nocardiaceae and Actinomycetales . Escherichia coli is particularly suitable.

The invention also relates to the provision of suitable gene structures and transformed cells which carry these gene structures and which make it possible to implement the process particularly successfully.

Within the context of the invention, novel gene structures are now made available, which gene structures, in recombinant form, either contain a gene which encodes a glucose-oxidizing enzyme a) together with a gene which encodes a gluconic acid- phosphorylating enzyme or b) together with a gene which encodes a transport protein for the PEP-independent uptake of a sugar or c) together with at least two of the three following genes encoding a gluconic acid- phosphorylating enzyme, a gluconolactonase or a transport protein for the PEP-independent uptake of a sugar.

In particular, the gene for the glucose- oxidizing enzyme encodes a glucose dehydrogenase and the gene for the gluconic acid-phosphorylating enzyme encodes a gluconate kinase.

The gene for the glucose dehydrogenase is preferably derived from Bacillus megaterium, while the gene for the gluconate kinase is preferably derived from Escherichia coli and the gene for gluconolactonase and the transport protein are preferably derived from Zymomonas mobilis. Gene structures in which the gene for the glucose dehydrogenase is the glucose dehydrogenase IV (gdhlV) from Bacillus mega terium, the gene gntK for the gluconate kinase is the GntK from Escherichia coli , and the genes for the transport protein and the gluconolactonase are the glf and gnl genes from Zymomonas mobilis are particularly advantageous. The isolation of the relevant genes, and - 17 -

the transformation of the cells, are effected in accordance with current methods: In the case, for example, of cloning the E. coli gluconate kinase gene gntK, the Bacillus megaterium glucose dehydrogenase IV (grd TV) gene, or the Zymomonas mobilis gluconolactonase {gnl ) gene or transport gene glf, the polymerase chain reaction (PCR) method for specifically amplifying the gene is, for example, suitable using chromosomal DNA from Escherichia coli K-12 { gntK) , Bacillus megaterium {gdhlV) and the Zymomonas mobilis strains ATCC 29191 or ATCC 31821 { gnl , glf) , respectively.

After amplifying the DNA and recombining it in vitro with known vectors (pGEM7, pUCBM20, pUC19 or others) , the host cell is transformed by means of chemical methods, electroporation, transduction or conjugation.

The complete nucleotide sequences of the gntK, gdhlV, gnl and glf genes from the 3 donor organisms are known and obtainable from generally accessible sources, for example deposited in databases such as in the EMBL/HUSAR in Heidelberg under access numbers D 84362 {gntK) , D 10626 {gdhlV) , X 67189 { gnl) and M 60615 {glf) . PCR for specifically amplifying the gene using chromosomal DNA from Escherichia coli K-12 strains and the gene sequences as described by Izu et al . Journal of Molecular Biology 267 (1997) 778- 793, and Tong et al . Journal of Bacteriology 178 (1996) 3260-3269 is suitable for cloning the Escherichia coli gntK gene. Chromosomal DNA from Bacillus megaterium is, for example, suitable for cloning the Bacillus megaterium gdhlV gene (Nagao et al . Journal of Bacteriology 174 (1992) 5013-5020) .

The isolated glucose dehydrogenase IV gene can be integrated, together with one or more of the genes described within the context of the invention, in any combination, into a gene structure or into several gene structures. Without considering the precise allocation to gene structures, this leads to combinations such as gdhlV + gntK, gdhlV + glf, gdhlV + gntK + glf; gdhlV + gntK + gnl ; gdhlV + gnl + glf; gdhlV + gntK + gnl + glf . In addition to the above- mentioned gene structures, any gene structure is meant which additionally includes one or more of the genes encoding for transketolase, transaldolase, glucokinase, DAHP- synthase, chorismate mutase / prephenate dehydratase, chorismate mutase / prephenate dehydrogenase, or for other enzymes positively influencing the synthesis of substances from aromatic metabolism.

