BR112016019081B1 - RECOMBINANT MICROBIAL HOST CELL AND METHOD FOR ANILINE PRODUCTION - Google Patents

Abstract

RECOMBINANT STRAIN PRODUCING O-AMINOBENZOATE AND FERMENTATIVE PRODUCTION OF ANILINE FROM RENEWABLE RESOURCES VIA 2-AMINOBENZOIC ACID. The invention provides a recombinant microbial host cell capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate (oAB). The invention further provides a method for the production of aniline, comprising the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate using a recombinant microbial host cell capable of biologically converting said raw material comprising at least one fermentable carbon substrate into o-aminobenzoate, wherein said o-aminobenzoate comprises anthranilate anion, b) converting said o-aminobenzoate from said anthranilate anion to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolution in an organic solvent and d) converting said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

Description

[001] The invention relates to the field of aniline production from raw material of renewable resources such as, for example, biomass, through a suitable microbial host, followed by chemical conversion of an intermediate product into aniline.

[002] Currently, aniline is produced in several million tons per year from fossil raw materials, for example, for the production of polyurethanes. A source of aniline based on renewable resources, also called "bioaniline", is intensely desired by the chemical industry, to become independent of fossil resources. Most importantly, there is a strong desire to reduce carbon dioxide (CO2) emissions, both from chemical processes and by increasing the use of renewable resources in raw materials. Bioaniline has a high potential to save CO2 emissions.

[003] The invention also refers to the engineering of microorganisms and the production of aromatic compounds from them. In particular, the invention relates to the field of producing o-aminobenzoate (oAB) from renewable sources such as, for example, biomass, in a suitable recombinant microbial host. Typically, a source is used that contains a significant proportion of fermentable sugars. These sugars can include polysaccharides, such as disaccharides, for example, sucrose, or trisaccharides, for example, ketose, as well as C-6 monosaccharides, such as glucose, fructose or mannose, and C-5 monosaccharides, such as xylose and arabinose. A recombinant microbial strain capable of converting sugar into o-aminobenzoate (2-aminobenzoate, ortho-aminobenzoate, o-aminobenzoate, oAB) would allow the production of o-aminobenzoate from a wide variety of renewable resources, including sugar beet and sugar cane, starch-containing plants such as maize, wheat and rye, as well as, for example, lignocellulose from straw, wood or bagasse.

[004] Currently, there is no commercially available renewable or biologically derived source for o-aminobenzoate or the corresponding acid, and no known example for large-scale biological production of o-aminobenzoate has been described. O-Aminobenzoate is a natural intermediate in the shikimic acid pathway and a precursor for the biosynthesis of the aromatic amino acid L-tryptophan. The biosynthetic pathway for o-aminobenzoate is relatively well understood, in both prokaryotes and eukaryotes. A chemical conversion of o-aminobenzoate to aniline can be achieved. Current aniline production methods rely on chemical synthesis from petroleum-derived raw materials. Such petroleum-derived raw materials are non-renewable, as opposed to raw materials that are renewable, such as the renewable resource "biomass". Several chemical steps involved in chemical synthesis result in high production costs for chemicals. Conventional chemical synthesis of aniline can be associated with hazardous intermediates, solvents and waste products, which can have a substantial impact on the environment. Non-specific side reactions of the aromatic ring result in reduced product yield. Petroleum-derived raw materials are influenced by cost fluctuations resulting from the global price of petroleum.

[005] WO 2013/103894 A1 discloses a method of producing aromatic amines through biologically derived p-aminobenzoic acid (4-aminobenzoate). However, this document discloses p-aminobenzoic acid production in either E. coli or S. cerevisiae and does not recognize the advantages of Corynebacterium glutamicum as a host. Furthermore, this document also does not disclose how to successfully combine the fermentation process with the downstream chemical process of converting the biologically derived p-aminobenzoic acid into aromatic amines.

[006] A direct fermentation of sugar to aniline, as a single-step conversion, was believed to be more cost-efficient if based on a biosynthetic pathway including an enzymatic in vivo decarboxylation of anthranilate to aniline as the final reaction step. Since an aminobenzoate decarboxylase could not be successfully identified or developed through protein engineering, the decarboxylation reaction of anthranilate to aniline could not be carried out by pure enzymatic means. Since such a one-step process was not technically possible, process alternatives for carrying out the final step of the decarboxylation reaction of anthranilate to aniline as the final step of the reaction, e.g. by a chemical step as opposed to an enzymatic step, were considered.

[007] Therefore, the technical problem of the invention is to provide an aniline production method, based on renewable resources, which is superior to existing fermentation and chemical methods and which achieves a great reduction in carbon dioxide emissions, independence from fossil and similar resources or lower production cost compared to established petroleum-based production processes.

[008] The invention solved said problem by providing a recombinant microbial host cell capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate.

[009] The invention further solved said problem by providing a method for the production of aniline, comprising the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate, using the recombinant microbial host cell of the invention as described herein and as claimed in the claims, which is capable of biologically converting said raw material comprising at least one fermentable carbon substrate into o-aminobenzoate, wherein said o-aminobenzoate comprises the anthranilate anion, b) converting said o-aminobenzoate from said anthranilate anion to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolution in an organic solvent, and d) converting said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

[010] The switch to aniline production based on renewable resources, for example, biomass or fermentable carbon sources, offers the advantages of significantly reducing CO2 emissions, allows independence from fossil resources and allows for a possible reduction in production cost. Another advantage of the invention is that the use of hazardous chemicals and the resulting waste is kept to a minimum. Furthermore, biologically derived o-aminobenzoate can be produced and converted to aniline in a process with much less overall impact on the environment.

[011] In particular, the invention provides a recombinant microbial host cell capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate. Such a recombinant microbial host according to the invention can be a microorganism genetically modified for the fermentative production of o-aminobenzoate from renewable sources such as, for example, biomass. For example, the invention provides genetically modified strains of Corynebacterium glutamicum as biocatalysts that are suitable for the efficient fermentative production of o-aminobenzoate from fermentable carbon sources.

[012] Corynebacterium glutamicum is a Gram-positive bacterium that lives in the soil. It belongs to the high GC Gram-positive actinobacteria. C. glutamicum is one of the most biotechnologically important bacterial species, with an annual production of more than two million tons of amino acids, mainly L-glutamate and L-lysine. Due to this great industrial importance, C. glutamicum has been studied extensively since soon after its discovery, in 1956. The genome of C. glutamicum was sequenced and published in 2003 (Ikeda M, Nakagawa S, Appl Microbiol Biotechnol, 2003, 62:99; Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, et al., J Biotechnol, 2003, 104: 5). C. glutamicum has a number of intrinsic attributes that are ideal as a microbial factory to produce not only amino acids but also other chemicals. A deep fundamental understanding of corinobacterial physiology and the post-genomic tools to model processes or design synthetic pathways in combinatorial approaches constitute a fundamental basis for designing efficient and versatile corinobacterial biorefineries. The C. glutamicum biosynthetic pathways leading to oAB have been extensively studied and can be divided into three main sections: central metabolism, the common aromatic pathway, and the L-tryptophan branch pathway. oAB is an intermediate in the biosynthesis of L-tryptophan (Figure 1). However, previous attempts to improve strains for amino acid production were based mainly on classical random selection and mutagenesis procedures, with the aim of eliminating competing pathways and eliminating feedback regulation (feedback) in biosynthetic pathways. The classical approach has limited utility, as complete downregulation of regulatory steps and enhancement of appropriate biosynthetic enzyme activity are difficult to obtain. Furthermore, random mutagenesis often produces unexpected mutations at some sites in the genome, along with desirable ones. As it is difficult to determine the influence of these unidentified mutations, further improvement of the strain would be affected. These problems exist largely in industrial strains constructed by random mutagenesis. Based on the reported literature, mainly during the last decade, current production with strains generated by the classical approach has yields, relative to sugar (% by weight), of approximately 20-23 for L-tryptophan and around 25 for L-phenylalanine. In contrast, much higher production yields relative to sugar (% by weight) have been reported for many other amino acids, eg L-lysine, 40-50; L-glutamate, 45-55; L-arginine, 30-40; L-threonine, 40-50; L-valine, 30-40; and L-alanine, 4555 ( Leuchtenberger W, Huthmacher K, Drauz K, Appl Microbiol Biotechnol, 2005, 69:1.; Ikeda M, Nakagawa S, Appl Microbiol Biotechnol, 2003, 62:99).

[013] Based on recent advances made in molecular biology and understanding the functionality of the biosynthetic pathways that lead to L-tryptophan (and oAB) from sugars, the invention provides a recombinant microbial host cell capable of converting, biologically, a raw material comprising a fermentable carbon substrate into o-aminobenzoate and, more specifically, an oAB producer from Corynebacterium glutamicum ATCC13032, based on the approach of directed mutagenesis and engineering metabolic. Some genes targeted for metabolic engineering to generate an oAB producer are located in the aromatic biosynthetic pathway that leads to oAB and, subsequently, to L-tryptophan (Figure 1). In C. glutamicum, the biosynthesis of L-tryptophan is strictly controlled in several steps. Therefore, overproduction of oAB necessitated the genetic removal of existing metabolic controls, both in the common aromatic pathway and in the L-tryptophan branch. Furthermore, amplification of DAHP synthase, which initiates the aromatic pathway, was an important strategy to increase the net flux of carbon along the common pathway, as another embodiment of the invention. These mutations were combined with the generation of bradytrophic strains for aromatic amino acids, to circumvent the constant need for aromatic amino acid supplementation. A balanced supply of precursors was directed towards achieving efficient production of the product, considering that the biosynthesis of 1 mol of oAB from glucose requires 1 mol of erythrose-4-phosphate (E4P) and 2 mol of phosphoenolpyruvate (PEP) as starting precursors and, in addition, consumes 1 mol of L-glutamine and releases 1 mol of pyruvate in its pathway.

[014] Next, the inventors focused on Corynebacterium glutamicum as a recombinant microbial strain for the biological production of o-aminobenzoate. Therefore, the invention provides a recombinant microbial host cell capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate. Said Corynebacterium glutamicum is preferably Corynebacterium glutamicum ATCC 13032, more preferably a recombinant Corynebacterium glutamicum ATCC 13032.

[015] According to other embodiments of the invention, the strain of Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC 13032, may comprise a genetic modification of the trpD gene (Cgl3032, SEQ ID NO: 1) encoding anthranilate phosphoribosyl transferase, wherein said genetic modification is preferably selected from the group consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, which have the effect of a reduced expression of the trpD gene, to obtain an auxotrophic strain for tryptophan. A single of the genetic modifications mentioned above already results in a recombinant strain of Corynebacterium glutamicum ATCC 13032 that produces o-aminobenzoate (oAB).

[016] Below are defined some terms used to describe the invention.

[017] The term "bioaniline", according to the invention, refers to aniline that is based on raw materials from renewable resources, such as sugar beet, sugar cane, plants containing starch, preferably corn, wheat and rye and lignocellulose, preferably straw, wood and bagasse, glycerol and C1 compounds, preferably CO, or such as fermentable sugars, preferably C-5 monosaccharides, monosaccharides C-6, disaccharides and trisaccharides, wherein the C5 monosaccharides are preferably xylose and arabinose and wherein the C-6 monosaccharides are preferably glucose, fructose or mannose, and wherein the disaccharide is preferably sucrose, and wherein the trisaccharide is preferably ketose.

[018] The "o-aminobenzoate", according to the invention, refers to ortho-aminobenzoate (o-aminobenzoate, "oAB"). The o-aminobenzoate can be present as the anthranilate salt comprising the anthranilate anion, C6H4COO-, and a suitable cation, such as NH4+ or Na+, or as anthranilic acid, which is the zwitterion ion C6H4COO-NH3+ and C6H4COO-NH2. "O-aminobenzoate" ("oAB") is different from "4-aminobenzoate" ("pAB") in that the amino group is attached to the benzene ring at the C4 (para) position, as opposed to the C2 (ortho) position in the case of o-aminobenzoate ("oAB").

[019] The term "host", within the meaning of the invention, can comprise any host that is capable of producing o-aminobenzoate by fermentation, either naturally or only after transformation as a "recombinant microbial host", or in addition to the o-aminobenzoate naturally present, either in the form of anthranilate anion or as anthranilic acid, after transformation. A "microbial host" according to the invention can be selected from the group consisting of bacteria, yeasts and fungi. Said host may be selected from the group consisting of bacteria, yeasts and fungi, wherein said bacteria is preferably an Escherichia coli strain, a Corynebacterium strain, or a Pseudomonas strain, wherein said Corynebacterium strain is preferably Corynebacterium glutamicum and wherein said Pseudomonas strain is preferably Pseudomonas putida. Preferably, said microbial host may be a recombinant microbial host. Such a recombinant microbial host can be E. coli W3110 trpD9923, as shown in Example 1. Such a recombinant host can be a Pseudomonas putida KT2440, as shown in Example 4. Such a recombinant host can also be a Corynebacterium glutamicum ATCC 13032, as shown in Example 3.

[020] The term "genetic modification", within the meaning of the invention, refers to changes in the nucleic acid sequence of a given gene of a microbial host, compared to the wild-type sequence. Such genetic modification may comprise deletions as well as insertions of one or more deoxyribonucleic acids. Such genetic modification may comprise partial or complete deletions as well as insertions introduced by transformations into the genome of a microbial host. Such genetic modification may produce a recombinant microbial host, wherein said genetic modification may comprise changes of at least one, two, three, four or more individual nucleotides compared to the wild-type sequence of the respective host microbial. For example, a genetic modification can be a deletion or insertion of at least one, two, three, four or more individual nucleotides or a transformation of at least one, two, three, four or more individual nucleotides. The genetic modification according to the invention can have the effect of, for example, a reduced expression of the respective gene or of, for example, an increased expression of the respective gene. In an example of such a genetic modification according to the invention, a recombinant microbial host, for example Corynebacterium glutamicum, may comprise a genetic modification of the trpD gene (Cgl3032, SEQ ID NO: 1) encoding the enzyme anthranilate phosphoribosyl transferase, wherein said genetic modification may have the effect of a reduced expression of the modified trpD gene. Such a modified trpD gene may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. A single of the genetic modifications mentioned above already results in a recombinant Corynebacterium glutamicum ATCC 13032 strain having reduced expression of the modified trpD gene, which produces o-aminobenzoate (oAB). In another embodiment of the invention, such genetic modification may also be a deletion of the trpD gene, for example as in Corynebacterium glutamicum ΔtrpD.

[021] The term "batch fermentation", within the meaning of the invention, refers to a single fermentation reaction that has a defined starting point and a defined end point. Batch fermentation can be used in step a) of the method according to the invention, in cases where the production rates of microorganisms cannot be maintained at a high value in continuous fermentation mode.

[022] The term "fed-batch fermentation", within the meaning of the invention, refers to an operational technique in biotechnological processes, where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain(s) in the bioreactor until the end of the batch. "Fed-batch fermentation" can be used in step a) of the method according to the invention, in cases where the production rates of microorganisms cannot be maintained at a high value in continuous fermentation mode.

[023] The term "continuous fermentation", within the meaning of the invention, refers to a fermentation method in which substrate is added and the product (i.e., o-aminobenzoate, oAB) is continuously removed during fermentation in step a) of the method according to the invention.

[024] In the following, the invention is described in more detail.

[025] The invention provides a recombinant microbial host cell capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate (oAB).

[026] Said host microbial cell may be selected from the group consisting of bacteria, yeasts and fungi, wherein said bacteria is preferably an Escherichia coli strain, a Corynebacterium strain, or a Pseudomonas strain, and wherein said Corynebacterium strain is preferably Corynebacterium glutamicum, more preferably Corynebacterium glutamicum ATCC 13032, and wherein the said Pseudomonas strain is preferably Pseudomonas putida, more preferably Pseudomonas putida KT2440.

[027] In another embodiment of the recombinant microbial host cell of the invention, the recombinant microbial host cell may be Corynebacterium glutamicum. In more specific embodiments of the invention, Corynebacterium glutamicum ATCC13032 is the preferred recombinant microbial host for o-aminobenzoate production, as the inventors have observed that the organism has a high tolerance for o-aminobenzoate, whereas for other microorganisms, for example Escherichia coli K12, low concentrations of o-aminobenzoate are already toxic.

[028] In another embodiment of the recombinant microbial host cell of the invention, said Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC 13032, may comprise a genetic modification of the trpD gene that encodes anthranilate phosphoribosyl transferase (Figure 3).

[029] Said genetic modification of the trpD gene (Cgl3032, SEQ ID NO: 1), which encodes anthranilate phosphoribosyl transferase, can be selected from the group consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 and wherein said genetic modification has the effect of a reduced expression of the trpD gene. More specifically, mutations in the aforementioned sequences can alter the spacer region between the ribosome binding site and the start codon of the trpD gene. Furthermore, a shift of the natural initiation codon of the trpD gene can lead to reduced translation of trpD and thus to a bradytrophic strain relative to L-tryptophan, which produces o-aminobenzoate on a multigram scale without supplementation with L-tryptophan. It follows that such a strain type is particularly preferred as a recombinant microbial host according to the invention.

[030] According to other embodiments of the recombinant microbial host cell of the invention, the microbial host cell may be Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC 13032, wherein said Corynebacterium glutamicum or Corynebacterium glutamicum ATCC 13032 may comprise a genetic modification of the csm gene (Cgl0853, SEQ ID NO: 9), which encodes chorismate mutase (Figure 3), wherein said genetic modification is preferably selected from the group consisting of sequences SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and wherein said genetic modification has the effect of a reduced expression of the csm gene. In particular, the csm gene can be manipulated to further improve o-aminobenzoate production rates by reducing L-phenylalanine and L-tyrosine production rates to a minimum.