When allocating the genes, glf is preferably introduced in low copy number x (such as x = 1 to 10) into the gene structure or the gene structures in order to avoid possible negative consequences arising from overexpressing a membrane protein.

Gene structures which contain at least one regulatory gene sequence, which is assigned to one of the genes, are advantageous.

Thus, regulatory elements can preferably be reinforced at the transcriptional level by, in particular, reinforcing the transcription signals. This can be effected, for example, by increasing the activity of the promoter or the promoters by altering the promoter sequences which are located upstream of the structure genes, or by completely replacing the promoters with more active promoters. Transcription can also be reinforced by appropriately influencing a regulatory gene which is assigned to the genes; in addition to that, however, it is also possible to reinforce the translation by, for example, improving the stability of - 19

the messenger RNA (mRNA) .

Furthermore, within the context of the invention, transformed cells which harbour a gene structure according to the invention in replicatable form are also made available. Within the meaning of the invention, a transformed cell is to be understood as being any microorganism which carries a gene structure according to the invention which brings about the increased formation in the cell of substances from aromatic metabolism. The host cells can be transformed by chemical methods (Hanahan J. Mol . Biol. 166 (1983) 557-580) , and also by electroporation, conjugation or transduction.

For the transformation, it is advantageous to employ host cells in which one or more enzymes, which are additionally involved in the synthesis of the substances, are deregulated and/or have had their activity increased. A microorganism strain, in particular Escherichia coli , which according to the invention produces an aromatic amino acid or another substance from aromatic metabolism is transformed with the gene structure which contains the relevant genes.

It is advantageous, for the transformation with the gene structures, to employ host cells in which, in addition, the PEP-dependent sugar uptake system, if present, has had its activity decreased or switched off.

In particular, transformed cells are made available which are able to produce an aromatic amino acid, with the aromatic amino acid preferably being

L-phenylalanine .

A process for microbially preparing substances from aromatic metabolism is consequently made available, which process employs transformed cells, as described above, which harbour gene - 20 -

structures, as described above.

In a particularly preferred embodiment of the process according to the invention, use is made of transformed cells which, apart from Ery4P, also contain other metabolites of central metabolism in increased availability. Examples of these metabolites are α-oxoglutarate or oxaloacetate , which result from intracellular synthetic processes, or else are made available to the growing cells by feeding in the corresponding compounds, or their precursors, such as fumarate or malate, as metabolites of the citric acid cycle .

The strain Escherichia coli AT247l/pGEM7gntKgdhIV was deposited in the DSMZ (German Collection of Microorganisms and Cell Cultures) on

15.04.1998 under the deposition number DSM 12118 and under the conditions of the Budapest Treaty.

The host organism employed, i.e. AT2471, was deposited by Taylor and Trotter (Bacteriol. Rev. 13 (1967) 332-53) in the CGSC under number 4510 and can be obtained without payment .

In that which follows, the materials and methods employed will be indicated and the invention will be supported by experimental examples and comparative examples:

General methods

Within the context of the genetic studies, E. coli strains were, unless otherwise mentioned, cultured on LB medium consisting of Difco Bacto tryptone (10 g-1^"1), Difco yeast extract (5 g-1^"1) and NaCl (10 g-1^"1) . Depending on the resistance properties of the strains employed, ampicillin (100 mg-1^"1) and chloramphenicol (17-34 mg-1^"1) were added, if necessary, to the medium. In this context, ampicillin was 21 -

previously dissolved in water and chloramphenicol previously dissolved in ethanol and then added to the already autoclaved medium after having been sterilized by filtration. Difco Bacto agar (1.5%) was added to the LB medium for preparing agar plates.