[031] The effect of reducing csm gene expression results in a bradytrophic strain with respect to L-tyrosine and L-phenylalanine, which produces o-aminobenzoate on a multigram scale without supplementation with L-tyrosine and L-phenylalanine. This avoids the reduction of o-aminobenzoate production due to feedback inhibition of enzymes that catalyze reactions in the aromatic acid pathway by L-tyrosine and/or L-phenylalanine, as is possible in auxotrophic strains for L-tyrosine and L-phenylalanine supplemented with L-tyrosine and/or L-phenylalanine. It follows that such a strain type is particularly preferred as a recombinant microbial host according to the invention. This effect may result in increased production of o-aminobenzoate.

[032] According to other embodiments of the recombinant microbial host cell of the invention, the Corynebacterium glutamicum microbial host cell, preferably Corynebacterium glutamicum ATCC 13032, may further comprise one or more deletions selected from the group consisting of hpr (Cgl1937 - SEQ ID NO: 17 or SEQ ID NO: 18), ptsG (Cgl1537 - SEQ ID NO: 19 or SEQ ID NO: 20), pepco (Cgl1523 - SEQ ID NO: 21 or SEQ ID NO: 22), pyk (Cgl2089 - SEQ ID NO: 23 or SEQ ID NO: 24) and gpi (Cgl0851 - SEQ ID NO: 27 or SEQ ID NO: 28).

[033] The hpr gene (Cgl1937 - SEQ ID NO: 17 or SEQ ID NO: 18) and the ptsG gene (SEQ ID NO: 19 or SEQ ID NO: 20) each encode a unit of the PEP-phosphotransferase (PTS) multienzyme complex system (Figures 1, 2). Both C. glutamicum PTS, Hpr, and PtsG units were manipulated and compared for effects on growth, cell viability, as well as oAB production, as shown in Example 3.

[034] The pepco gene (Cgl1523 - SEQ ID NO: 21 or SEQ ID NO: 22) encodes the enzyme phosphoenolpyruvate carboxylase. Phosphoenolpyruvate carboxylase consumes PEP (phosphoenolpyruvate), transforming it into oxaloacetate (Figures 1, 2). This reaction is the main oxygen-starved anaplerotic reaction, whereas under standard conditions of oxygen supply, pyruvate kinase consumes most of the PEP to generate pyruvate, with a gain of one ATP. The C. glutamicum pepco gene was manipulated and compared for effects on growth, cell viability, as well as oAB production, with other C. glutamicum strains, as shown in Example 3.

[035] The pyk gene (Cgl2089 - SEQ ID NO: 23 or SEQ ID NO: 24) encodes the enzyme pyruvate kinase. Pyruvate kinase consumes PEP (phosphoenolpyruvate) by producing pyruvate and ATP (Figures 1, 2). This reaction is part of glycolysis in C. glutamicum. The gene encoding Pyk, noted in the C. glutamicum genome, has been deleted as described in Example 3.

[036] The gpi gene (Cgl0851 - SEQ ID NO: 27 or SEQ ID NO: 28) encodes the enzyme glucose-6-phosphate isomerase. The enzyme catalyses the first step of glycolysis of transforming glucose-6-phosphate into fructose-6-phosphate (Figures 1, 2).

[037] In more specific embodiments of the recombinant microbial host cell of the invention, the microbial host cell Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC 13032, may further overexpress one or more of the genes selected from the group consisting of galP (SEQ ID NO: 30), iolT2 (SEQ ID NO: 31), ppgk (SEQ ID NO: 32), pps (SEQ ID NO: 30), pps (SEQ ID NO: 33-35), ppk (SEQ ID NO: 36), zwf1 (SEQ ID NO: 37), opcA (SEQ ID NO: 3839), tktCg (SEQ ID NO: 40), tktEc (SEQ ID NO: 41), talCg (SEQ ID NO: 42), talEc (SEQ ID NO: 43), a gene encoding TrpEGS38F (trpEGS38 F, SEQ ID NO: 50), a gene encoding TrpEGS38R (trpEGS38R, SEQ ID NO: 51), a gene encoding TrpEGS40R (trpEGS40R, SEQ ID NO: 52), a gene encoding TrpEGS40F (trpEGS40F, SEQ ID NO: 53), the gene encoding AroGD146N (aroGD146N, SEQ ID NO: 55), Escherichia coli LJ110 aroL (SEQ ID NO: 93), aroK (SEQ ID NO: 94), glnA (SEQ ID NO: 95) and qsuA (SEQ ID NO: 96).

[038] The expression of feedback-resistant trpEG genes (anthranilate synthase genes), a gene encoding TrpEGS38F (trpEGS38F - SEQ ID NO: 50), a gene encoding TrpEGS38R (trpEGS38R - SEQ ID NO: 51), a gene encoding TrpEGS40R (trpEGS40R - SEQ ID NO: 52), a gene encoding tr pEGS40F (trpEGS40F - SEQ ID NO: 53), in the microbial host Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC 13032, is a preferred embodiment of the invention, alone or in combination with any of the other genetic modifications of the invention. All genes share the common feature that they encode mutated versions of the anthranilate synthase unit, which releases a pyruvate molecule with the formation of o-aminobenzoate and L-glutamate (Figure 3). These are feedback-resistant versions of TrpEG, which is why the corresponding mutant genes are referred to as trpEGfbr. GD146N - SEQ ID NO: 55), is another preferred embodiment of the invention, alone or in combination with any of the other genetic modifications of the invention. The gene encoding AroGD146N (aroGD146N - SEQ ID NO: 55) encodes a mutated version of AroG, which is an enzyme that catalyzes the reaction from erythrose-4-phosphate (E4P) to 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) in Escherichia coli (Figure 3).

[039] The galP gene (Cgl2409 - SEQ ID NO: 30) which encodes the galactopermease enzyme. Glucose uptake can be restored after PTS disruption through the expression of a galactopermease (Figures 1, 2). A gene has been identified in C. glutamicum and annotated as galactopermease and therefore this is a good candidate for such expression. The candidate gene is expressed in C. glutamicum strains with disrupted PTS, as shown in Example 3.

[040] The iolT2 gene (Cgl3058 - SEQ ID NO: 31) encodes the T2 inositolpermease unit. Glucose uptake can be restored after PTS disruption through the expression of a galactopermease (Figures 1, 2). As an alternative approach to overexpression of a galactopermease, to restore glucose uptake after PTS breakdown in C. glutamicum, overexpression of the T2 inositolpermease unit of the inositolpermease is performed. Characteristics of the resulting strain are compared to those of strains that overexpress a galactopermease, as shown in Example 3.

[041] The ppgk gene (Cgl1910 - SEQ ID NO: 32) encodes the enzyme polyphosphoglucokinase. The galactopermease, as well as the T2 inositolpermease unit, can only absorb glucose but cannot phosphorylate the sugar molecule (Figures 1, 2). This phosphorylation, however, is essential to allow the metabolism of the glucose molecule. Therefore, the expression of the different permeases is combined with the expression of a glucose phosphorylating enzyme. This Ppgk is co-expressed in the different PTS-deficient strains of the invention that express permease, as described in Example 3.

[042] The pps genes (Cgl0551 and Cgl0552 - SEQ ID NO: 33-35) encode the enzyme PEP (phosphoenolpyruvate) synthase. In addition to disrupting the reactions that consume PEP, biosynthetic steps leading to the generation of PEP are promoted. PEP synthase catalyzes PEP formation through the recycling of pyruvate and consumption of ATP to AMP, as part of gluconeogenesis in C. glutamicum (Figures 1, 2). Overexpression of the pps gene results in an increase in the oAB group in C. glutamicum strains, as described in Example 3.

[043] The ppk gene (Cgl2862 - SEQ ID NO: 36) encodes the enzyme PEP carboxykinase. As part of the glyoxylate pathway, oxaloacetate can be recycled to PEP by a PEP carboxykinase (Figures 1, 2). Overexpression of the gene may be another pathway to a larger PEP pool available for oAB biosynthesis, as described in Example 3.

[044] The zwf1 gene (Cgl1576 - SEQ ID NO: 37) and the opcA gene (Cg1577 - SEQ ID NO: 38-39) encode the enzyme glucose-6-phosphate dehydrogenase (Figures 1, 2). Increased production of the enzyme that catalyzes the first reaction of the pentose phosphate (PPP) pathway (formation of ribulose-5-phosphate from glucose-6-phosphate) (Figures 1, 2) may result in increased flux to the PPP, leading to the production of greater amounts of E4P and production via fructose-6-phosphate (Frc-6-P) and may, in addition, increase cells in the PEP group, ultimately resulting in an increase in the oAB group in C. glutamicum. This effect is achieved when this manipulation is applied to C. glutamicum strains and an expression of the Zop enzyme is performed, as described in Example 3.

[045] The tktCG gene (Cgl1574 - SEQ ID NO: 40) and the tktEC gene (ECDH10B_3110 - SEQ ID NO: 41) encode the transketolase enzyme. Increased flux through PPP and a larger E4P group, and even increased production of oAB and aromatic amino acids by expression of a transketolase in C. glutamicum strains, are observed (Figures 1, 2). An E. coli transketolase gene was overexpressed, as well as the transketolase encoded in C. glutamicum, as described in Example 3.

[046] The talCG gene (Cgl1575 - SEQ ID NO: 42) and the talEC gene (ECDH10B_2629 - SEQ ID NO: 43) encode the transaldolase enzyme. A comparable favorable effect on E4P production, such as by overexpression of transketolase, can be seen for overexpression of transaldolases in E. coli (Figures 1, 2). As the sequences of the E. coli and C. glutamicum transaldolases differ significantly, the inventors continued to express the described E. coli gene and the natural C. glutamicum gene in C. glutamicum strains of the invention, as described in Example 3. The genes encoding the transaldolase and transketolase are combined into a single vector and co-expressed in C. glutamicum strains, to further improve the characteristics of the oAB-producing strains, as described in Example 3.

[047] The qsuA gene (Cgr0492 - SEQ ID NO: 96) encodes the drug-resistant transport protein QsuA. Gene expression in C. glutamicum strains results in optimized oAB transport, as described in Example 3.

[048] The aroL gene (B0388 - SEQ ID NO: 93) from Escherichia coli LJ110 encodes an enzyme that catalyzes the reaction from shikimate (SHI) to shikimate-3-phosphate (SHI3P)) in Escherichia coli. The E. coli aroL gene is expressed in C. glutamicum strains as described in Example 3.

[049] The aroK gene (Cgl1622 - SEQ ID NO: 94) encodes an enzyme that catalyzes the reaction from shikimate (SHI) to shikimate-3-phosphate (SHI3P) in Corynebacterium glutamicum ATCC 13032 (Figure 3). The aroK gene is expressed in C. glutamicum strains as described in Example 3.

[050] The glnA gene (Cgl2214 - SEQ ID NO: 95) encodes the glutamine synthetase enzyme in Corynebacterium glutamicum ATCC 13032 (Figure 3). The glnA gene is expressed in C. glutamicum strains as described in Example 3.

[051] In particularly preferred embodiments of the invention, the recombinant microbial host cell can be C. glutamicum ΔtrpD, C. glutamicum ΔtrpD::trpD5, C. glutamicum ΔtrpD::trpD5Δpepco, C. glutamicum ΔtrpD::trpD5Δgpi, C. glutamicum ΔtrpD::trpD5Δpyk, C. glutamicum ΔtrpD::trpD5/pEKEx2-trpEGS40F, C. glutamicum ΔtrpD::trpD5/pEKEx2-aroGD146N, C. glutamicum ΔtrpD::trpD5/pEKEx2-aroGD146N-trpEGS40F, C. glutamicum ΔtrpD::trpD5/p EKEx2-aroL, C. glutamicum ΔtrpD::trpD5/pSB072, C. glutamicum ΔtrpD::trpD5/pSB073, C. glutamicum ΔtrpD::trpD5/pSB074, C. glutamicum ΔtrpD::trpD5/pSB075, C. glutamicum ΔtrpD::trpD5/ pSB076, C. glutamicum ΔtrpD::trpD5/pSB077, C. glutamicum ΔtrpD::trpD5/pSB078, C. glutamicum ΔtrpD::trpD5/pSB085, or C. glutamicum ΔtrpD::trpD5/pSB096, as further described in Example 3 and Table 4.

[052] Other recombinant microbial hosts of the invention are described below.

[053] In yet another embodiment of the recombinant microbial host cell of the invention, the microbial host cell may be Pseudomonas putida, preferably Pseudomonas putida KT2440.

[054] In another embodiment of the recombinant microbial host cell of the invention, said Pseudomonas putida, preferably Pseudomonas putida KT2440, may comprise a deletion of the trpDC gene encoding anthranilate phosphoribosyl transferase and indole-3-glycerol phosphate synthase (PP_0421 and PP_0422 - SEQ ID NO: 63-64), or a deletion of the pheA gene encoding chorismate mutase and prephenate dehydratase (PP_1769 -SEQ ID NO: 65-66), or both, as described in Example 4 (Figure 4).

[055] In another embodiment of the recombinant microbial host cell of the invention, said Pseudomonas putida, preferably Pseudomonas putida KT2440, wherein said Pseudomonas putida or Pseudomonas putida KT2440 can express one or more of the genes selected from the group consisting of the gene encoding TrpEGS40F (trpEGS40F - SEQ ID NO: 53) and the gene encoding AroGD146N (aroGD146N - SEQ ID NO: 55) (Figure 4). In certain embodiments of the recombinant microbial host cell of the invention, the one or more expressed genes mentioned above can be integrated into said P. putida host microbial cells by plasmid transformation or by chromosomal transformation, as described in Example 4.

[056] The raw material comprising a fermentable carbon substrate, which is biologically converted by the recombinant microbial host cell into o-aminobenzoate (oAB), may be selected from the group consisting of sugar beet, sugar cane, plants containing starch, preferably corn, wheat and rye, and lignocellulose, preferably, straw, wood and bagasse, glycerol and C1 compounds, preferably CO.

[057] According to other embodiments of the invention, the fermentable carbon substrate that is contained in the raw material can be selected from the group consisting of C-5 monosaccharides, C-6 monosaccharides, disaccharides and trisaccharides, where the C-5 monosaccharides are preferably xylose and arabinose and where the C-6 monosaccharides are preferably glucose, fructose or mannose and where the disaccharide wherein the trisaccharide is preferably sucrose and wherein the trisaccharide is preferably ketose.

[058] The invention further solved the above problem by providing a method for the production of aniline, comprising the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate using the recombinant microbial host cell, as described herein and as claimed in the claims, capable of biologically converting said raw material comprising at least one fermentable carbon substrate into o-aminobenzoate, wherein said o-aminobenzoate comprises the anion an tranylate, b) converting said o-aminobenzoate from said anthranilate anion to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolution in an organic solvent and d) converting said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

[059] The method according to the invention provides the technical advantages of aniline production while simultaneously obtaining a large reduction in CO2 emissions compared to methods based on fossil resources, independence from such fossil resources, as well as obtaining similar or lower production costs when compared to established production processes for petroleum-based aniline.

[060] The method according to the invention comprises two main parts, wherein the first part, step a), is based on (biotechnological) fermentation and comprises converting the raw material comprising at least one fermentable carbon substrate into o-aminobenzoate by fermentation by a suitable recombinant microbial host, wherein said o-aminobenzoate comprises anthranilate anion (C6H4COO-NH2).

[061] The second part is more chemical in nature and is downstream of step a), comprising the following steps b) to d) and optionally e): b) conversion of said o-aminobenzoate from said anthranilate anion into anthranilic acid by acid protonation, c) recovery of said anthranilic acid by precipitation or by dissolution in an organic solvent, and d) conversion of said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

[062] In summary, the invention therefore provides a method for the production of aniline that combines a first step a), of biotechnological fermentation, with a subsequent chemical process, downstream, comprising at least steps b), c) and d), with an optional step e).

[063] In the following, the invention is described in more detail. The general concept of the method according to the invention is represented in Figure 5 and a more detailed description is presented in Figure 6.

[064] The invention additionally provides a method for the production of aniline, comprising the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate using the recombinant microbial host cell, as described herein and as claimed in the claims, capable of biologically converting said raw material comprising at least one fermentable carbon substrate into o-aminobenzoate, wherein said o-aminobenzoate comprises the anthranyl anion to, b) converting said o-aminobenzoate from said anthranilic acid to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolution in an organic solvent, and d) converting said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

[065] The fermentation of step a) may be a batch fermentation, a fed-batch fermentation or a continuous fermentation. In the case of a continuous fermentation in step a), a cell retention device can be used to separate the host from an aqueous phase used in fermentation step a) and to retain the host in a fermenter used to carry out step a). The cell retention device can be incorporated into such a fermenter or, as a preferred option, it can be located outside the fermenter as a separate device. Such separation can be carried out by centrifugation, for example in a disk separator or in a decanter, by gravity in a decantation device, or by a filtration technique such as cross-flow filtration. The separation can be designed with a limited retention time, so that the host, for example bacteria, yeast and fungal cells, spends a limited time outside the fermenter and does not lose its ability to grow or produce anthranilate while in the separation device. Continuous fermentation with cell retention allows fermentation step a) to be carried out with a high cell density and thus with a high space-time yield. Most importantly, the fermentation in step a) can be carried out with a very low growth rate. This results in a higher overall product yield (g of product per g of raw material) as less sugar is used for biomass production and more for oAB production.

[066] An additional advantage of continuous fermentation over batch fermentation is that the so-called “downtime” of the fermentor can be reduced to a minimum. Continuous fermentation has a much longer production phase than batch fermentation, so the time spent cleaning, sterilizing, filling and collecting is proportionately much shorter. This aspect contributes to the increase of the space-time yield and, therefore, reduces the investment costs for a given capacity of the plant, which is used to execute the method according to the invention.

[067] If step a) of the method is carried out as a batch or fed-batch fermentation, the total fermentation capacity can be divided between several fermenters and intermediate tanks can be used to allow a continuous supply of fermentation material from step a) to the downstream section, comprising steps b) to d), or even e). Batch or fed-batch fermentation can be used in step a) of the method according to the invention, in cases where the production rates of microorganisms cannot be maintained at a high value in continuous fermentation mode.

[068] Step a) of the method according to the invention provides o-aminobenzoate, comprising the anthranilate anion, by fermentation of a raw material comprising fermentable sugars using a host, preferably a recombinant host, which is capable of converting sugars into o-aminobenzoate by fermentation. Preferably, a fermentation reactor comprising such a host is cultivated with the addition of a carbon source, for example, corn syrup, sugar cane juice, molasses and the like, and a nitrogen source, for example, ammonia gas, ammonium hydroxide solution, ammonium sulfate, ammonium nitrate, corncin and the like, as well as the respective micronutrients necessary for the survival of the microorganism. The fermentation pH in step a) can be in the range of 3 to 9, preferably between pH 4 to 8, more preferably maintained at a value between 6.5 and 7.5. The pH in step a) can be adjusted, for example, by adding a base, for example ammonia gas, ammonium hydroxide or sodium hydroxide.

[069] In another embodiment of the method according to the invention, at least step a), of producing o-aminobenzoate by fermentation, and step b) of converting said o-aminobenzoate from said anthranilate anion into anthranilic acid by acid protonation, can be carried out continuously. In this embodiment, a fermenter in which step a) is carried out is operated continuously, in which a fermentation broth produced in step a) can be continuously withdrawn and processed through a device for separating the biomass, comprising the cultured host to a certain density, for example, a filter, a centrifuge, membranes, etc. This biomass can be recycled to the fermenter used in the fermentation of step a) after purging a small portion of the biomass comprising the recombinant microbial host. Such a biomass purge stream can be useful to prevent biomass accumulation. A portion of the microbial host cells that multiply in the fermenter and the dead cells can thus be removed, to keep the concentration of live host cells in the reactor of step a) of fermentation within defined limits, more preferably constant. This may be different in the case of fed-batch fermentation, where the recombinant host cells and the fermentation product(s) remains in the bioreactor until the end of the batch, which is therefore not a continuous fermentation, but a fed-batch fermentation. Sufficient oxygen can be added to the reactor used in step a), either as pure oxygen, as air, or as enriched air. The cell-free fermentation broth of fermentation step a) can essentially be a solution of an anthranilate salt comprising the anthranilate anion and a suitable counter cation such as NH4+ or Na+. The anthranilate solution produced in fermentation step a) can have a concentration of between 5 g/l and 500 g/l, preferably between 20 g/l and 200 g/l, more preferably between 50 g/l and 150 g/l of anthranilate salt.

[070] In another embodiment of the method according to the invention, the recombinant microbial host used in the fermentation of step a) is removed before carrying out step b) of converting said o-aminobenzoate from said anthranilate anion into anthranilic acid. Preferably, the removed recombinant microbial host is fed back to the fermentation of step a). This technique has the advantage of allowing a continuous fermentation process, which can be particularly efficient and cost-effective.

[071] The o-aminobenzoate (oAB) is an amino acid; thus, its ionization state and solubility in water are pH dependent, with the zwitterion ion being the least soluble form. At pH 7, which prevails in the fermenter that can be used in step a) of fermentation, oAB exists as anions buffered with cations such as Na+ or NH4+. Therefore, adding an acid such as HCl until the pH drops to the isoelectric point causes oAB crystals to precipitate. Therefore, step b), of the method according to the invention, comprises converting the anthranilate anion into anthranilic acid by acid protonation, thus forming a precipitate comprising crystals of anthranilic acid. For this reason, step b) of the method may also be referred to as "crystallization". Specifically, the anthranilate solution from step a) can be mixed with an acid, preferably HCl. Thus, the pH can be reduced to a value between 2 and 4, preferably between 3.2 and 3.6. This causes a change in solubility and results in precipitation of anthranilate crystals. The anthranilate crystals can be recovered, preferably by filtration, in the subsequent step c) ("recovery"). Residual moisture and inorganic salt contents in precipitated anthranilate crystals (“pie”) depend on the solid-liquid separation operation.

[072] When carrying out step b) of the method, the anthranilate salt in the cell-free aqueous fermentation broth produced in step a) of fermentation is first protonated to anthranilic acid by reaction with strong acid, preferably HCl, in which anthranilic acid precipitates and is subsequently recovered as a solid material in the method of step c) (“recovery”). The anthranilic acid is then converted to aniline in a subsequent step d) of the method, by thermal decarboxylation.

[073] In a preferred embodiment of the method according to the invention, the conversion of o-aminobenzoate from anthranilate anion to anthranilic acid, by acid protonation in step b) of the method, can be carried out by adding HCl, preferably at a pH of 2.5 to 4.5, more preferably at a pH of 3-4, even more preferably between 3.2 and 3.6.

[074] In another embodiment of the method according to the invention, the recovery of anthranilic acid by precipitation, in step c) of the method, comprises filtration, thus generating a suspension comprising said recovered anthranilic acid, wherein said suspension comprising said recovered anthranilic acid is preferably dissolved in an organic solvent. Preferably, the organic solvent used in this step is aniline or 1-dodecanol, or a mixture thereof.

[075] In another embodiment of the method according to the invention, recovering anthranilic acid by dissolving said anthranilic acid in an organic solvent in step c) of the method comprises adding said organic solvent, preferably aniline, or 1-dodecanol, or a mixture thereof to said anthranilic acid, such that said anthranilic acid is recovered as a solute in the organic solvent.

[076] Preferably, the recovery of step c) of the method according to the invention can be followed by washing and drying the anthranilic acid precipitate recovered, before carrying out the thermal decarboxylation of step d).

[077] In another embodiment of the method according to the invention, the thermal decarboxylation of step d) can be carried out in the presence of a catalyst. Such a preferred catalyst may be a zeolite catalyst, wherein said zeolite catalyst is preferably H-Y zeolite (e.g. supplied by Zeolyst International, catalog number CBV600). Zeolite H-Y acid catalyst (SiO2/Al2O3 can be between 5 and 7, preferably 5.5) has a particularly high acid character and has a larger pore size (0.7-0.8 nm) than, for example, ZSM5-27 zeolite (e.g. as supplied by Clariant SuedChemie, catalog number MFI-27, SiO2/Al2O3 = 27), which also has a strong acidic character, but it has the smallest pore size (0.5 nm), so that AA molecules cannot effectively penetrate it and, consequently, do not have easy access to the active sites of the acid catalyst, thus reducing its effectiveness.

[078] Step d) of the method comprises converting said anthranilic acid from step c) into aniline by thermal decarboxylation in an organic solvent. Anthranilic acid, for example, in the form of anthranilate crystals with or without residual moisture, is thermally decarboxylated, preferably by feeding the anthranilic acid into a decarboxylation reactor, in which step d) can be carried out. The thermal decarboxylation of step d) can be carried out at a temperature between 150 °C and 250 °C, preferably between 160 °C and 220 °C, more preferably between 180 °C and 200 °C. The thermal decarboxylation of step d) can be carried out for a time sufficient for the anthranilic acid to react with aniline. Preferably, step d) can be carried out for 0.5 hours to 3 hours.

[079] The thermal decarboxylation of step d) can be performed in the presence of an acid catalyst, in order to accelerate the thermal decarboxylation.

[080] The thermal decarboxylation of step d) can be carried out in a solvent such as water, aniline, or in 1-dodecanol, preferably in 1-dodecanol, or in a mixture of 1-dodecanol and aniline.

[081] Furthermore, the thermal decarboxylation of step d) can be carried out in a reactor, where the pressure in the reactor can be selected as a function of the amount of the liquid phase that is allowed to evaporate during the reaction and leaves the reactor with carbon dioxide (CO2), which is a product of the decarboxylation.

[082] In addition, the thermal decarboxylation of step d) can produce a mixture of organic solvent and aniline, which can then be distilled with aniline and any water carried or dissolved in it. Any organic solvent used in recovery step c) can be recovered as "top product". The solvent can then be cooled and recycled to distillation. The aniline-containing overhead stream can then be fed to a distillation step, for example a heteroazeotropic distillation.

[083] In another embodiment of the method according to the invention, the thermal decarboxylation of step d) can be followed by an additional step e) of purifying the aniline, preferably by distillation.

[084] In one embodiment of the method according to the invention, following step c) of recovery, the solution (“mother liquor”) may still have up to 3-10 g/l of anthranilate. Thus, in another embodiment of the method according to the invention, following step c) of recovery, the residual anthranilate anion can be recovered by adsorption on an ion exchange resin or on an activated carbon material or a zeolite, preferably a zeolite modified with Fe3+ or Ca2+, preferably an Fe-Y zeolite, which can be produced by ion exchange of commercially available H-Y zeolites (e.g. as supplied by Zeolyst International - CBV600), for example, as described in Example 6, such as Fe-Y, followed by desorption of the recovered anthranilate anion and feedback of said anthranilate anion to the conversion step b) for acid protonation. The desorption of the recovered anthranilate anion can preferably be carried out at a pH of 5 to 10. For example, the desorption can be carried out with water having a pH of 5 to 10 or, for example, water having a neutral or alkaline pH can be used. The solution after desorption can be recycled upstream of acid addition in conversion step b) and downstream of biomass removal after fermentation step a).

[085] After recovery step c), the residual anthranilate anion can be recovered by adsorption to an ion exchange resin or to an activated carbon or zeolite material. A preferred zeolite is modified with Fe3+ or Ca2+, preferably an Fe-Y zeolite. Such Fe-Y can be prepared as described in Example 6. After adsorption of residual anthranilate anion, desorption of recovered anthranilate anion in liquid phase follows. Such desorption of the recovered anthranilate anion can be in an organic solvent phase or in an aqueous phase. Figure 7 shows a mass profile of the decomposition of AA adsorbed on Fe-Y zeolite. Figure 8 shows the desorption of AA from the Fe-Y zeolite into the organic solvent 1-dodecanol, which is the preferred organic solvent because of the high solubility of AA in it and because of its low miscibility in water (0.004 g/l). Figure 9 shows the desorption of anthranilic acid (AA) from the adsorbent into the liquid phase.

[086] It is preferable that the recovered and desorbed residual anthranilate anion is, at least partially, fed back to the conversion step b) for the conversion of said anthranilate anion to anthranilic acid by acid protonation. This has the technical advantage of improving the efficiency of the method even further, leading to the associated economic benefit.

[087] In a particularly preferred embodiment of the method according to the invention, after recovering the residual anthranilate anion by adsorption, following recovery step c) a stream of water free of the adsorbed anthranilate anion can be, at least partially, fed back to fermentation step a). Again, this has the technical advantage of improving the efficiency of the method even further.

[088] In a preferred embodiment of the invention, steps a) to d) can be carried out continuously, as an integrated process. In another preferred embodiment of the invention, steps a) to e) can be carried out continuously, as an integrated process. In such an integrated and continuous process, the fermentation of step a) can be carried out continuously, with cell retention and continuous removal of anthranilate from the fermentation broth, from the fermentation of step a), followed by continuous processing to aniline by thermal decarboxylation in subsequent steps b) to d), or b) to e). Therefore, the method according to the invention can be an integrated and continuous process, which is optimized with respect to production cost. The method according to the invention therefore offers significant technical advantages that lead to a higher aniline yield with the corresponding economic benefit, compared to the more traditional methods of the state of the art, which work non-continuously.

[089] In another embodiment of the method according to the invention, the raw material of step a) of fermentation can be selected from the group consisting of sugar beet, sugar cane, starch-containing plants, preferably corn, wheat and rye and lignocellulose, preferably, straw, wood and bagasse, glycerol and C1 compounds, preferably CO.

[090] In another embodiment of the method according to the invention, such fermentable carbon substrate from step a) of fermentation can be selected from the group consisting of C-5 monosaccharides, C-6 monosaccharides, disaccharides and trisaccharides, where the C-5 monosaccharides are preferably xylose and arabinose and where the C-6 monosaccharides are preferably glucose, fructose or mannose and where the disaccharide wherein the trisaccharide is preferably sucrose and wherein the trisaccharide is preferably ketose.

[091] In another embodiment of the method according to the invention, such recombinant microbial host of step a) of fermentation can be selected from the group consisting of bacteria, yeasts and fungi, wherein said bacteria is preferably a strain of Escherichia coli, a strain of Corynebacterium, or a strain of Pseudomonas and wherein said strain of Corynebacterium is preferably Corynebacterium glutamicum, more preferably Corynebacterium glutamicum ATCC 13032 and wherein said Pseudomonas strain is preferably Pseudomonas putida, more preferably Pseudomonas putida KT2440. Essentially, any of the recombinant microbial host cells of the invention described above can be used in fermentation step a) of the method according to the invention.

[092] In a different embodiment of the invention, an additional method for the production of aniline is provided, comprising the steps of: a) supplying o-aminobenzoate, wherein said o-aminobenzoate comprises the anthranilate anion and a suitable cation, b) converting said anthranilate anion into aniline, by thermal decarboxylation in the presence or absence of a catalyst, c) extracting the aniline produced in step b) in an organic solvent, at least once, and d ) purification of the aniline produced in steps b) and c) by distillation, said distillation producing aniline and an aqueous phase.

[093] In this embodiment, said o-aminobenzoate in step a) can be supplied chemically or biologically produced, preferably, it is produced biologically by fermentation of a raw material comprising at least one fermentable carbon substrate, using a recombinant microbial host cell capable of converting said raw material, which comprises a fermentable carbon substrate, into o-aminobenzoate by fermentation, wherein said o-aminobenzoate comprises anthranilate anion and a suitable cation.

[094] Said fermentation step a) may be a batch fermentation, a fed-batch fermentation or a continuous fermentation. In a preferred embodiment, step a) through step d) can be carried out continuously. The suitable cation from step a) can be NH4+ or Na+. Recombinant microbial host from step a) may be removed prior to the subsequent conversion of said anthranilate anion to aniline by thermal decarboxylation in step b), wherein said removed recombinant microbial host may preferably be fed back to fermentation step a). The catalyst used in this embodiment of the invention may be a heterogeneous acid catalyst, preferably a zeolite, more preferably an H-Y zeolite. However, said catalyst may also be a heterogeneous basic catalyst, preferably a double-layer hydrotalcite, more preferably Mg-Al hydrotalcite.

[095] In a preferred embodiment of the additional method according to the invention, the extraction of aniline in an organic solvent in step c) can be carried out more than once, for an additional pre-concentration of the aniline, before distillation.

[096] In a preferred embodiment of the additional method according to the invention, the method may comprise the recovery of the organic solvent used in the extraction of step c), in which said recovery can be carried out, preferably, by distillation, in which said recovered organic solvent, preferably, can be fed back to step c) to be reused again for the extraction of the aniline produced in step b). Said organic solvent may be selected from the group consisting of alcohols, phenols, amides, ethers and aromatic hydrocarbons, wherein said alcohol is preferably 1-dodecanol. In another embodiment, this additional method of the invention may comprise another step e) of feeding back the aqueous phase from the extraction carried out in step c) and/or feeding back the aqueous phase from the distillation carried out in step d), to the fermentation in step a). The NH4+ cation can be recovered in the form of NH3, subsequent to the distillation of step d) and can be fed back to the fermentation of step a).

[097] Finally, the invention also provides the use of aniline produced according to the method of the invention, in which the aniline produced in step d) and/or in step e) is further converted into methylenedianiline (MDA) with formaldehyde in the presence of water and catalyst. The MDA produced in this way can be additionally converted to methylenediisocyanate (MDI) with phosgene.

[098] It will be apparent to those skilled in the art that various modifications can be made to the methods and recombinant host strains of the invention. Thus, it is intended that the present invention cover modifications and variations, provided they are within the scope of the appended claims and their equivalents.

Figures and Tables List of Figures

[099] Figure 1 shows a schematic overview of the biosynthetic pathway from glucose to oAB (anthranilate) in C. glutamicum, showing the strategy to increase precursor supply from central metabolism (PTS: phosphotransferase system; PEP: phosphoenolpyruvate; TCA: tricarbonic acid; IolT2: T2 inositolpermease unit; PorA/H: PorA and PorH complex; MFS: superfamily lead facilitator). Thick arrows show the biosynthetic steps that are amplified and crosses mark the biosynthetic steps that are stopped.

[100] Figure 2 is a schematic overview of central carbon metabolism in C. glutamicum. The phosphotransferase (PTS) system for glucose uptake, glycolysis, the pentose phosphate pathway and relevant anaplerotic reactions are shown. The TCA (tricarbonic acid) cycle and the shikimic acid pathway are indicated to explain the positioning and transitions for these pathways (GAPDH = glyceraldehyde-3-phosphate dehydrogenase; G6PDH = glucose-6-phosphate dehydrogenase; 6PGD = phosphogluconate dehydrogenase; PEP = phosphoenolpyruvate).

[101] Figure 3 is a schematic overview of the shikimic acid pathway in C. glutamicum, from precursor molecules to aromatic amino acids. Structural formulas of the central intermediates are presented.

[102] Figure 4 shows the genetic background of aromatic amino acid biosynthesis in P. putida KT2440. The genetic manipulations carried out are highlighted in grey.

[103] Figure 5 shows the general concept of the method according to the invention, which comprises the conversion of raw materials into anthranilate in the fermentation step, followed by a chemical conversion and purification to aniline in downstream processing.

[104] Figure 6 shows the general concept of the method according to the invention in more detail. Both NaOH and NH3 can be used as a buffer in the fermenter.

[105] Figure 7 shows a mass profile of the decomposition of anthranilic acid adsorbed on Fe-Y in the temperature range 100-550°C.