Plasmid DNA from E. coli was isolated by means of alkaline lysis using a commercially available system (Qiagen, Hilden) . Chromosomal DNA was isolated from E. 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 phosphatase, RNase and T4 DNA ligase were used in accordance with the producers' (Boehringer, Mannheim, Germany or Promega, Heidelberg, Germany) instructions. For restriction analysis, the DNA fragments were fractionated in agarose gel (0.8%) and isolated from the agarose by extraction using a commercially available system (QuiaExII, Hilden, Germany) . Before being transformed, the cells were incubated at 37°C and 200 rpm for 2.5-3 h in LB medium (5 ml tubes) . At an optical density (620 nm) of approx. 0.4, the cells were centrifuged down and taken up in a tenth of the volume of TSS (LB medium containing 10% (w/v) PEG 8000, 5% (v/v) DMSO and 50 mM MgCl₂) . After having been incubated for 30 minutes at 4°C with from 0.1 to 100 ng of DNA and subsequently incubated at 37°C for 1 h, the cells were plated out on LB medium containing the appropriate antibiotic.

Example I

Preparing pGEM7σntKσdhIV as a model of plasmid-based αene structures according to the invention

The Bacillus megaterium DSM 319 gdhlV gene, encoding glucose dehydrogenase IV, was cloned following - 22 -

specific amplification of the chromosomal DNA of Bacillus megaterium DSM 319 by the polymerase chain reaction (PCR) on the basis of the known DNA sequence of the gene, which was described by Nagao et al . Journal Bacteriology 15 (1992) 5013-5020. The PCR oligonucleotide primers were provided with cleavage sites for the restriction enzymes BamHI (5' end) and Sad (3' end). Primer 1 (BamHI) consisted of 5' ATG GAT CCA TGA AAA CAC TAG GAG GAT TTT 3' . Primer 2 (Sad) consisted of 5' GCC AGA GCT CTT TTT TCC ACA TCG ATT AAA AAC TAT 3 ' , and was complementary to the 3 ' end of the gdhlV gene. The resulting DNA amplification product, of approx. 800 base pairs, was restricted with BamHI plus Sad and then ligated into the vector pGEM7 , which had been treated in the same way (see Tab. 1) . Transformation was effected into the strain JM109DE3, with selection on LB agar plates containing X-Gal and ampicillin. Successful cloning was detected by determining the DNA sequence of the cloned gdhlV gene. This vector (pGEM7gdhIV) enabled glucose dehydrogenase IV activity to be expressed even in the absence of the T7 polymerase system (strain JM109DE3) (see Tab. 2) . The gntK gene for the Escherichia coli K-12 gluconate kinase, was cloned by specific DNA amplification using the strain E. coli K-12 W3110 as the chromosomal template. The sequence of the gntK gene has been described by Tong et al . Journal of Bacteriology 178 (1966) 3260-3269. For amplification by PCR, oligonucleotide primers were selected which were additionally provided with restriction cleavage sites for EcoRI (5') and BamHI (3') . Primer 1 consisted of 5 ' CCG AAT TCT TGT ATT GTG GGG GCA C 3 ' and binds 5 ' upstream of the gntK gene; primer 2 consisted of 5' CCG GAT CCG TTA ATG TAG TCA CTA CTT A 3' and is complementary to the 3' end of the gntK gene. The 23 -

amplification product of approx. 600 base pairs was purified, restricted with EcoRI plus BamHI and ligated into vector pGEM7 , which had been opened as well. Transformation was effected into the strain JM109DE3, with selection on LB agar plates containing X-Gal and ampicillin. Successful cloning was detected by determining the DNA sequence of the cloned gntK gene. This vector (pGEM7gntK) enabled the gluconate kinase activity to be expressed even in the absence of the T7 polymerase system (strain JM109DE3) (see Tab. 2) .

The gntK and gdhlV genes were combined by opening vector pGEM7gntK by subjecting it to double restriction with BamHI plus Sad . An 800 base pair fragment, which had been obtained after restricting vector pGEM7gdhIV and which contained the gdhlV gene, was then ligated into this vector which had been opened in this way. Transformation was again carried out with selection on ampicillin. The new gene structure pGEM7gntKgdhIV, in accordance with the invention, mediates T7 polymerase- independent expression of the enzyme activities for glucose dehydrogenase IV and for gluconate kinase GntK (see Tab. 2) .

The transformants which had been obtained were stored on LB medium in the form of glycerol cultures (30%) at -80°C. When required, the glycerol cultures were thawed directly before use.