[106] Figure 8 shows the desorption of anthranilic acid to 1-dodecanol from Fe-Y. The experiment was carried out by suspending 0.2 g of Fe-Y containing 10.8 mg of anthranilic acid in 2 ml of 1-dodecanol. The suspension was stirred for 0.5 h in the temperature range of 25120 °C. The desorption results are shown in Figure 2, where a maximum of 27.8% anthranilic acid can be desorbed into the 1-dodecanol phase at 120°C.

[107] Figure 9 shows the desorption of anthranilic acid (AA) from the adsorbent into the liquid phase. The desorption test of anthranilic acid in water, from FeY, showed that the adsorption of anthranilic acid on metal substituted zeolite in aqueous solution is reversible. The desorption of anthranilic acid in an organic solvent was tested. 1-dodecanol was selected as the organic solvent due to the high solubility of anthranilic acid in it and also due to its very low miscibility in water (0.004 g/l).

[108] Figure 10 shows the decomposition of anthranilic acid in organic medium with a catalyst. The kinetics of the decarboxylation of anthranilic acid dissolved at 3% by weight in 1-dodecanol in the presence of zeolite Y at 160 °C and 180 °C is shown. Anthranilic acid (3% by weight) was dissolved in 1-dodecanol. At this concentration, the acid was perfectly soluble in the organic solvent, even at room temperature. 80 ml of the above solution was then transferred to a 160 ml autoclave, 1.5 g of zeolite catalyst was added and the mixture was heated to 160°C or 180°C. For comparison, a blank test, without catalyst (blank), with an alkaline hydrotalcite (HTC), H-ZSM5, with sodium-doped zeolite Y (NaY), the ammoniacal form of zeolite Y (NH4-Y), was also tested. Samples were collected at different time intervals and analyzed by HPLC methods to determine the rate of aniline formation.

[109] Figure 11 shows the optical density at 600 nm (OD600), followed for 33 h (triple determination), of cultures in 100 ml shake flasks, at pH 5 (with MES buffer) of strains of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas putida and Saccharomyces cerevisiae. C. glutamicum was further cultured at pH 7 (buffer supplemented with MOPS).

[110] Figure 12 shows the optical density at 600 nm (OD600), followed for 25 h (double determination), of cultures in 100 ml shake flasks, at pH 7 (with MOPS buffer) of strains of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas putida and Saccharomyces cerevisiae.

[111] Figure 13 shows the optical density at 600 nm (OD600), accompanied for 25 h (double determination), 100 ml agitation vials, in pH 7 (with mops) supplemented with 3 g/l of oab of strains of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas Putido and Saccharomy cherry VISIAE.

[112] Figure 14 shows the optical density at 600 nm (OD600), followed for 25 h (double determination), of cultures in 100 ml shake flasks, at pH 7 (with MOPS buffer) unsupplemented (black) or supplemented with 3 g/l oAB (grey) of Corynebacterium glutamicum strain.

[113] Figure 15 shows the optical density at 600 nm (OD600), followed for 25 h (double determination), of cultures in 100 ml shake flasks, at pH 7 (with MOPS buffer) unsupplemented (black) or supplemented with 3 g/l oAB (grey) of strain Pseudomonas putida.

[114] Figure 16 shows the optical density at 600 nm (OD600), accompanied for 27 h (double determination), 100 ml agitation vials at pH 7 (with mops) supplemented with 3 g/l of Escherichia coli strains, bacillus subtilis, Corynebacterium glutamicum, pseudomonas pitted and saccharomy cere VISIAE.

[115] Figure 17 shows the optical density at 600 nm (OD600), followed for 25 h (double determination), of cultures in 100 ml shake flasks, at pH 7 (with MOPS buffer) supplemented with 0.1 g/l of octanol (black and gray duplicates) of a strain of Corynebacterium glutamicum.

[116] Figure 18 shows the optical density at 600 nm (OD600), followed for 25 h (double determination), of cultures in 100 ml shake flasks, at pH 7 (with MOPS buffer) supplemented with 0.1 g/l octanol (black and gray duplicates) of a strain of Pseudomonas putida.

[117] Figure 19 shows the optical density at 600 nm (OD600), followed for 25 h, of cultures in 100 ml shake flasks, pH 7 (with MOPS buffer) of Corynebacterium glutamicum (supplemented as indicated in the graph; duplicates).

[118] Figure 20 shows the optical density at 600 nm (OD600), followed for 25 h, of 100 ml shake flask cultures at pH 5 of Corynebacterium glutamicum (supplemented as indicated in the graph; duplicates).

[119] Figure 21 shows the optical density at 600 nm (OD600), followed for 28 h, of cultures in 100 ml shake flasks, at pH 7 (MOPS) and pH 5 (MES) of Corynebacterium glutamicum (supplemented as indicated in the graph; duplicates).

[120] Figure 22 shows the optical density at 600 nm (OD600), followed for 50 h, of pH 7 fermentation cultures of Corynebacterium glutamicum supplemented after 3, 4, and 5 h to a final concentration of 0 g/l supplement (square), 7 g/l oAB (circle), 7 g/l pAB (cross), 40 mg/l dodecanol (triangle ).

[121] Figure 23 shows the HPLC chromatogram at 254 nm of Corynebacterium glutamicum fermentation culture supernatants with 7 g/l oAB after 2 h (before oAB supplementation), 5 h (after 7 g/l oAB supplementation), 25 h and 49.5 h. More oAB standard.

[122] Figure 24 shows the optical density at 600 nm (OD600), followed for 58 h, of pH 7 fermentation cultures of Corynebacterium glutamicum supplemented after 3, 4, 5, 6 and 7 h to a final concentration of 0 g/l supplement (squares), 15 g/l oAB (triangles), 35 g/l oAB (crosses) and 80 g/l of oAB (circles).

[123] Figure 25 shows the optical density at 600 nm (OD600), followed for 58 h, of pH 7 fermentation cultures of Corynebacterium glutamicum supplemented after 3, 4, 5, 6 and 7 h to a final concentration of 35 g/l oAB.

[124] Figure 26 shows the optical density at 600 nm (OD600), followed for 58 h, of pH 7 fermentation cultures of Corynebacterium glutamicum supplemented after 3, 4, 5, 6 and 7 h to a final concentration of 80 g/l oAB.

[125] Figure 27 shows a schematic of the knockout process based on the pEMG vector system.

[126] Figure 28 shows the genetic map of the P. putida KT2440 trpEGDC cluster (top) and the P. putida KT2440 trpDC deletion genetic map (bottom) (gph: phosphoglycolate phosphatase (partial); trpE: anthranilate synthase component I; GDSL: lipase of the GDSL family (not related to L-tryptophan biosynthesis); hyp: hypothetical protein; trpG: anthranilate synthase component II; crg: cAMP regulatory protein (partial); TS1: suppression flank 1; TS2: suppression flank 2).

[127] Figure 29 shows the genetic map of the P. putida KT2440 serCpheAtyrA cluster (top) and the P. putida KT2440 pheA deletion genetic map (bottom) (gyrA: DNA gyrase subunit A (partial); serC: phosphoserine aminotransferase; pheA: chorismate mutase/prephenate dehydratase; tyrA: prephenate dehydrog enase/3-phosphoshikimate 1-carboxyvinyltransferase; cmk: cytidylate kinase; TS1: suppression flank 1; TS2: suppression flank 2).

[128] Figure 30 shows the integration of a cross-flow ultrafiltration module (750 kDa cut-off value) for continuous fermentations with cell retention in a 1L laboratory scale bioreactor.

[129] Figure 31 shows the integration of a centrifuge (Centritech Lab III) for continuous fermentations with cell retention in a 1L laboratory scale bioreactor.

[130] Figure 32 shows the fermentation of C. glutamicum ΔtrpD for the production of oAB, during batch mode and continuous mode, using a culture volume of 1 l, a dilution rate of 0.05 l/h, culture temperature of 30 °C at pH 7 controlled with 1 M NH4OH, air flow of 0.2 l/min for aeration, pO2 controlled at 30% air saturation adjusting stirring speed between 200 and 1200 rpm. Cell retention obtained by cross-flow ultrafiltration.

[131] Figure 33 shows the fermentation of C. glutamicum ΔtrpD::trpD5Δgpi for the production of oAB, during batch mode and continuous mode, using a culture volume of 1 l, a dilution rate of 0.01 l/h, culture temperature of 30 °C at pH 7 controlled with 1 M NH4OH and 1 M HCl, air flow rate of 0.2 l/min for aeration, pO 2 controlled at 30% air saturation by adjusting the stirring speed between 200 and 1400 rpm. Cell retention obtained by centrifugation.

[132] Figure 34 shows fermentation of C. glutamicum ΔtrpD for continuous production of oAB, using a culture volume of 1 L, a dilution rate of 0.025 L/hr or 0.05 L/hr as listed in the diagram, culture temperature 30 °C at pH 7 controlled with 1 M NH4OH, air flow rate 0.2 L/min for aeration, pO2 controlled at 30% air saturation by adjusting the stirring speed between 200 and 1200 rpm. Cell retention obtained by cross-flow ultrafiltration.

[133] Figure 35 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) to aniline with a catalyst. The decarboxylation kinetics of anthranilic acid (AA) dissolved at 40% by weight in aniline in the presence of zeolite Y at 160°C, 180°C and 200°C is shown.

[134] Figure 36 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) to aniline with a catalyst. The kinetics of the decarboxylation of anthranilic acid (AA) dissolved at 40% by weight in aniline and in 10% water-90% aniline in the presence of zeolite Y at 200°C is shown.

[135] Figure 37 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) to aniline with a catalyst. The kinetics of the decarboxylation of anthranilic acid (AA) dissolved in 40% by weight in aniline in the presence of zeolite Y at 180°C is shown.

[136] Figure 38 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) to aniline with a catalyst. The kinetics of the decarboxylation of anthranilic acid (AA) dissolved in 40% by weight in aniline in the presence of zeolite Y at 180°C is shown.

[137] Figure 39 shows the IR spectrum of a freshly prepared H-Y catalyst and another after the reaction as described above using AA isolated from hydrolyzed corn starch. Typical bands for aniline are marked. Absorption of this highly basic species occurs. The OH region is also highlighted. The reduction of the OH signal is compatible with some partial ion exchange that could occur with present trace elements, however the OH groups do not seem to be active in the catalysis, but the AL-O-Si bridge is probably responsible.

List of Tables

[138] Table 1 shows the vectors and plasmids used and/or generated in this study.

[139] Table 2 shows the bacterial strains used and/or generated in this study.

[140] Table 3 shows the primers used in this study.

[141] Table 4 shows the characteristics regarding oAB production of bacterial strains used and/or generated in this study (CDW: dry weight of cells; Y: yield; μmax: maximum growth rate; STY: space-time yield).

[142] Table 5 shows the biochemical characteristics of crude extracts of C. glutamicum ΔtrpD producing different TrpEG variants with respect to TrpEG activity.

[143] Table 6 shows the adsorption of 0.5% AA in water by HAP, zeolite Y (GO257 and GO55) and ZSM5 (ZSM5-27 and ZSM5-55), as described in Example 6.

[144] Table 7 shows the AA adsorption capacities of zeolite substituted with metal GO257 in g of AA/kg of adsorbent, in distilled water and buffered aqueous solution after 10 and 60 minutes, as described in Example 6.

Examples Example 1 - Production of o-aminobenzoate with E. coli

[145] E. coli strain W3110 trpD9923 (Escherichia coli::trpD9923; Table 2) was purchased from the Center for Genetic Resources in E. coli at Yale University. The strain was created by random mutagenesis and contained a mutant trpD gene called trpD9923. An inactivation of the enzyme was achieved by a random point mutation (G^T) in the related gene, resulting in a nonsense mutation in the region encoding the transferase activity. As a result, only seven amino acids of the original transferase domain are translated (Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615). The related truncated enzyme of the trpD9923 gene loses its ability to catalyze the anthranilate phosphoribosyl transferase reaction, but retains its anthranilate synthase activity. Therefore, the strain can synthesize anthranilate but cannot metabolize it beyond L-tryptophan and is therefore auxotrophic for L-tryptophan. This leads to anthranilate buildup.

[146] The strain was grown in 50 ml shake flasks with a culture volume of 10 ml at 28 °C and 140 rpm. The medium used was mineral medium M9 (1 g/l of (NH4)2Cl, 0.5 g/l of NaCl, 0.05 g/l of thiamine, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.247 g/l of MgSO4 • 7H2O (Merck, Darmstadt), 0.015 g/l of CaCl2 and 10 g/l of glucose). The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution with 20 mg/l of L-tryptophan. The strain produced 60 mg/L of anthranilate after 25.5 h as measured by HPLC-DAD (254 nm) (Table 4 and Example 3). The strain was further optimized by inactivating the phosphotransferase (PTS) system using knockout suppression. Therefore, the resulting PTS-deficient Escherichia coli::trpD9923Δhpr strain (Table 2) was generated and adapted for growth in glucose and tested for anthranilate production using a 25 ml fermentation shake flask at 37°C and 150 rpm with a culture volume of 10 ml. The same medium as for the pts positive strain was used. It produced 69 mg/L of anthranilate after 25 hours as measured by HPLC-DAD (254 nm) (Table 4 and Example 3).

Example 2 - Strain selection for development of a microbial strain that produces o-aminobenzoate

[147] Several strains commonly used in industrial biotechnology have been investigated for their natural tolerance to oAB and the organic solvents in question for further product extraction (eg, 1-dodecanol or octanol). Furthermore, the influence of the pH value used was investigated. It was known from previous experiments that, at least for E. coli the toxicity of aromatic compounds strongly depends on the pH value, probably because protonated acids can penetrate the cell membrane much more efficiently and, consequently, their toxicity is greatly increased. Extraction of oAB in an organic phase supposedly works best at low pH values, close to the isoelectric point of oAB. The most relevant host strains considered were: Escherichia coli K12, Corynebacterium glutamicum DSM 20300 (= ATCC 13032), Pseudomonas putida KT2440, Bacillus subtilis subsp. 168, Saccharomyces cerevisiae DSM 70449 and Pichia pastoris X33 (DSM 70382).

[148] To investigate the chosen strains with respect to the mentioned characteristics, shake flasks and small-scale fermentation experiments were used and the organisms were supplemented with toxic substances (aminobenzoates, solvents) at different concentrations, different pH values, and the behavior of the strains was studied.

[149] The organisms (obtained commercially by order from DSMZ, German collection of Microorganisms and cell cultures, Braunschweig) were first cultured in shake flasks and then each was added to each of them with published standard minimal medium to characterize their growth under standard culture conditions (C. glutamicum: Preculture: BHI medium (37 g/l; Brain Heart infusion; Becton Dickenson and Company, Heidelberg); main culture pH value 7: CGX medium II-MOPS (42 g/l MOPS buffer, 20 g/l (NH4)2SO4, 5 g/l urea (Fisher Scientific, Schwerte), 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g/l MgSO4•7H2O (Merck, Darmstadt), 0.01 g/l CaCl2 and 10 g/l glucose (sterile) autoclaved separately)). The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution. The following components were added after sterile filtration: 2 mg/l Biotin, 0.01 g/l MnSO4^7H2O (Merck, Darmstadt), 0.01 g/l FeSO4^7H2O (Merck, Darmstadt), 1 mg/l ZnSO4^7H2O, 0. 2 mg/l of CuSO4^5H2O (Merck, Darmstadt), 0.02 mg/l of NiCl2^6H2O (Merck, Darmstadt) and 0.03 g/l of 3,4-dihydroxybenzoic acid (Acros Organics, Nidderau); Main culture pH = 5: CGXII-MES medium (39 g/l MES buffer, 20 g/l (NH4)2SO4, 5 g/l urea (Fisher Scientific, Schwerte), 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g/l MgSO4 • 7H2O (Merck, Darmstadt), 0.01 g/l Ca Cl2 and 10 g/l of glucose (autoclaved separately). The pH was adjusted to 5 with 5 mol/l sodium hydroxide solution. The following components were added after sterile filtration: 2 mg/l of biotin, 0.01 g/l of MnSO4^H2O (Merck, Darmstadt), 0.01 g/l of FeSO4^7H2O (Merck, Darmstadt), 1 mg/l of ZnSO4^7H2O, 0.2 mg/l CuSO4^5H2O (Merck, Darmstadt), 0.02 mg/l NiCl2^6H2O (Merck, Darmstadt) and 0.03 g/l 3,4-dihydroxybenzoic acid (Acros Organics, Nidderau) E. coli: Preculture: LB Medium (Luria-Bertani; Roth, Karlsruhe); pH of culture Major 7: M9 medium (1 g/l (NH4)2Cl, 0.5 g/l NaCl, 0.05 g/l thiamine chloride, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.247 g/l MgSO4 • 7H2O (Merck, Darmstadt), 0.015 g/l CaCl2 and 10 g/l glucose). The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution; Main culture pH 5: M9-MES medium (19.5 g/l MES buffer, 1 g/l (NH4)2Cl, 0.5 g/l NaCl, 0.05 g/l thiamine chloride, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.247 g/l MgSO4 • 7H2O (Merck, Darmstadt), 0.015 g/ l of CaCl2 and 10 g/l of glucose. The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution; 8. subtilis: Preculture: LB medium; main culture pH 7: M9-SL medium (1 g/l of (NH4)2Cl, 0.5 g/l of NaCl, 0.05 g/l of thiamine, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.247 g/l MgSO4^7H2O, 0.015 g/l CaCl2, 1.0 mg/l MnCl^O, 1.35 mg/l FeCl-6H2O, 1.7 mg/l ZnSO4^7H2O, 0.04 mg/l CuCl-2H2O, 0.06 mg/l CoCl2^6H2O, 0.06 mg/l of Na2MoO4 • 2H2O and 10 g/l of glucose. The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution; main culture pH 5: M9-SL-MES medium (19.5 g/l of MES buffer, 1 g/l of (NH4)2Cl, 0.5 g/l of NaCl, 0.05 g/l of thiamine, 1 g/l of KH2PO4, 1 g/l K2HPO4, 0.247 g/l MgSO4^7H2O, 0.015 g/l CaCl2, 1.0 mg/l MnCl-H2O, 1.35 mg/l FeCl-6H2O, 1.7 mg/l ZnSO4^7H2O, 0.04 mg/l CuCl-2H2O, 0.06 mg/l CoCl 2^6H2O, 0.06 mg/l of Na2MoO4 • 2H2O and 10 g/l of glucose. The pH was adjusted to 5 with 5 mol/l sodium hydroxide solution); P. putida: Preculture: LB medium; Main culture pH 7: Brunner medium (0.5 g/l (NH4)2SO4, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.2 g/l MgSO4^7H2O, 0.05 g/l CaCl2, 5.0 mg/l EDTA, 2.0 mg/l FeSO4^7H2O, 0.03 mg/l MnCl ^O, 0.1 mg/l ZnSO4^7H2O, 0.01 mg/l CuCl-2H2O, 0.2 mg/l CoCl2^6H2O, 0.03 mg/l Na2MoO4 • 2H2O, 0.3 mg/l H3BO3, 0.02 mg/l NiCl2^6H2O and 10 g/l glucose. The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution; main culture pH 5: Brunner-MES medium (19.5 g/l MES buffer, 0.5 g/l (NH4)2SO4, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.2 g/l MgSO4^7H2O, 0.05 g/l CaCl2, 5.0 mg/l EDTA, 2.0 mg/l FeSO4^7H2O, 0.03 mg/l MnCl^O, 0.1 mg/l ZnSO4^7H2O, 0.01 mg/l CuCl-2H2O, 0.2 mg/l CoCl2^6H2O, 0.03 mg/l Na2MoO4 • 2H2O, 0.3 mg/l H3BO3, 0.02 mg /l NiCl2^6H2O and 10 g/l glucose. The pH was adjusted to 5 with 5 mol/l sodium hydroxide solution) S. cerevisiae: Preculture: YPD medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose); Main culture pH 5: SCM medium (5 g/l (NH4)2SO4, 3 g/l KH2PO4, 2.5 g/l MgSO4^7H2O, 0.5 g/l NaCl, 20 mg/l inositol, 5 mg/l thiamine, 1.32 mg/l riboflavin, 17.8 mg/l calcium pantothenate, 18.4 mg /l of nicotinic acid, 5.16 mg/l of pyridoxine-HCl, 0.18 mg/l of biotin, 0.12 mg/l of folic acid, 40.6 mg/l of FeCl3^6H2O, 6.0 mg/l of ZnCl2^4H2O, 3.0 mg/l of CaCl2^2H2O, 5.76 mg/l of CuSO4^5H2O, 6.0 mg/l of CoCl2^6H2O, 6.0 mg/l of Na2MoO4 • 2H2O, 1.56 mg/l of H3BO3 and 5 g/l of glucose. The pH was adjusted to 5 with 5 mol/l sodium hydroxide solution.) (Sambrock J; Fritsch, EF, and Maniatis, T: Molecular Cloning - a laboratory manual. Cold Spring Larbor Laboratory Press; 1989; New York; Harwood CR, Cutting SM: Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons Ltd; 1990; Keilhauer, C, Eggeling, L, Sahm, H: J Bacteriol, 1993, 175(17): 5595 ). These experiments were carried out with a pH of the medium equal to 7 and 5, to evaluate the difference in toxicity of the aminobenzoate at low pH values. Yeast strains were only investigated at pH values below 7 (5.5 and 3.5). The different pH values were stabilized by adding different buffer systems (MOPS, MES).

[150] 100 ml shake flasks for strain cultivation, pH 5 (with MES buffer), were incubated for 33 h and growth was followed by measuring optical density at 600 nm (OD600) over time (triple determination). C. glutamicum was further cultured at pH 7 (with MOPS buffer). Figure 11 shows the results.

[151] The data obtained showed that C. glutamicum has the best growth at pH 5, among all the investigated organisms. E. coli, P. putida and S. cerevisiae grew very weakly at pH 5. However, in some of the cultures, due to metabolic activity, the pH fell further below 4. To exclude growth effects due to metabolic activities, experiments were carried out in fermenters with automatic pH regulation. As expected, it was already visible that culture at low pH decreases the growth rate of all tested organisms. However, for the comparison of the growth of all considered organisms under standard culture conditions at pH 7, the experiment was repeated with all minimal media supplemented with MOPS buffer. Furthermore, the cultures were similarly supplemented with oAB (final concentration of 3 g/l, added at the beginning of the exponential growth phase in three portions (after 2, 3 and 4 h of incubation time)). 100 ml shake flasks for strain cultivation at pH 7, with or without supplementation with 3 g/l of oAB, were incubated for 25 h and growth was monitored by measuring optical density at 600 nm (OD600) over time (double determination) (Figure 12 and Figure 13). C. glutamicum reached the highest OD600 values under the chosen culture conditions and, moreover, its growth was not inhibited by the addition of 3 g/l of oAB, whereas the growth of the other organisms, e.g. B. subtilis, was greatly diminished at 3 g/l of oAB. From these results, C. glutamicum was identified as the preferred candidate for oAB production at pH 7 (Figure 14 and Figure 15).

[152] To investigate the resistance of the considered strains to para-aminobenzoate (PAB) and solvents (1-octanol and dodecanol), they were cultured at pH 7 (S. cerevisiae at pH 5.5), as described above, and supplemented with 3 g/l of pAB (Figure 16). The growth of C. glutamicum, E. coli, P. putida and P. pastoris was not inhibited by pAB under these conditions, whereas the growth of S. cerevisiae and B. subtilis was severely reduced. The toxicity of the octanol extraction solvent was investigated. The solubility of octanol in water is about 0.1 g/l, therefore cultures were supplemented with such amount of octanol and additionally with 3 g/l. It was shown that already at 0.1 g/l, but even more clearly at 3 g/l of octanol, all organisms were able to grow. Only P. putida showed lower growth activity after the addition of octanol (Figure 17 and Figure 18).

[153] As in all experiments carried out, C. glutamicum had shown the highest cell densities and best resistance capacities, this strain was similarly cultivated with 5 g/l of oAB and 5 g/l of pAB at pH 5 and pH 7, respectively. No growth inhibition was detected under these conditions (Figure 19 and Figure 20). At pH 5, cell density is reduced by half, which results in the conclusion that fermentation should be carried out, more preferably, at pH 7.

[154] Dodecanol has been investigated as an alternative extraction solvent. Dodecanol has a solubility in water of about 40 mg/l. C. glutamicum was cultured as described above, at pH 5 and 7 with 40 mg/l 1-dodecanol supplementation. Dodecanol does not influence strain growth under these conditions (see Figure 19 and Figure 20).

[155] Results obtained from shake flask experiments with C. glutamicum have been replicated in small-scale fermenters (Figure 21). To further characterize the resistance of C. glutamicum against aminobenzoates and 1-dodecanol stress at pH 7, small-scale fermentations of the organism were carried out using a 1 L - 4 times - fermentation unit (HiTecZang) with regulated pH, agitation, oxygen supply, temperature and glucose feed. The glucose feed allowed the strain to grow to higher cell densities.

[156] The fermentation experiment was performed with C. glutamicum in minimal medium at pH 7 (as described above, but without the addition of MOPS, biotin, protocatechuate, and urea) and in a fermenter without supplementation (positive control), a fermenter with 7 g/l oAB, a fermenter with 7 g/l pAB, and a fermenter with 40 mg/l 1-dodecanol. The resulting growth curves show that the growth of C. glutamicum is not influenced by the addition of 1-dodecanol. This confirmed the results obtained in the shake flasks (see Figure 22). Supplementation with oAB and pAB, respectively, resulted in a prolonged latency phase, but eventually the same cell densities were achieved (see Figure 22). It is concluded that 1-dodecanol is a suitable solvent for oAB extraction.

[157] The aminobenzoate concentration was further increased to find the boundary at which it significantly inhibited the growth of C. glutamicum. It had to be excluded that aminobenzoates were degraded by the body and therefore growth is restored after the prolonged latency phase. To investigate the stability of the aminobenzoate in the supernatant of fermenter cultures, samples were collected during fermentation and analyzed by HPLC-DAD (254 nm) (see Figure 23 and Example 3). The aminobenzoate was not significantly degraded during culture.

[158] In an additional experiment, the concentration of oAB was increased to investigate its effect on the C. glutamicum strain. The four fermenters were supplemented with 0 g/l, 15 g/l, 35 g/l and 80 g/l of oAB. To achieve acid solubility at such high concentrations at pH 7, the acid was titrated with ammonium hydroxide to form the corresponding ammonium salt of oAB in standard solutions and then added to the fermenters in five portions over 5 h (after culturing for 3, 4, 5, 6, and 7 h). The remaining culture conditions were maintained as described above. Addition of 80 g/l of oAB caused death (lysis) of the cells after 54 hours and addition of 35 g/l of oAB resulted in an inhibition of growth (see Figure 24, Figure 25 and Figure 26). Cultures supplemented with 15 g/l of oAB demonstrated an extensive lag phase, greater than 34-48 h, before growth recovered and the cells entered the exponential phase (see Figure 24). A degradation of oAB during fermentation was again excluded by HPLC measurements of culture supernatants (data not shown).

[159] Taken together, it was concluded that the production of oAB should be done, even more preferably, with a strain of Corynebacterium glutamicum ATCC 13032. In addition, it was also concluded that the fermentation process should preferably be carried out at pH between 5-7 and more preferably at pH 7, and the extraction (if necessary) should preferably be carried out with 1-dodecanol as the extraction solvent. Octanol is not a suitable extraction solvent.

Example 3 - Production of o-aminobenzoate with C. glutamicum

[160] A bacterial strain has been developed that produces anthranilate on a g/l scale. This work was based on genetic manipulations of tryptophan producers (e.g., in E. coli and C. glutamicum) and included the development and implementation of a strategy to genetically manipulate central metabolism, common aromatic biosynthesis, and the L-tryptophan branch pathway, in order to obtain an anthranilate (oAB) producer. Based on the results described above, the metabolic engineering was preferably based on Corynebacterium glutamicum ATCC 13032.

General culture of strains based on Escherichia coli DH5α

[161] Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (Sternheim) and all applied enzymes from New England Biolabs (Schwalbach). E. coli strains were grown under sterile conditions on LB medium (Luria Bertani; Roth, Karlsruhe) or on LB-agar plates (Abcr GmbH & Co. KG, Karlsruhe) at 37 °C. Selective culture was obtained by adding antibiotics (100 mg/l ampicillin sodium salt; 50/25 mg/l kanamycin sulfate, 100 mg/l spectinomycin sulfate). For plasmid preparation, strains were cultivated in 15 ml tubes for 14-16 h, with constant agitation at 200 rpm, in 3 ml of LB medium and a selective antibiotic, at 37 °C (Kuhner Shaker ISF-4-W; Adolf Kühner AG, Basel (Switzerland)).

General methods of molecular biology

[162] PCR reactions were generally performed using Platinum® Taq DNA Polymerase (5 U/μL; Life technologies, Darmstadt) with about 50 ng of PCR template and 10 pmol of response primer pair (see Table 3). PCR conditions: 98 °C 5 min, 98 °C 30 s, 56 °C 30 s, 72 °C 1 min/kb, 30x, 16 °C hold (Mastercycler® nexus - Cycler; Eppendorf, Hamburg). Plasmid DNA purification was performed using the NucleoSpin® Plasmid Pure kit (Macherey & Nagel, Düren), according to the manufacturer's instructions. Plasmid DNA digestion was performed in 20 µl scales, with 5 U of the related restriction enzyme(s) and about 1 µg of plasmid DNA in buffer, recommended by the enzyme supplier, and incubated for about 2 h at 37 °C and then analyzed by agarose gel electrophoresis. Agarose gels were prepared with 1% agarose (Abcr GmbH & Co. KG, Karlsruhe) dissolved in 1x TAE electrophoresis buffer (Promega, USA Fitchburg). The supplied electric field was 90 V-120 V (EPS300; Pharmacia Biotech; VWR International GmbH, Darmstadt) and DNA was detected using ethidium bromide (0.3-0.5 mg/l) or Midori Green (Midori Green Advance DNA strain; NIPPON Genética EUROPE GmbH, Düren). Gel documentation: Gene Genius®; VWR; Darmstadt. For DNA extraction in agarose gel, a NucleoSpin® Gel and PCR Clean-up kit (Macherey & Nagel, Düren) was used, according to the manufacturer's specification. For DNA ligation, a T4-DNA ligase (20 U) was used in the buffer provided by the manufacturer. The mass ratio of insert used to plasmid was 4:1. The mixture was incubated for about 15 h at 16 °C and then incubated at 65 °C for 10 min. Plasmids and ligation reactions were introduced into electro-competent Escherichia coli DH5a cells (ElectroMAX™ DH5α-E™ Competent Cells; Life Technologies, Darmstadt). After electrotransformation (conditions: ~20 ms pulse with exponential decay, 2.5 kV/cm, 25 F, 200 Q) in 0.2 cm gap electroporation crucibles (BioRad, Hercules, CA) using a Gene Pulser Xcell system (BioRad, Hercules, CA), 800 μl of LB regeneration medium was immediately added and the suspension was transferred to a microcentrifuge tube ga of 1.5 ml. After 1 h at 37 °C, a cell suspension was spread onto LB-agar plates, supplemented with appropriate antibiotics and incubated at 37 °C overnight. Clones were collected and correct transformation was confirmed by restriction analysis, colony PCR and/or sequencing. For PCR analysis of colonies, a colony was picked with a sterile toothpick, transferred to a separate plate (master plate), dissolved in 1 μl of DMSO and boiled for 10 min at 98 °C before being added to a PCR master mix. Correct cloning of all plasmids and generation of related mutants was confirmed by PCR and/or DNA sequencing.

General Culture of strains based on Corynebacterium glutamicum ATCC13032

[163] C. glutamicum strains were grown under sterile conditions in tubes of BHI medium (37 g/l; Brain-Heart infusion; Becton Dickenson and Company, Heidelberg) and tubes, shake flasks or on agar plates at 30 °C. Selective culture was obtained by adding antibiotics (15 mg/l kanamycin sulfate, 100 mg/l spectinomycin sulfate).

[164] For strain characterization, the first preculture was started from an isolated colony and grown for 10 hours with constant agitation at 400 rpm, in 4 ml of BHI medium and, depending on the strain, supplemented with an aromatic amino acid or suitable antibiotics. The second preculture was grown in 50 ml CGXII-MOPS medium (42 g/l MOPS buffer, 20 g/l (NH4)2SO4, 5 g/l urea (Fisher Scientific, Schwerte), 3.7 g/l BrainHeart infusion, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g/l MgSO4 • 7H2O (Merck, Darmstadt), 0.01 g/l CaCl2 and 10 g/l glucose (autoclaved separately). The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution. The following components were added after sterile filtration: 2 mg/l biotin, 0.01 g/l MnSO4^H2O (Merck, Darmstadt), 0.01 g/l FeSO4^ 7H2O (Merck, Darmstadt), 1 mg/l ZnSO4^7H2O, 0.2 mg/l CuSO4^5H2O (Merck, Darmstadt), 0.02 mg/l NiCl2^6H2O (Merck, Darmstadt) and 0.03 g/l 3,4-dihydroxybenzoic acid (Acros Organics, Nidderau) in 250 ml shake flasks, after inoculation with 1 ml of the first pre-culture grown for 16-18 h with constant stirring at 400 rpm. The fermentation culture was grown in 100 ml of CGXII-MOPS medium (20 g/l of (NH4)2SO4, 5 g/l of urea, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.25 g/l of MgSθ4'7H2θ , 0.01 g/l of CaCl2, 100 μl/l of polypropylene glycol (autoclaved separately) and 18 g/l of glucose (autoclaved separately). The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution. The following components were added after sterile filtration: 2 mg/l Biotin, 0.01 g/l MnSO4^H2O, 0.01 g/l FeSO4^7H2O, 1 mg/l ZnSO4^7H2O, 0.2 mg/l CuSO4^5H2O, 0.02 mg/l NiCl2^6H2O and 0.03 g/l acid 3 ,4-dihydroxybenzoic acid. Each bioreactor (DasBox, Eppendorf, Hamburg, Germany) was inoculated with the second preculture to a final OD600 of 0.5. The initial stirring speed was set to 100 rpm and air was supplied at 2.2 l/h (0.37 V/(V*min)). Dissolved oxygen was continuously monitored (OxyFermFDA 120; Hamilton, Bonaduz (Switzerland)) and maintained at 30% air saturation by automatic adjustment of stirring speed. The pH was measured (EasyFermPlus K8 120; Hamilton, Bonaduz (Switzerland)) and maintained at 7 by automatic addition of 1 M NaOH and the temperature was maintained at 30 °C. To avoid foaming, 10% polypropylene glycol solution was added automatically by a conductivity sensor measuring the conductivity of the above fermentation broth. Fermentation was carried out as a repeated batch process. An additional feeding of 18 g/l of glucose (sterilized in an autoclave separately) was performed three times. During dry cell weight (BDW) fermentation, glucose concentration and anthranilate concentrations were measured off-line from 1.5 ml samples from the fermentor. A 1 ml aliquot of each sample was centrifuged for 5 min at 16000 x g (5415R; Eppendorf, Hamburg, Germany) in a weighed reaction tube. For determination of dry weight of cells, the pellet was dried for at least 72 h at 60 °C (hybridization oven; Appligene Oncor LifeScreen, Watford (UK)). Then an analytical balance (La 230 S; Satorius AG, Gottingen) was used to determine the exact weight of the pellet. The supernatant was analyzed by HPLC-DAD (1100; Agilent Technologies, Santa Clara (USA)) and YSI (YSI-Select 2700; Kreienbaum Neoscience GmBH, Langenfeld). The collected data are shown in Table 4.