Exampl e II : determining the enzyme activities of σlucose dehydrogenase and σluconate kinase

For determining the enzyme activities in crude bacterial extracts, the E . coli cells, and the cells of plasmid-harbouring mutants, were cultured in mineral medium. This medium consisted of sodium citrate-3H₂0 (1.0 g-1^"1), MgS0₄ ^• 7H₂0 (0.3 g-1^"1), KH₂P0₄ - 24

(3.0 g-1^"1), K₂HP0₄ (12.0 g-1^"1), NaCl 0.1 (g-1^"1), (NH₄)₂S0₄ (5.0 g-1^"1), CaCl₂ ^■ 2H₂0 (15.0 mg-1^"1), FeS0₄ • 7H₂0 (0.075 g-1^"1), and L-tyrosine (0.04 g-1^"1). Further minerals were added in the form of a trace element solution (1 ml-1^"1) which was composed of Al₂ (S0₄) ₃ ^■ 18H₂0 (2.0 g-1^"1), CoS0₄-6H₂0 (0.7 g-1^"1), CuS0₄ ^• 5H₂0 (2.5 g-1^"1), H₃BO3 (0.5 mg-1^"1), MnCl₂-4H₂0 (20.0 g-1^"1), Na₂Mo0₄-2H₂0 (3.0 g-1^"1), NiS0₄-3H₂0 (2.0 g-1^"1) and ZnS0₄-7H₂0 (15.0 g-1^"1). Vitamin Bl (5.0 mg-1^"1) was dissolved in water and was added, after having been sterilized by filtration, to the medium after the latter had been autoclaved, just as were ampicillin and/or ampicillin and chloramphenicol when required. Glucose (30 g-1^"1) was autoclaved separately and likewise added to the medium after the latter had been autoclaved.

The harvested cells were washed in 100 mM tris/HCl buffer (pH 8.0). The cells of the sediment were disrupted by ultrasound (Branson sonifier 250 fitted with a microtip) , in a sonication cycle of 25% and with an intensity of 40 watts, for 4 min per ml of cell suspension. After centrifuging for 30 min at 18,000 g and 4°C, the supernatant (crude extract) was used for measuring the activity of the glucose dehydrogenase and/or gluconate kinase.

The activity of the glucose dehydrogenase was determined in accordance with Harwood & Cutting, Molecular Biological Methods for Bacillus, John Wiley & sons. Glucose dehydrogenase catalyzes the oxidation of glucose to gluconolactone. The activity of the enzyme was determined photometrically at a wavelength of 340 nm by means of the increase in the concentration of the reduced cofactor NADH + H⁺. The determination was performed in quartz cuvettes having a total volume of 1 ml . The reaction mixture consisted of Tris HC1 (final - 25

concentration 250 mM, pH 8.0), 2.5 mM sodium EDTA, 100 mM KCl and 2 mM NAD. Crude extract was preincubated at 25°C for 5 minutes in the buffer. Glucose (final concentration, 100 mM) was added to start the detection reaction. The increase in extinction was monitored at 340 nm. A mixture without glucose served as the control in each case. The specific glucose dehydrogenase activity is given in U/mg, defined as the formation of 1 μmol of NADH per minute and per mg of protein, which is set at being equivalent to the conversion of 1 μmol of glucose per minute and per mg of protein.

The gluconate kinase was determined in the crude extract as described by Izu et al . , FEBS Letters 394 (1996) 14-16. Gluconate kinase catalyzes the ATP- dependent phosphorylation of gluconate to 6- phosphogluconate . In the enzyme test, the 6- phosphogluconate formed is determined photometrically at a wavelength of 340 nm by means of the increase in NADPH concentration when using the NADP-dependent auxiliary enzyme 6-phosphogluconate dehydrogenase (Boehringer Mannheim, No. 108 405) . In this context, the formation of 1 μmol of NADPH corresponds to the phosphorylation of 1 μmol of gluconate. The enzymic detection was carried out at 25 °C in quartz cuvettes having a total volume of 1 ml. The reaction mixture contained 50 mM Tris HC1, pH 8.0, 100 mM ATP, 0.25 mM NADP, 1.2 units of the auxiliary enzyme 6-phosphogluconate dehydrogenase and variable quantities of crude extract. The mixtures were preincubated at 25 °C for 5 minutes, and the reaction was started by adding gluconic acid (pH 6.8; final concentration in the mixture, 10 mM) . Mixtures to which gluconic acid was not added served as the controls. The protein concentration in the crude 26