General manipulation of Corynebacterium glutamicum ATCC13032

[165] Plasmid DNA was transferred into Corynebacterium glutamicum ATCC13032 by electroporation. Para a transformação, foram coletadas células de uma cultura de 200 ml cultivada em meio BHI e na fase de crescimento exponencial (DO600 = 1,75-2,0) por centrifugação (4000 g, 10 min, 4 °C (5810R; Eppendorf, Hamburgo)) e lavadas três vezes com 20 ml de tampão TG gelado (Tris-HCl 1 mM, glicerina a 10 %, pH 7,0). The pellet was resuspended in 2 ml of 10% glycerin solution and used directly for transformation, or stored until use at -80 °C. For transformation, plasmid DNA (about 1 µg) was pipetted first into a 0.2 cm gap electroporation crucible, followed by 150 µl of cell suspension. After incubation on ice for 5 min, electroporation was performed (conditions: ~ 20 ms pulse with exponential decay, 2.5 kV/cm, 25 F, 200 Q). The suspension was transferred to reaction tubes with 500 μl of pre-warmed BHI medium (46 °C) and incubated at 30 °C for 1 h, with constant stirring at 400 rpm. Cells were spread on agar plates with BHI medium containing the corresponding antibiotic.

[166] For expression of the designated target genes, the pEKEx2 bridge vector from E. coli-C was used. glutamicum (Eikmanns BJ, Kleinertz E, Liebl W, Sahm H, Gene, 1991, 102:93). The 8.16 kb vector is derived from the pU18 vector and encodes a kanamycin-resistant cassette, a pBL1 point of origin V (oriV) for proliferation in C. glutamicum, a pU18 oriV for proliferation in E. coli, a strong tac promoter (inducible by isopropyl β-d-1-thiogalactopyranoside (IPTG)), and a lacIQ gene. Alternatively, the bridge vector pCRB210 from E. coli-C was used. glutamicum (Yukawa H, Inui M, US20130302860 (A1), 2013), which is compatible for cotransformation with pEKEx2. The 4.96 kb vector encodes a spectinomycin-resistant cassette, a pCASE1 point of origin V (oriV) for proliferation in C. glutamicum, an oriV for proliferation in E. coli, and a gapA promoter for constitutive gene expression. Genetic fragments selected for overexpression in C. glutamicum or its mutants were synthesized by MWG Operon GmbH, by removing all naturally occurring SbfI, BamHI, BglII, NcoI and NdeI restriction sites by individual nucleotide exchanges (silent mutations) and inserting a ribosomal binding site upstream of the corresponding ORF (in addition to the aroG, aroK, glnA and aroL gene sequences, which were amplified from their natural loci by PCR, see primers (Table 3)). In the case of the trpEG and aroG genes, individual nucleotide changes were included in the gene to access the feedback resistant version of the related enzymes. The resulting sequences were cloned into the multiple cloning site of the pEKEx2 vector by restriction and religation of SbfI and BamHI. With the exception of the glnA genetic sequence, this was cloned into the pCRB210 vector via NcoI restriction and religation, yielding pCRB-glnA (Table 1). The synthesized gene fragments basically have the structure: "SbfI-BglII-RBS-gene-BamHI". This allows for the interactive ligation of target genes (such as BglII-BamHI fragments) into the vector through unique BamHI restriction sites. Plasmids were sequenced to confirm correct cloning and sequence. C. glutamicum transformants (and their mutants) were screened for antibiotic resistance and the correct generation of related mutants was confirmed by PCR.

[167] For the deletion of target genes, as well as for the insertion of fragments of genes or other sequences into the genome of C. glutamicum, the pK19mobsacB bridge vector from E. coli-C was used. glutamicum (Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Pühler A, Gene, 1994, 145:69). The 5.72 kb vector is derived from the pU18 vector and encodes a kanamycin resistant cassette, no point of origin for proliferation in C. glutamicum, an oriV pU18 for proliferation in E. coli, a lacZα gene, and a sacB gene. The target sequences were synthesized by MWG Operon GmbH (with the exception of trpD and csm, which were amplified by PCR from the genome of C. glutamicum ATCC13032 with specific primers (Table 3)), removing all naturally occurring HindIII and EcoRI restriction sites by single nucleotide exchanges (silent mutations) and cloned into the multiple cloning site of the vector by restriction and religation of HindIII and EcoRI. For gene deletion (or integrations), the cloned DNA sequences consist of 300-500 bp of the 5' flanking region of the target gene (including the first 6 codons of the gene), 21 bp of the foreign missense sequence (or a sequence to be integrated into the genome), and 300-500 bp of the 3' flanking region of the target gene (including the last 6 codons of the gene of interest). C. glutamicum transformants were selected for individual crossover events by integrating the plasmids into the genome by kanamycin selection, as the resulting plasmids cannot proliferate in C. glutamicum by themselves. BHI medium agar plates (containing 15 mg/l kanamycin) carrying the transformants were cultured at 30°C for 1-7 days. Then, the isolated clones were classified for double crossover events, resulting in the deletion of the target genes in the structure, leaving only the extraneous missense sequence of 21 bp (or else, sequences to be integrated in the genome), by selection with sucrose (the colonies were cultivated during 4-6 h in 5 ml of BHI medium and then plated in two different dilutions (1:100 and 1:10000) in agar plates with BHI medium containing 10% (w/ v) of sucrose. The sacB gene in the vector encodes a levansucrase, which transforms sucrose into a lethal product and, as a result, prevents the growth of all clones that still carry the plasmid sequences in their genome. All colonies that grew on plates containing sucrose were analyzed by cotransfer on agar plates with BHI medium supplemented with 15 mg/l of kanamycin, as well as agar plates with BHI medium and grown at 30 °C for 1 -7 days Colonies that grew only on plates without kanamycin supplementation were analyzed by colony PCR to confirm correct recombination. Correct cloning of all plasmids and generation of related mutants was confirmed by PCR and/or DNA sequencing.

High Performance Liquid Chromatography

[168] An Agilent 1100 series HPLC-DAD system (with diode array detector, Agilent Technologies, Santa Clara (USA)) was used to quantify oAB concentrations in culture supernatants. As a stationary phase, a C18 Luna® HPLC column (4.6 x 250 mm, 3 μm; Phenomenex) was used at 20°C with a binary solvent system consisting of methanol (solvent B) and water containing 0.1% formic acid (solvent A). 10 μl of diluted culture supernatants were injected. The following gradient was applied with a flow rate of 0.5 ml/min: 0-1 min, 2% B; 1-2 min, 2-10% B; 2-12 min, 10-70% B; 12-23 min, 7090% B; 23-25 min, 90-98% B, 25-27 min, 98% B; 27-27.5 min, 98% to 2% B; 27.5-30 min, 2% B. The oAB concentration was determined from signal integration at 254 nm (retention time: 18.9 min) using an external calibration curve.

Engineering of the trpD gene in C. glutamicum strains

[169] The Corynebacterium glutamicum strain ΔtrpD (see Table 2) was created by in-frame deletion of the trpD gene (encoding anthranilate phosphoribosyl transferase; Cgl3032; SEQ ID NO: 1) using the bridge-like vector pK19mobsacB from E. coli-C. glutamicum. The 5' flanking region of the trpD open reading frame (including the first 7 codons of the gene) and the 3' flanking region of the target gene (including the last 8 codons of the trpD open reading frame) were amplified from genomic DNA isolated from Corynebacterium glutamicum ATCC 13032 by PCR using primer pairs DeL-trpD-1 and DeL-trpD-2, and DeL-tr pD-3 and DeL-trpD-4, respectively (Table 3, SEQ ID NO: 66-69). The two resulting fragments were combined by Crossover PCR technique, using the primer pair DeL-trpD-1 and DeL-trpD-4. The resulting fragment was cloned into the multiple cloning site of the pK19mobsacB vector via SmaI restriction and religation. The resulting vector was applied for the in-frame deletion of trpD from the genome of Corynebacterium glutamicum ATCC 13032 and introduced in-frame a strange missense sequence of 24 bp in the genome, which was used for the Crossover PCR technique (resulting sequences in the trpD locus: SEQ ID NO: 2). The correct mutants were isolated as described above and the correct gene deletion verified by PCR. This yielded an auxotrophic strain for L-tryptophan, with oAB accumulation. The strain was characterized with respect to its properties as an oAB producer as described above with the addition of 0.1 mM L-tryptophan or 0.1 mM indole (see Table 4).

[170] Six versions of the trpD gene were cloned into the pEKEx2 vector by restriction and religation of SbfI and BamHI as described above. Six different versions of the trpD gene were generated with different initiation codons and spacer lengths between the ribosomal binding site and the trpD initiation codon (SEQ ID NO: 3-8) by PCR using C. glutamicum genomic DNA as a template. The primers applied for the generation of the six fragments were forward primers (Ex-trpD-1 to -6, Table 2, SEQ ID NO: 7075), which include the altered initiation codon and the spacer between the ribosome binding site and the initiation codon, together with the altered reverse primer for each version (Ex-trpD-rev, Table 2, SEQ ID NO: 76). This method was chosen in order to obtain a reduction in the translation of the TrpD protein in the produced strains. The Corynebacterium glutamicum ΔtrpD strain (Table 2) was transformed with the resulting plasmids (Table 1) and the resulting Corynebacterium glutamicum ΔtrpD/pEKEx2-trpD1-6 strains (Table 2) were grown without the addition of L-tryptophan and were therefore characterized with respect to their properties as an oAB producer, as described above, with supplementation of 2 5 mg/l of kanamycin and 1 µM of IPTG (isopropyl-β-d-thiogalactopyranoside) in the medium (see Table 4). Corynebacterium glutamicum ΔtrpD/pEKEx2-trpD5-6 strains also produced enough L-tryptophan to allow biomass formation; they accumulated significant amounts of oAB (Table 4). Furthermore, the trpD, trpD1-3, trpD5 and trpD6 gene versions were integrated into the genome of the Corynebacterium glutamicum ΔtrpD strain (Table 2) by homologous recombination using the pK19mobsacB vector, as described above. The 5' flanking region of the trpD gene versions and the 3' flanking region of the target gene were amplified from genomic DNA isolated from Corynebacterium glutamicum ATCC 13032 by PCR using primer pairs DeL-trpD-1 and Ko-trpD-1, as well as DeL-trpD-3 and DeL-trpD-4, respectively (Table 3, SEQ ID NO: 66, 77, 68 and 69). The two resulting fragments were combined with the different variants of trpD by Crossover PCR technique using primer pairs Ex-trpD-1-3, -5 or -6 and Ko-trpD-2 (Table 3, SEQ ID NO: 70-72, 74, 75 and 78). The resulting five fragments were cloned into the multiple cloning site of the pK19mobsacB vector by SmaI restriction and religation. The resulting plasmids (Table 1) were applied to introduce the six versions of trpD into the genome of the Corynebacterium glutamicum ΔtrpD strain. Furthermore, 10 foreign nucleotides (GCCCTGCAGG, SEQ ID NO: 92) were integrated into the genome as a result of the cloning procedure, upstream of the ribosomal binding site of the trpD gene, which were used for Crossover PCR. Correct mutants were isolated as described above and integrations were verified by sequencing the trpD region. The resulting Corynebacterium glutamicum ΔtrpD::trpD1-3, trpD5 or trpD6 strains (Table 2) grow without the addition of L-tryptophan or kanamycin and therefore have been characterized with respect to their properties as an oAB producer as described above without additional medium supplements (see Table 4).

[171] The described method for reducing gene expression can be applied to other genes instead of performing gene deletions.

Engineering of the csm gene in C. glutamicum strains

[172] Corynebacterium glutamicum Δcsm, Corynebacterium glutamicum ΔtrpDΔcsm, and Corynebacterium glutamicum ΔtrpD::trpD5Δcsm strains (Table 2) were created by in-frame deletion of the csm gene (which encodes chorismate mutase; Cgl0853) using the E-bridge vector pK19mobsacB. . coli-C. glutamicum. The 5' flanking region of the csm open reading frame (including the first 6 codons of the gene) and the 3' flanking region of the target gene (including the last 6 codons of the csm open reading frame) were amplified from genomic DNA isolated from Corynebacterium glutamicum ATCC 13032 by PCR, using primer pairs DeL-csm-1 and DeL-csm-2, and DeL- csm-3 and DeL-csm-4, respectively (Table 3, SEQ ID NO: 79-82). The two resulting fragments were combined by Crossover PCR technique, using the primer pair DeL-csm-1 and DeL-csm-4. The resulting fragment was cloned into the multiple cloning site of the pK19mobsacB vector by SmaI restriction and religation as described above. The resulting vector was applied for in-frame suppression of csm from the genome of Corynebacterium glutamicum ATCC 13032, C.glutamicum ΔtrpD::trpD5 and the strain Corynebacterium glutamicum ΔtrpD, respectively, and introduced in-frame a strange missense sequence of 24 bp in the genome, which was used for the Crossover PCR technique (resulting sequences at the csm locus: SE Q ID NO: 10). The correct mutants were isolated as described above and the correct gene deletion verified by PCR. This produced the auxotrophic strains for L-tyrosine and L-phenylalanine C. glutamicum Δcsm and C. glutamicum ΔtrpD::trpD5Δcsm, as well as the auxotrophic strain for L-tyrosine, L-phenylalanine and L-tryptophan C. glutamicum ΔtrpDΔcsm. The strains were characterized in relation to their properties as oAB-producing strains, as described above, with supplementation of the medium with 0.1 mM L-tyrosine and 0.1 mM L-phenylalanine for the C. glutamicum Δcsm strain and with supplementation of the medium with 0.1 mM L-tyrosine, 0.1 mM L-phenylalanine and 0.1 mM L-tryptophan for the C. glutamicum strain ΔtrpDΔcsm (Table 2). The generated strains had a decreased growth rate compared to the wild-type strain as well as the C. glutamicum ΔtrpD strain and, under standard culture conditions, they did not accumulate significant amounts of oAB (Table 4).

[173] Six versions of the csm gene were cloned into the pEKEx2 vector by restriction and religation of SbfI and BamHI as described above. The six different versions of the csm gene were generated with different initiation codons and spacer lengths between the ribosomal binding site and the csm initiation codon (SEQ ID NO: 11-16), by PCR using C. glutamicum genomic DNA as a template. The primers applied for the generation of the six fragments were forward primers (Ex-csm-1 to -6), which include the altered initiation codon and the spacer between the ribosomal binding site and the initiation codon, together with the unaltered reverse primer for each version (Ex-csm-rev, Table 3, SEQ ID NO: 89). This method was chosen in order to obtain a reduction in the translation of the Csm protein in the produced strains. The C. glutamicum ΔtrpD strain (Table 2) was transformed with the resulting plasmids (Table 1) and the resulting C. glutamicum ΔtrpD/pEKEx2-csm1-6 strains (Table 2) were grown without the addition of L-tyrosine, L-phenylalanine and L-tryptophan and were therefore characterized with regard to their properties as an oAB producer as described above , supplemented with 25 mg/l of kanamycin and 1 μM IPTG (isopropyl-β-d-thiogalactopyranoside) in the medium (see Table 4). The C. glutamicum ΔtrpD::trpD5Δcsm strain (Table 2) was transformed with the empty control vector pEKEx2 (Table 1) and the resulting C. glutamicum ΔtrpD::trpD5Δcsm/pEKEx2 strain (Table 2) was grown without the addition of L-tryptophan and was therefore characterized with respect to its properties as an oAB producer, as described above, supplemented with 25 mg/l of kanamycin and 0.1 mM IPTG in the medium (Table 4). The generated strains produced enough aromatic amino acids to allow the formation of biomass, but under standard culture conditions they do not accumulate significant amounts of oAB (Table 4).