extract was determined in accordance with Bradford M.M. (Anal. Biochem. 72 (1976) 248-254) using a commercially available colour reagent . Bovine serum albumin was used as the standard. Table 2 shows the results of the enzyme measurements when using the host strain E. coli W3110 and its mutants harbouring the plasmids pGEM7gdhIV, pGEM7gntK or pGEM7gntKgdhIV. It was found that, when the gene structures which have been described and which are in accordance with the invention were used, it was possible to express the enzymes in a functional manner in cells according to the invention.

Exampl e III : Producing substances using strains which exhibit increased glucose dehydrogenase activity.

The synthetic efficiency of Escherichia coli AT2471 and Escherichia coli AT247l/pGEM7ghdIV was determined in the mineral medium described in Example II. For this, shaking flasks (1000 ml containing 100 ml of medium) were inoculated with 2 ml of glycerol culture and incubated on an orbital shaker at 37 °C and 150 rpm for 72 h. The pH of the cultures was measured at intervals of approximately 12 h and, as required, restored to the starting value of 7.2 by adding KOH

(45%) . In addition, samples (2 ml) were taken after 24 and 48 h for determining the optical density and the concentrations of glucose and L-phenylalanine.

The concentration of phenylalanine was ascertained by means of high pressure liquid chromatography (HPLC, Hewlett Packard, Munich, Germany) in combination with detection by fluorescence (extinction 335 nm, emission 570 nm) , A nucleosil-120-8 C18 column (250-4.6 mm) was used as the solid phase,- the elution was performed using a gradient (eluent A: 27

90% 50 mM phosphoric acid, 10% methanol, pH 2.5; eluent B: 20% 50 mM phosphoric acid, 80% methanol, pH 2.5; gradient: 0-8 min, 100% A, 8-13 min, 0% A, 13-19 min, 100% A) . The elution rate was set at 1.0 ml -min^"1 and the column temperature at 40°C. The post-column derivatization was performed using o-phthaldialdehyde in a reaction capillary (14m- 0.35 mm) at room temperature. Under the conditions described, L-phenylalanine was found to have a retention time of 6.7 min.

By measuring the glucose concentration using enzymic test strips (Diabur, Boehringer Mannheim, Germany) and, independently of the results, subsequently metering in 2 ml of a concentrated glucose solution (500 g-1^"1), care was taken to ensure that glucose did not become limiting in the experimental mixtures .

Simply introducing the plasmid pGEM7gdhIV resulted, after an incubation time of 48 h, in an index value, which described the phenylalanine concentration, of 145 being achieved, as compared with a (phenylalanine) index value of 100 for the host strain E. coli AT2471. This result demonstrates the aromatic compound synthesis-increasing effect, according to the invention, of increasing the activity of a glucose dehydrogenase in substance-producing microorganisms.

Example IV:

Producing substances using strains in which a PEP- independent sugar uptake system is expressed in addition to the activity of the glucose dehydrogenase being increased.

The Zymomonas mobilis glf gene was amplified using the plasmid pZY600 (Weisser et al . , J. Bacteriol 177 (1995) 3351-3345) as the template. At the 28 -

same time, the choice of the primers resulted in a BamHI cleavage site and a Kpnl cleavage site being introduced. Using these unique cleavage sites, the gene was inserted into the vector pUCBM20 (Boehringer Mannheim) , which had likewise been opened with BamHI and Kpnl. A DNA fragment of 1.5 kb in size was isolated from this vector (pBM20glf) by restricting it with BamHI and Hindlll and ligated to the vector plasmid pZY507 (Weisser et al . , J. Bacteriol 177 (1995) 3351- 3345) , which had likewise been opened with the restriction enzymes BamHI and Hindlll. The recombinant plasmid pZY507glf was obtained after transforming E. coli and cloning the transformants . This vector confers resistance to chloramphenicol , contains the lacl^q-tac promoter system and has a low copy number.

Vector pZY507glf was transformed into the host strain AT2471 together with the gene structures of the invention obtained as described in Example I . Following the experimental conditions described for Example III, the mutants E. coli

AT2471glf, E. coli AT2471glf/pGEM7 , E. coli AT2471glf/pGEM7gntK and E. coli

AT2471glf/pGEM7gntKgdhIV were in each case cultured in two parallel mixtures. After 48 h, the concentration of L-phenylalanine in the medium was determined.

In comparison with the starting strain E . coli AT2471glf, which achieves an L-phenylalanine concentration corresponding to the index value of 100, the presence of the vector pGEM7 resulted in an index value of 96, and consequently virtually identical concentrations, being achieved. By contrast, the use of E. coli AT247glf/pGEM7gntK resulted in an L-phenylalanine concentration which, by comparison with the previously mentioned strains, corresponded to an index value of 179. Expression of both the alternative - 29

metabolism genes, i.e. glucose dehydrogenase and gluconate kinase, in E. coli AT2471glf/pGEMgntKgdhlV, made it possible to achieve a further increase, to a phenylalanine concentration-representing index value of 195.

This result demonstrates that expression of an alternative metabolic pathway by introducing the activity of the glucose dehydrogenase and increasing the activity of the gluconate kinase has a positive influence on L-phenylalanine synthesis specifically in those microorganisms in which, at the same time, a PEP- independent sugar uptake system is transformed and expressed.

Example V:

Producing substances using PTS- mutants in which a PEP- independent sugar uptake system is expressed in addition to the glucose dehydrogenase activity being

In order to integrate the glf gene into the genes which encode components of the E . coli PTS system, the plasmid pPTSl was digested with Bgll l and treated with Klenow fragment . The unique cleavage site lies in the ptsl gene. The glf gene was isolated as a BamHI /Kpnl fragment from the plasmid pBM20glfglk and likewise treated with Klenow fragment. Clones which carry the glf gene in the same orientation as the ptsHI genes were obtained by blunt end ligation. A 4.6 kb Pstl fragment, which carries the 3' region of the ptsH gene and also ptsl with an integrated glf and err, was obtained from the resulting plasmid pPTSglf. This fragment was ligated into the EcoRV cleavage site of the vector pGP704. Since this vector is only able to be replicated in λpir strains, transformants which do not 30

harbour this phage have integrated the vector into the chromosome if they are able to grow on carbenicillin. The integration was checked by Southern analysis (Miller V.L. et al . , J. Bacteriol. 170 (1988) 2575-83). In addition to the glf gene, the resulting trans - formants also contained the complete PTS genes.

The vector moiety can be recombined out in a second homologous crossover, leading to loss of the resistance to carbenicillin. Since the pts genes are interrupted by the insertion of the glf gene in this case, the PTS is not functionally expressed in these mutants. The desired PTS^" mutants were selected as follows: After repeatedly subinoculating the still PTS⁺ transformants onto LB medium without antibiotics, aliquots of the cell suspension were plated out on LB plates containing 100 μg-1^"1 phosphomycin. PTS^" mutants are able to grow on these plates . Growing clones were streaked out on LB plates containing either phosphomycin or 20 μg-1^"1 carbenicillin. Chromosomal DNA was isolated from clones which exhibited renewed growth on the phosphomycin plates but not on the carbenicillin plates . Integration of the glf gene into the genes encoding the PTS system was confirmed by Southern analysis. Corresponding mutants were identified as being phenotypically PTS-deficient .