Engineering of the common aromatic pathway and the L-tryptophan branch of C. glutamicum strains Engineering of the trpEG gene in C. glutamicum strains

[174] The aromatic biosynthetic pathway of C. glutamicum has been elucidated and the genes related to the coding enzymes for oAB biosynthesis are known (Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615). oAB is an intermediate in the L-tryptophan biosynthetic pathway and is derived from chorismate (CHO) by an anthranilate synthase, which consists of an amidotransferase (TrpE; donor: L-glutamine) and an anthranilate synthase unit (TrpEG), which releases a pyruvate molecule with the formation of oAB and L-glutamate. Increased expression of the genes encoding TrpEG has been shown to lead to an accumulation of L-tryptophan in Escherichia coli strains ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615 ). However, the enzyme has been reported to be highly inhibited by L-tryptophan feedback (Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615), resulting in the application of feedback-resistant versions of the TrpEG proteins. Feedback-resistant versions of the TrpEG proteins based on trpEG sequences from Brevibacterium lactofermentum (C. glutamicum), E. coli, and Salmonella typhimurium have been reported (Calguri et al., J Bio Chem, 1991, 8328-8335; Kwak et al., J Biochem Mol Bio, 1999, 20-24, Ikeda M, Appl Microbio l Biotechnol, 2006, 69:615 and Matsui et al., J Bac, 1987, 5330-5332). The most favorable trpEG versions based on E. coli genes were expressed in C. glutamicum strains, as well as selected amino acid changes were implemented in the trpEG sequence of the natural C. glutamicum TrpEG protein ATCC13032 and expressed in C. glutamicum strains. Taken together, four TrpEG proteins resistant to E. coli TrpEG-based feedback: TrpEGS40R and TrpEGS40F (SEQ ID NO: 50-51); and based on TrpEG from C. glutamicum: TrpEGS38R and TrpEGS38F (SEQ ID NO: 52-53) were characterized in comparison with the natural TrpEG protein from C. glutamicum. The five versions of the trpEG genes were synthesized by Eurofins MWG Operon GmbH and cloned into the pEKEx2 vector by restriction and religation of SbfI and BamHI as described above, and correct cloning was verified by sequencing. C. glutamicum strains ATCC13032 and C. glutamicum ΔtrpD::trpD5 (Table 2) were transformed with the resulting plasmids (Table 3) (C. glutamicum ΔtrpD::trpD5 with plasmid pEKEx2-trpEGS40F only) and the resulting strains C. glutamicum/pEKEx2-trpEG, C. glutamicum /pEKEx-trpEGS38F, C. glutamicum/pEKEx2-trpEGS38R, C. glutamicum/pEKEx2-trpEGS40F, C. glutamicum /pEKEx2-trpEGS40R, C. glutamicum ΔtrpD::trpD5/pEKEx2-trpEGS40F (Table 2) were characterized with respect to their properties as a producer of oAB as described above, supplemented with 25 mg/l kanamycin and 0.1 mM IPTG in medium (see Table 4). In addition, cell lysates from the five C. glutamicum ΔtrpD-based strains carrying the pEKEx2-trpEG variants were analyzed for anthranilate synthase activity, according to Caliguri and Bauerle (1991). Cell lysates were produced from cell pellets generated from 30 ml cultures of C. glutamicum ΔtrpD-based strains carrying pEKEx2-trpEG variants in 300 ml shake flasks, with 30 ml of BHI medium supplemented with 25 mg/l of kanamycin and 1 mM IPTG, and grown at 30 °C with shaking at 150 rpm (OD600 final 4). Cell pellets were lysed using 0.1 mm zirconium beads (Zymo Research Corporation, Irvine, CA) and resuspended in assay buffer (50 mM KH2PO4/K2HPO4, 20 mM L-glutamine, 10 mM MgCl2 and 25 µM chorismate), according to Caliguri and Bauerle (1991) at 25 °C. The emission was measured at 390 nm using a spectrofluorometer (excitation: 390 nm). All tested variants showed a higher specific activity for anthranilate synthase, compared to the cell lysate with the natural TrpEG protein from C. glutamicum. Three biological replicates were used for measurements. Both E. coli TrpEG protein variants had a higher activity and lower KM compared to C. glutamicum TrpEG protein variants (Determination of KM and Vmax values was performed with Graphpad Prism (La Jolla, CA)). The cell lysate of the C. glutamicum strain Δ trpD / pEKEx2-trpEGS40F showed the best performance (see Table 5). Table 5 - Biochemical characteristics of crude extracts of C. glutamicum ΔtrpD producing different TrpEG variants in relation to TrpEG activity.

Engineering of the aroG gene in C. glutamicum strains

[175] It has been reported that in both C. glutamicum and E. coli, carbon flux through the common aromatic pathway to chorismate is mainly controlled in the first reaction of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (Ikeda M, Appl Microbiol Biotechnol 2006, 69:615). In C. glutamicum there are two types of DS with different subunit sizes. One is an L-tyrosine sensitive DS with a predicted molecular mass of 39 kDa (DS type I; the aro product; Cgl0950) and the other is an L-phenylalanine and L-tyrosine sensitive DS with a predicted molecular mass of 51 kDa (DS type II; the aroII product; Cgl2098). Type II DS forms a polypeptide complex with chorismate mutase, which converts chorismate to prephenate. Type II DS exhibits its activity by itself, whereas CM activity requires the presence of type II DS protein ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615 ). A feedback resistant AroI DS (AroIS187C) has been described ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69: 615 ). The feedback-resistant DS equivalent of AroII, AroGfbr (AroGD146N; SEQ ID NO: 55) from E. coli, which is not inhibited by aromatic amino acids, was expressed in C. glutamicum strains to increase carbon flux through the aromatic pathway common to CHO and oAB. The aroGD146N gene was amplified by PCR using Ex-aroG-1 and Ex-aroG-2 primers (Table 3, SEQ ID NO: 109-110), which included the restriction sites and ribosomal binding site, as described above. The resulting DNA fragment was cloned into the pEKEx2 vector by restriction and religation of SbfI and BamHI as described above, and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5 strain (Table 2) was transformed with the resulting plasmid (Table 1) and the resulting C. glutamicum ΔtrpD::trpD5/pEKEx2-aroGD146N strain (Table 2) was characterized with respect to its properties as an oAB producer, as described above, with 25 mg/l kanamycin supplementation and IPTG 0 .1 mM in the medium (Table 4). Engineering of the trpEG and aroG genes in C. glutamicum strains

[176] To allow investigation of the influence of the combined expression of AroGD146N and TrpEGS40F in C. glutamicum strains, the DNA sequences encoding these proteins were fused into the pEKEx2 vector. The aroGD146N sequence was fused with pEKEx2-trpEGS40F to generate pEKEx2-trpEGS40F-aroGD146N (Table 1) and the trpEGS40F gene was fused with plasmid pEKEx2-aroGD146N to generate pEKEx2-aroGD146N-trpEGS40F (Table 1), by restriction and religation of BglII and BamHI as described above, and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5 strain (Table 2) was transformed with each of the resulting plasmids and the C. glutamicum ΔtrpD:: trpD5/pEKEx2-aroGD146N-trpEGS40F and C. glutamicum ΔtrpD::trpD5/pEKEx2-trpEGS40F-aroGD14 strains The results (Table 2) were characterized for their oAB-producing properties, as described above, with 25 mg/l kanamycin supplementation and 0.1 mM IPTG in the medium (see Table 4). Furthermore, the C. glutamicum ΔtrpD::trpD5Δcsm strain (Table 2) was transformed with the plasmid pEKEx2-aroGD146N-trpEGS40F and the resulting C. glutamicum ΔtrpD::trpD5Δcsm /pEKEx2-aroGD146N-trpEGS40F strain (Table 2) was characterized in relation to its properties as an oAB producer, as described above, with supplementation of kanamycin 25 mg/l, L-tyrosine 0.1 mM, L-phenylalanine 0.1 mM and IPTG 0.1 mM in medium (see Table 4). Engineering of the aroK and aroL gene in C. glutamicum strains

[177] To prevent an accumulation of intermediates in the common aromatic amino acid biosynthetic pathway of C. glutamicum strains, the aroK gene (encoding shikimate kinase; Cgl1622; SEQ ID NO: 94) from C. glutamicum and the aroL gene (encoding shikimate kinase; CP000948; SEQ ID NO: 93) from E. coli were investigated. The aroK and aroL genes were amplified separately by PCR, using primers Ex-aroK-1 and Ex-aroK-2 (Table 3, SEQ ID NO: 101-102) and Ex-aroL-1 and Ex-aroL-2 (Table 3, SEQ ID NO: 99-100), respectively. The primers included the restriction sites and the ribosomal binding site as described above. The resulting DNA fragments were separately integrated into the pEKEx2 vector via SbfI and BamHI restriction and religation, as described above, to generate plasmids pEKEx2-aroK and pEKEx2-aroL, and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5 strain (Table 2) was transformed with the resulting plasmids (Table 3) and the resulting C. glutamicum ΔtrpD::trpD5/pEKEx2-aroL and C. glutamicum ΔtrpD::trpD5/pEKEx2-aroK strains (Table 2) were characterized in relation to their properties as a producer s of oAB as described above with 25 mg/l kanamycin supplementation and 0.1 mM IPTG in medium (Table 4). In the C. glutamicum ΔtrpD::trpD5/pEKEx2-aroL strain, no significant accumulation of shikimate and 3-dehydroshikimate was detected by analysis of culture supernatants by HPLC-DAD.

Engineering of the glnA gene in C. glutamicum strains

[178] To analyze the impact of L-glutamine synthetase (GlnA; Cgl2214, SEQ ID NO: 95) activity on oAB production by C. glutamicum strains, the L-glutamine synthetase gene, glnA, was expressed in C. glutamicum strains. The glnA gene was amplified by PCR using primers Ex-glnA-1 and Ex-glnA-2 (Table 3, SEQ ID NO: 9798) which included the restriction sites and ribosomal binding site as described above. The resulting DNA fragment was cloned into the pCRB210 vector via NcoI restriction and religation as described above, and correct cloning was verified by sequencing. CEPA C. glutamicum ΔTRPD :: TRPD5 was transformed with the resulting plasmid (Table 3) and the resulting strain C. glutamicum ΔTRPD :: TRPD5/PCRB-GLNA TTABELA 2) was characterized in relation to its properties as an OAB producer, as described above, with Canamicin supplementation at 25 mg/L and IPTG 0.1 mm in half (Table 4). As a control, the C. glutamicum strain ΔtrpD::trpD was transformed with the empty control vector pCRB210 (Table 1) and the resulting C. glutamicum strain ΔtrpD::trpD5/pCRB210 (Table 2) was characterized in relation to its properties as an oAB producer, as described above, with kanamycin supplementation at 25 mg/l and IPTG 0.1 mM in the medium ( Table 4). Although strain growth was reduced, no beneficial effect on C. glutamicum ΔtrpD::trpD5 anthranilate accumulation could be observed. It cannot be excluded that increased GlnA activity will increase anthranilate accumulation in a more advanced oAB-producing strain, as has been observed in E. coli (Sun et al., Appl Environ Microbiol, 2013, 4024-4030).

Engineering of the central metabolism of C. glutamicum strains Engineering of the pyk gene in C. glutamicum strains

[179] Pyruvate kinase (Pyk) consumes phosphoenolpyruvate for the production of pyruvate and ATP. The effect of an in-frame deletion of the pyk gene (SEQ ID NO: 23) in C. glutamicum strains was investigated. The C. glutamicum ΔtrpD::trpD5Δpyk strain (Table 2) was created by in-frame deletion of the pyk gene (which encodes pyruvate kinase; Cgl2089) using the E. coliC bridge-like vector. glutamicum pK19mobsacB as described above. The resulting plasmid pSB082 (Table 1) was applied for in-frame deletion of the pyk gene from the genome of C. glutamicum ΔtrpD::trpD5 and introduced in-frame into the genome a 24 bp foreign missense sequence (resulting sequence at the pyk locus: SEQ ID NO: 24). The correct mutants were isolated as described above and the correct gene deletion verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5Δpyk strain, which was characterized with respect to its properties as an oAB-producing strain, as described above (Table 4).

gpi gene engineering in C. glutamicum strains

[180] In order to direct carbon flux into the pentose phosphate pathway, the effect of an in-frame deletion of the gene encoding glucose-6-phosphate isomerase (Gpi) (SEQ ID NO: 27) in C. glutamicum strains was investigated. The C. glutamicum strain ΔtrpD::trpD5Δpyk (Table 2) was created by in-frame deletion of the gpi gene (which encodes glucose-6-phosphate isomerase; Cgl0851) using the E. coli-C. glutamicum pK19mobsacB as described above. The resulting plasmid pSB064 (Table 1) was applied for in-frame deletion of the gpi gene from the genome of C. glutamicum ΔtrpD::trpD5 and introduced in-frame into the genome a 24 bp foreign missense sequence (resulting sequence at the gpi locus: SEQ ID NO: 28). The correct mutants were isolated as described above and the correct gene deletion verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5Δgpi strain, which was characterized with respect to its properties as an oAB-producing strain, as described above (Table 4).

Engineering of the pepco gene in C. glutamicum strains

[181] Phosphoenolpyruvate carboxylase (Pepco) consumes PEP, turning it into oxaloacetate. In order to access an extended PEP pool, an in-frame deletion of the gene encoding phosphoenolpyruvate carboxylase (SEQ ID NO: 21) in C. glutamicum strains was investigated. The C. glutamicum ΔtrpD::trpD5Δpepco strain (Table 2) was created by in-frame deletion of the pepco gene (which encodes phosphoenolpyruvate dcarboxylase; Cgl1585) using the E. coli-C. glutamicum pK19mobsacB as described above. The resulting plasmid pSB061 (Table 1) was applied for in-frame deletion of the pepco gene from the genome of C. glutamicum ΔtrpD::trpD5 and introduced in-frame into the genome a 24 bp foreign missense sequence (resulting sequence at the pepco locus: SEQ ID NO: 22). The correct mutants were isolated as described above (with the additional supplementation of 1% acetate to the agar plates with BHI medium) and the correct gene deletion verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5Δpepco strain, which was characterized for its properties as an oAB-producing strain as described above (Table 4).

Engineering of ptsG and hpr genes in C. glutamicum strains

[182] Glucose and fructose uptake by C. glutamicum is primarily utilized by the phosphotransferase (PTS) system. This multienzyme complex translocates glucose via a phosphoenolpyruvate-mediated phosphorylation cascade with concomitant phosphorylation of glucose to glucose-6-phosphate across the cytoplasmic membrane (Siebold et al., 2001). The system consists of two soluble components, enzyme I and histidine-rich protein (Hpr) and a membrane-bound/associated enzyme complex (Tanaka et al., 2008). C. glutamicum ΔtrpD::trpD5Δhpr and C. glutamicum ΔtrpD::trpD5ΔptsG strains (Table 2) were created by inframe deletion of the hpr gene (which encodes the histidine-rich protein; Cgl1937, SEQ ID NO: 17) and the ptsG gene (which encodes the G unit of the phosphotransferase system; Cgl1360, SEQ ID NO: 19), respectively, using the E. coliC bridge-type vector. glutamicum pK19mobsacB as described above. The resulting plasmids pSB060 and pSB079 (Table 1) were applied for in-frame deletion of the hpr gene and the ptsG gene, respectively, from the genome of C. glutamicum ΔtrpD::trpD5, and introduced in-frame into the genome of a 24 bp foreign missense sequence (resulting sequence at the hpr/ptsG locus: SEQ ID NO: 18; pt locus sG: 20). The correct mutants were isolated as described above (with the additional supplementation of 5 g/l of ribose to the agar plates with BHI medium) and the correct gene deletion verified by PCR. This produced the C. glutamicum strain ΔtrpD::trpD5Δhpr and the C. glutamicum strain ΔtrpD::trpD5ΔptsG, which were characterized with respect to their properties as oAB-producing strains, as described above (Table 4). Both strains did not show significant growth under standard fermentation conditions, as described above, and as a result did not accumulate oAB.

Engineering of the ppk gene in C. glutamicum strains

[183] Phosphoenolpyruvate carboxykinase (Ppk) can, as part of the glyoxylate pathway, recycle oxaloacetate to PEP. In order to access an extended PEP pool, a plasmid-based expression of the gene encoding phosphoenolpyruvate carboxykinase (Cgl2862; SEQ ID NO: 36) in C. glutamicum strains was investigated. The ppk gene was synthesized by Eurofins MWG Operon GmbH and cloned into the pEKEx2 vector via restriction and religation of SbfI and BamHI, as described above, resulting in plasmid pSB072 (Table 1) and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5/pSB072 strain (Table 2) was created by transforming the C. glutamicum ΔtrpD::trpD5 strain with plasmid pSB072 as described above. Correct mutants were isolated as described above and successful transformation with the target gene was verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5/pSB072 strain, which was characterized with respect to its properties as an oAB-producing strain, as described above (Table 4).

Pps gene engineering in C. glutamicum strains

[184] The catalysis of phosphoenolpyruvate synthase (Pps), as part of gluconeogenesis, the formation of PEP by recycling of pyruvate and consumption of ATP in AMP. In order to access an extended PEP pool, a plasmid-based expression of the gene encoding phosphoenolpyruvate carboxykinase synthase together with the gene encoding the PEP synthase binding site (Cgl0551 and Cgl0552; SEQ ID NO: 33-35) in C. glutamicum strains was investigated. The pps genes were jointly synthesized by Eurofins MWG Operon GmbH and cloned into the pEKEx2 vector via restriction and religation of SbfI and BamHI, as described above, resulting in plasmid pSB073 and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5/pSB073 strain (Table 2) was created by transforming the C. glutamicum ΔtrpD::trpD5 strain with plasmid pSB073 as described above. Correct mutants were isolated as described above and successful transformation with the target gene was verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5/pSB073 strain, which was characterized for its properties as an oAB-producing strain, as described above (Table 4).

Engineering zwf1 together with the opcA gene in C. glutamicum strains

[185] In Zymomonas mobilis and E. coli, increased production of the enzyme that catalyzes the first reaction of the pentose phosphate (PPP) pathway (formation of ribulose-5-phosphate from glucose-6-phosphate by glucose-6-phosphate dehydrogenase (Zwf1 and OpcA)) has been reported to result in increased carbon flux to PPP and, as a result, an increase in erythrose-4-phosphate (E4P) pool. In order to access an extended E4P pool, a plasmid-based expression of the gene encoding glucose-6-phosphate dehydrogenase (zwf1 (Cgl1576) and opcA (Cgl1577; SEQ ID NO: 37-39)) in C. glutamicum strains was investigated. The genes were synthesized together by Eurofins MWG Operon GmbH and cloned into the pEKEx2 vector via restriction and religation of SbfI and BamHI as described above, resulting in plasmid pSB078 (Table 1) and correct cloning was verified by sequencing. described above and successful transformation with the target gene proved by PCR.This produced the C. glutamicum strain ΔtrpD::trpD5/pSB078, which was characterized with respect to its properties as an oAB-producing strain, as described above (Table 4).

Tal and tkt gene engineering in C. glutamicum strains

[186] Enhanced carbon flux through PPP and an enhanced E4P pool have been described by increased expression of transketolase and transaldolase in E. coli. The genes encoding transketolase (Tkt) and transaldose (Tal) from E. coli (tktEC (ECDH10B_3110), and talEC (ECDH10B_2629); SEQ ID NO: 41 and 43), as well as the equivalent genes from C. glutamicum (tktCG (Cgl1574) and talCG (Cgl1575); SEQ ID NO: 40 and 42) were expressed on a plasmid basis in C. glutamicum strains. The four sequences were synthesized by Eurofins MWG Operon GmbH and separately integrated into the pEKEx2 bridge vector via restriction and religation of SbfI and BamHI, as described above, resulting in plasmids pSB074-pSB077 (Table 1), and correct cloning was verified by sequencing. The C. glutamicum strain ΔtrpD::trpD5/pSB074 - pSB077 (Table 2), was created by transforming the C. glutamicum strain ΔtrpD::trpD5 with each of the plasmids, as described above. Correct mutants were isolated as described above and successful transformation with the target gene was verified by PCR. This produced C. glutamicum ΔtrpD::trpD5/pSB074 - pSB077 strains, which were characterized with respect to their properties as oAB-producing strains, as described above (Table 4).