One clone was selected as the host organism E. coli AT2471glfintPTS^" and used for the transformations (see above) with plasmid pGEM7gntKgdhIV. Following the experimental conditions described for Example III and IV, the PTS-negative mutant E. coli AT2471glfintPTS^"/pGEM7gntKgdhIV, and the corresponding host strain AT2471glfintPTS^" , were in each case cultured in two parallel mixtures. After incubating for 48 h, the integral, biomass-specific productivity was calculated from the results. - 3 1 -

As compared with the host strain E. coli AT2471glfintPTS^", whose integral biomass-specific productivity is represented by an index value of 100, the mutant AT2471glfintPTS^"/pGEM7gntKgdhIV achieved an integral biomass-specific productivity which had an index value of 133.

This result demonstrates that introducing the activity of the glucose dehydrogenase and increasing the activity of the gluconate kinase has a positive influence on phenylalanine synthesis productivity specifically in those microorganisms which are characterized by a PTS system whose activity has been diminished or completely switched off and in which, at the same time, a PEP-independent sugar uptake system has been integrated.

32

Table 1

Strains Genotype/characteris ics Source or reference

Bacillus donor for gdhlV gene Nagao et al . J . megaterium Bacteriol. 174,

DSM 319 5013-5020

E. coli tyrA4, relAl, spoTl, Taylor and Trotter,

AT2471 thi-1 Bacteriol. Rev. 13 (1967) 332-53

JM109DE3 A (pro- lac) /F'pro⁺ Promega Co . lacZΔM15; carries gene for T7 RNA polymerase

E . coli K-12 F-, prototrophic wild- Coli Genetic Stock

W3110 type strain, thi-1; Center, Yale donor for gntK gene University, New Haven, CT, U.S.A.

Plasmid Cm² Weisser et al . , J. pZY507 Bacteriol 177 (1995) 3351-4 pZY507glf Z . mobilis glf-gene in Weisser et al . , J. pZY507 Bacteriol 177 (1995) 3351-4

PGEM7 ApR; T7 and SP6 Promega Co . promoters

PGEM7gntK pGEM7 containing E. coli this application gntK gene

PGEM7 gdhlV pGEM7 containing this application Bacillus magaterium gdhlV gene pGEM7gntKgdhIV pGEM7 containing gntK this application and gdhlV genes 33

Table 2 :

Determination of the glucose dehydrogenase and gluconate kinase activities in crude extracts of Escherichia coli harbouring various gene structures

Strain specific activity of specific activity glucose dehydrogenase of gluconate kinase W3110/pGEM7 n.d.a. n.d.a

W3110/pGEM7gdhXV 0.4 U/mg n.d.

W3110/pGEM7gnti^; n.d.a. 0.9 U/mg

W3110/pGEM7gn tKgdhlV 1.0 U/mg 0.9 U/mg

n.d.a. = no detectable activity; n.d. = not determined

Claims

- 34C L A I M S

1. Process for microbially preparing substances from aromatic metabolism in which, in a microorganism which is producing these substances, glucose- containing substrates are transformed as the result of increasing the activity of a glucose- oxidizing enzyme.

2. Process according to Claim 1, characterized in that the activity of a glucose dehydrogenase is introduced into and/or increased in the microorganism.

3. Process according to Claim 2, characterized in that the activity of a Bacillus mega terium glucose dehydrogenase is introduced into and/or increased in the microorganism.

4. Process according to Claim 2 or 3 , charac-terized in that the activity of the Bacillus megaterium glucose dehydrogenase IV is introduced into and/or increased in the microorganism.

5. Process according to one of Claims 1 to , characterized in that the activity of a gluconic acid-phosphorylating enzyme is additionally increased.

6. Process according to Claim 5, characterized in that the activity of a gluconate kinase is increased.

7. Process according to Claim 5 or 6 , charac-terized in that the activity of an Escherichia coli gluconate kinase is increased.

8. Process according to one of Claims 2 to 7, characterized in that the activity of a gluconolactonase, in particular a Zymomonas mobilis gluconolactonase, is additionally 877

35

increased.

9. Process according to one of Claims 1 to 8 , characterized in that the activity of a transport protein for the PEP- independent uptake of a sugar is additionally increased.

10. Process according to Claim 9, characterized in that the transport protein is a facilitator.

11. Process according to Claim 9 or 10, characterized in that the facilitator is the Zymomonas mobilis glucose facilitator protein (Glf) .

12. Process according to one of Claims 2 to 11, characterized in that the activity of a glucose- oxidizing enzyme or the activity of a glucose- oxidizing enzyme and, in addition, at least one activity from gluconic acid-phosphorylating enzyme, gluconolactonase and transport protein for PEP- independent sugar uptake is increased a) by introducing the genes b) and/or by increasing the gene copy number c) and/or by increasing gene expression d) and/or by increasing the endogenous activity of the said enzymes e) and/or by altering the structure of the enzymes f) and/or by using deregulated enzymes g) and/or by introducing genes which encode deregulated enzymes .

13. Process according to Claim 12, characterized in that the increase in the activity is achieved by the gene or the genes being integrated into a gene structure or into several gene structures, with the gene or the genes being introduced into the gene structure as single copies or in increased copy number.

14. Process according to one of Claims 9 to 13, - 36

characterized in that the activity of a PEP- dependent sugar uptake system, if present, is additionally decreased or switched off.

15. Process according to one of Claims 1 to 14, characterized in that use is made of a microorganism in which one or more enzymes, which are additionally involved in the synthesis of the substances, is/are deregulated and/or have had its/their activity increased.

16. Process according to Claims 1 to 15, characterized in that the substance which is prepared is an aromatic amino acid.

17. Process according to Claim 16, characterized in that the aromatic amino acid is L-phenylalanine.

18. Process according to one of Claims 1 to 17, characterized in that the microorganism employed belongs to the genera Escherichia, Serratia, Bacillus, Corynebacterium or Brevibacterium.

19. Process according to Claim 18 characterized in that the microorganism is Escherichia coli .

20. Gene structure which contains, in recombinant form, either a gene encoding a glucose-oxidizing enzyme together with a gene encoding a gluconic acid-phosphorylating enzyme, or a gene encoding a glucose-oxidizing enzyme together with a gene encoding a transport protein for the PEP-independent uptake of sugars, or a gene encoding a glucose-oxidizing enzyme together with at least two of the three following genes, encoding a gluconic acid-phosphorylating enzyme, encoding a gluconolactonase or encoding a transport protein for the PEP- independent uptake of sugars .

21. Gene structure according to Claim 20, charac- terized in that the gene for the glucose- 37

oxidizing enzyme encodes a glucose dehydrogenase and the gene for the gluconic acid- phosphorylating enzyme encodes a gluconate kinase .

22. Gene structure according to Claim 20 or 21, characterized in that the gene for the glucose dehydrogenase is derived from Bacillus megaterium, the gene for the gluconate kinase is derived from Escherichia coli and the gene for the gluconolactonase and the transport protein are derived from Zymomonas mobilis .

23. Transformed cells which harbour a gene structure according to Claims 20 to 22 in replicatable form.

24. Transformed cell according to Claim 23, characterized in that, in the cell, one or more enzymes which is/are additionally involved in the synthesis of the substances is/are deregulated and/or have had its/their activity increased.

25. Transformed cell according to Claim 23 or 24, characterized in that the cell is an Escherichia coli cell.

26. Transformed cell according to one of Claims 23 to

25, characterized in that the PEP-dependent sugar uptake system, if present, has additionally had its activity reduced or switched off.

27. Transformed cell according to one of Claims 23 to

26, characterized in that it is able to produce an aromatic amino acid.

28. Transformed cell according to Claim 27, characterized in that the aromatic amino acid is L-phenylalanine .

29. Process for microbially preparing substances according to one of Claims 1 to 19, characterized in that use is made of transformed cells - 38 -

according to one of Claims 23 to 28 in which a gene structure according to one of Claims 20 to 22 is present.