[187] To allow investigation of the effect of combined transketolase and transaldoase expression in C. glutamicum strains, the DNA sequences encoding these proteins were fused into the pEKEx2 vector. The talCG sequence was fused to pSB075 to generate pSB085 (Table 1) and the talEC gene was fused to plasmid pSB077 to generate pSB086 (Table 1), both via restriction and religation of SbfI and BamHI, as described above, and correct cloning was verified by sequencing. The C. glutamicum ΔtrpD::trpD5 strain was transformed with each of the resulting plasmids and the resulting C. glutamicum ΔtrpD::trpD5/pSB085 and C. glutamicum ΔtrpD::trpD5/pSB086 strains (Table 2) were characterized with respect to their properties as oAB-producing strains, as described above, with kanamycin supplementation at 2 5 mg/l and 0.1 mM IPTG in medium (Table 4).

Engineering of galP, iolT2 and ppgk genes in C. glutamicum strains

[188] In order to restore efficient glucose uptake in Pts-deficient strains of C. glutamicum, expression of the gene encoding galactopermease (galP, Cgl2409; SEQ ID NO: 30) in combination with a gene encoding a polyphosphoglycokinase (a glucose phosphorylating enzyme) (ppgk (Cg1910; SEQ ID NO: 32) in strains of C. glutamicum. In addition, the gene encoding inositolpermease T2 (iolT2, Cgl3058; SEQ ID NO: 31) and the T2 unit (inositolpermease) were investigated in combination with a gene encoding polyphosphoglycokinase (ppgk (Cg1910; SEQ ID NO: 32) in C. glutamicum strains. The four genes were synthesized by Eurofins MWG Operon GmbH and each integrated into the pEKEx2 vector via restriction and religation of SbfI and BamHI as described above, resulting in plasmids pSB068, pSB070 and pSB071 (Table 1) and correct integration was verified by sequencing.The ppgk sequence was fused with the galP and iolT2 sequences, respectively, in the pEKEx2 vector. The galP sequence was fused to pSB071 to generate pSB083 (Table 1) and the iolT2 gene was fused to plasmid pSB071 to generate pSB084 (Table 1), both via restriction and religation of SbfI and BamHI as described above, and correct cloning was verified by sequencing. C. glutamicum ΔtrpD::trpD5ΔptsG and C. glutamicum ΔtrpD::trpD5Δhpr strains (Table 2) were each transformed with plasmids pSB083 and pSB084, respectively, and the resulting C. glutamicum ΔtrpD::trpD5ΔptsG/pSB083, C. glutamicum strains ΔtrpD::trpD5ΔptsG/pSB084, C. glutamicum ΔtrpD::trpD5Δhpr/pSB083, and C. glutamicum ΔtrpD::trpD5Δhpr/pSB084 (Table 2) were characterized with respect to their properties as oAB-producing strains, as described above, with kanamycin supplementation at 25 mg/l and IPTG 0.1 mM in medium (Table 4). The ΔtrpD::trpD5Δhpr/pSB083 strain did not show significant growth under the standard fermentation conditions as described above and, as a result, did not accumulate oAB.

Engineering oAB export from C. glutamicum strains

[189] As the production rate of oAB increases, the export of the product from producing cells can easily become a limiting step in production. Furthermore, the intracellular accumulation of oAB may very likely have a significant toxic effect on cells. So far, no oAB exporter has been described. Recently, a shikimate permease (QsuA), belonging to the family of major facilitator systems, was described by Kubota et al., which facilitates the import of shikimate and quinate (Kubota et al., Molecular Microbiol, 2014, 92(2), 356) in C. glutamicum. To test the effect of shikimate permease on intra- and extracellular concentrations of oAB in C. glutamicum strains, the related gene (qsuA, Cgl0492; SEQ ID NO: 96) was synthesized by Eurofins MWG Operon GmbH and integrated into the pEKEx2 vector via restriction and religation of SbfI and BamHI, as described above, resulting in plasmid pSB096 (Table 1), and correct cloning was performed. verified by sequencing. The C. glutamicum ΔtrpD::trpD5/pSB096 strain (Table 2) was created by transforming the C. glutamicum ΔtrpD::trpD5 strain with plasmid pSB096 as described above. Correct mutants were isolated as described above and successful transformation with the target gene was verified by PCR. This produced the C. glutamicum ΔtrpD::trpD5/pSB096 strain, which was characterized with respect to its properties as an oAB-producing strain, as described above (Table 4).

Example 4 - Production of o-aminobenzoate with P. putida

[190] Pseudomonas putida KT2440-derived strains have been developed that produce oAB. To achieve an accumulation of oAB by P. putida strains, the trpDC genes (PP_0421 and PP_0422; SEQ ID NO: 63), which encode anthranilate phosphoribosyl transferase and indole-3-glycerol phosphate synthase, were chosen to be disrupted in Pseudomonas putida KT2440. The construction of the deletion mutant P. putida ΔtrpDC, based on the knockout plasmid pEMG-del-trpDC (Table 1), was approached by the method described by Martinez-Garcia et al. ( Martinez-Garcia et al., Environ Microbiol 2011, 13(10), 2702 ) using the pEMG vector. This approach results in the removal of the genetic sequences (gene of interest, GOI) without scarring or residual cloning markers. The concept of the method is shown in Figure 27. In step A - B, the engineered pEMG knockout vector, containing the TS1 and TS2 breakout flanks, a kanamycin marker and the I-SceI sites, is integrated into the genome of P. putida. In step C double strand breakage is induced by transformation of a second plasmid (PSW-2) containing the I-SceI meganuclease genes. The break is repaired in vivo by homologous recombination of the TS1 or TS2 region, resulting in both wild-type P. putida and P. putida ΔtrpDC (step D). Step E shows the PCR sorting, which is necessary to distinguish and isolate the different strains. The deletion flanks were chosen in such a way that the open reading frame of the genes is removed, but keeping the neighboring genes intact (Figure 28). This strategy can lead to greater strain stability under long-term continuous culture conditions. The 800 bp TS1 and TS2 breakout flanks were amplified using a Phusion®-High Fidelity DNA polymerase (New England Biolabs). A Phusion PCR was performed according to the manufacturer's manual (25 cycles, 61.8 °C (TS1), 70.2 °C (TS2) 1:30 minutes). The TS1 PCR fragment was digested with BamHI and XhoI and the TS2 PCR fragment was digested with XhoI and SbfI. Plasmid pEMG and the PCR-fused TS1-TS2 product were digested with BamHI and SbfI. All restriction mixes were purified using a High Pure PCR Product Purification Kit (Roche). Ligation of pEMG-Del-trpDC, using the TS1 and TS2 individual flanks, was performed with T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer's instructions. The ligation mixture was chemically transformed into the competent strain E. coli DH5α Àpir. Positive plasmids from the three-point ligation mix, consisting of the two individual flanks and the digested pEMG vector were identified by restriction analysis and verified by sequencing, which confirmed the successful construction of pEMG-Del-trpDC (Table 1). The knockout vector pEMG-Del-trpDC was integrated into the genome of P. putida (Figure 27, step A-B) by means of tri-parental crossing, using the acceptor strain P. putida, the donor strain E. coli DH5α Àpir/pEMG-Del-trpDC and an E. coli helper strain HB101 pRK2013 (Martinez-Garcia et al, Environ Microbiol, 201 1, 13(10), 2702). P. putida /pEMG-Del-trpDC was isolated from the cell mixture using 50 mg/l kanamycin cetrimide agar plates and individual colonies were reseeded onto LB-kanamycin plates. Genomic integration of the knockout vector was confirmed in individual colonies by colony PCR. To introduce the double-strand break into the genome (Figure 27, Step C), a second plasmid (PSW-2) containing the I-Scel meganuclease gene was transformed into the newly constructed strain. Therefore, electrocompetent P. putida /pEMG-Del-trpDC cells were obtained according to Choi et al. (Choi et al, J Microbiol Methods, 2005, 64(3), 391 ). Electroporation was performed using a Biorad Gene Pulser Xcell electroporator (2.5 kV, 200 ohms, 25 μF) and the cell suspension was seeded on LB-gentamicin plates (30 mgl) and LB-kanamycin-gentamicin plates (30 mg/l and 50 mg/l, respectively). Following the knockout process protocol described by Martinez-Garcia et al., an I-Scel meganuclease induction is required. However, this step was omitted due to practical experience suggesting faulty expression of I-SceI meganuclease. To distinguish the desired P. putida ΔtrpDC knockout strain from the P. putida and P. putida/pEMG-Del-trpDC strains, individual colonies were streaked onto LB and LB-kanamycin plates. Kanamycin sensitive colonies were checked by colony PCR as described above. In addition, kanamycin-sensitive colonies were checked for auxotrophy to L-phenylalanine on minimal medium plates with 20 mM glucose, with and without 0.1 mM L-tryptophan. Gene deletions were verified by PCR analysis and sequencing. The resulting P. putida ΔtrpDC strain (Table 2) was unable to grow on minimal medium without L-tryptophan supplementation.

[191] In addition, the pheA gene (PP_1769, SEQ ID NO: 64), which encodes chorismate mutase, was disrupted in the Pseudomonas putida strain ΔtrpDC. The pheA gene encodes the first step in the biosynthesis of L-phenylalanine and L-tyrosine, which probably competes with anthranilate production. The construction of the P. putida ΔtrpDCΔpheA deletion mutant, based on the pEMG-Del-pheA knockout plasmid, was approached by the method described by Martinez-Garcia et al. (Martinez-Garcia et al., Environ Microbiol 2011, 13(10), 2702 ) using the pEMG vector as described above. Suppression flanks were chosen such that the gene's open reading frame is removed, while neighboring genes are kept intact (Figure 29). This strategy can lead to greater strain stability under long-term continuous culture conditions. In the case of pheA this means that the 5' eight nucleotides of the gene remained, due to an overlap with the serC gene. To construct the 800 bp TS1 and TS2 breakout flanks, the following primers were used: JK038f, JK039r, JK040f and JK041r (Table 3, SEQ ID NO: 103-106). The TS1 PCR fragment was digested with BamHI and XhoI and the TS2 PCR fragment was digested with XhoI and SbfI. The pEMG plasmid and the TS1-TS2 PCR fused product were digested with BamHI and SbfI. All restriction mixes were purified using a High Pure PCR Product Purification Kit (Roche). Ligation of pEMG-Del-pheA, using the individual TS1 and TS2 flanks, was performed with a T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer's instructions. The ligation mixture was chemically transformed into the competent strain E. coli DH5α Àpir. Positive plasmids from the three-point ligation mixture, consisting of the two individual flanks and the digested pEMG vector were identified by restriction analysis and verified by sequencing, which confirmed the successful construction of pEMG-Del-pheA (Table 1). The pEMG-Del-pheA knockout vector was integrated into the genome of P. putida ΔtrpDC (Figure 27, step A-B) by means of tri-parental crossing, using the acceptor strain P. putida ΔtrpDC, the donor strain E. coli DH5α Àpir/pEMG-Del-pheA, and a helper strain E. coli HB101 pRK2013 (Martinez-Garcia et al, Environ Microbiol, 2011, 13(10), 2702). P. putida ΔtrpDC/pEMG-Del-pheA was isolated from the cell mixture using 50 mg/l kanamycin cetrimide agar plates and individual colonies were reseeded onto LB-kanamycin plates. Genomic integration of the knockout vector was confirmed in individual colonies by colony PCR. To introduce the double-strand break into the genome (Figure 27, Step C), a second plasmid (PSW-2) containing the I-Scel meganuclease gene was transformed into the newly constructed strain. Therefore, electrocompetent P. putida ΔtrpDC /pEMG-Del-pheA cells were obtained according to Choi ( Choi et al, J Microbiol Methods, 2005, 64(3), 391 ). Electroporation was performed using a Biorad Gene Pulser Xcell electroporator (2.5 kV, 200 ohms, 25 μF) and the cell suspension was seeded on LB-gentamicin plates (30 mgl) and LB-kanamycin-gentamicin plates (30 mg/l and 50 mg/l, respectively). Following the protocol of the knockout process described by Martinez-Garcia et al., an induction of I-Scel meganuclease is required. However, this step was omitted due to practical experience suggesting a faulty expression of meganuclease I-SceI. To distinguish the desired P. putida ΔtrpDCΔpheA knockout strain from the P. putida ΔtrpDC and P. putida ΔtrpDC/pEMG-Del-pheA strains, individual colonies were streaked onto LB and LB-kanamycin plates. Kanamycin sensitive colonies were checked by colony PCR as described above. In addition, kanamycin-sensitive colonies were checked for L-phenylalanine auxotrophy on minimal medium plates with 20 mM glucose, 0.1 mM L-tryptophan, with and without 1 mM L-phenylalanine. Gene deletions were verified by PCR analysis and sequencing. Since P. putida KT2440 has an L-phenylalanine-4-hydroxylase (PheAA) which converts L-phenylalanine to L-tyrosine, auxotrophs for L-tyrosine were rescued by addition of L-phenylalanine. Since both L-tryptophan and L-phenylalanine auxotrophs were obtained by single gene disruptions, it was assumed that there are no redundant genes encoding alternative enzymes in P. putida KT2440.

[192] The first dedicated reaction of the aromatic amino acid biosynthetic pathway is catalyzed by the DAHP synthase isoenzymes. It has been shown in several organisms that overexpression of a feedback-insensitive mutant of this enzyme leads to increased flux through the biosynthetic pathway of aromatic compounds ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615 ). Therefore, in order to increase anthranilate production, a feedback-insensitive 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase encoded by the aroGD146N gene (SEQ ID NO: 55) was expressed in P. putida KT2440, P. putida ΔtrpDC and P. putida ΔtrpDCΔpheA strains via the pSEVA234 vector. (with control of the lacIq-Ptrc system, which encodes a kanamycin resistance and a pBBR1 origin of replication) (Silva-Rocha et al, Nucleic Acids Research, 2013, D666-75). The aroGD146N gene was restricted to the donor plasmid (pCAS-2JF-aroGD146N) using restriction enzymes EcoRI and BamHI and standard conditions as described above (Example 3), resulting in a fragment of 1117 bp. The pSEVA234 vector was restricted with the same enzymes. The fragments were purified using a High Pure PCR Product Purification Kit (Roche). Ligation of the fragment to generate pSEVA234-aroGD146N (Table 1) was performed with a T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer's instructions. The ligation mix was introduced into electro-competent Escherichia coli DH5α cells (ElectroMAX™ DH5α-E™ Competent Cells; Life Technologies, Darmstadt). After electrotransformation (conditions: ~20 ms pulse with exponential decay, 2.5 kV/cm, 25 M, 200 Q) in 0.2 cm gap electroporation crucibles (BioRad, Hercules, CA) using a Gene Pulser Xcell system (BioRad, Hercules, CA), 800 μl of LB regeneration medium (Luria-Bertani; Roth, Karls, CA) were immediately added. ruhe) and the suspension was transferred to a 1.5 ml microcentrifuge tube. After 1 h at 37 °C, a cell suspension was spread onto LB-agar plates (Abcr GmbH & Co. Kg, Karlsruhe), supplemented with appropriate antibiotics and incubated at 37 °C overnight. Clones were collected and correct transformation was confirmed by restriction analysis and sequencing. For plasmid preparation, strains were cultivated in 15 ml tubes for 14-16 h, with constant stirring at 200 rpm, in 3 ml of LB medium and 50 mg/l of kanamycin at 37 °C (Kuhner Shaker ISF-4-W; Adolf Kühner AG, Basel (Switzerland)). Plasmid DNA purification was performed using the NucleoSpin® Plasmid Pure kit (Macherey & Nagel, Düren), according to the manufacturer's instructions. Electrocompetent P. putida KT2440, P. putida ΔtrpDC and P. putida ΔtrpDCΔpheA cells were obtained according to Choi et al ( Choi et al, J Microbiol Methods, 2005, 64(3):391 ). Electroporation was performed using a Biorad Gene Pulser Xcell electroporator (2.5 kV, 200 ohms, 25 μF) and the cell suspension was seeded on LB-agar plates supplemented with 50 mg/l of kanamycin. This resulted in the generation of P. putida KT2440/pSEVA234-aroGD46N, P. putida ΔtrpDC/pSEVA234-aroGD46N and P. putida ΔtrpDCΔpheA/pSEVA234-aroGD46N strains (Table 2). In addition, the empty pSEVA234 vector was transformed into P. putida ΔtrpDC and P. putida ΔtrpDCΔpheA strains to generate P. putida ΔtrpDC/pSEVA234 and P. putida ΔtrpDCΔpheA/pSEVA234 strains (Table 2).

[193] oAB is an intermediate in the L-tryptophan biosynthetic pathway and is derived from chorismate by an anthranilate synthase, which consists of an amidotransferase (TrpE; donor: L-glutamine) and an anthranilate synthase (TrpEG) unit, which releases a pyruvate molecule with the formation of oAB and L-glutamate. Increased expression of the genes encoding TrpEG has been shown to lead to an accumulation of L-tryptophan in Escherichia coli strains ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69:615 ). However, the enzyme has been reported to be highly feedback-inhibited by L-tryptophan, resulting in the delivery of feedback-resistant versions of the TrpEG proteins. Feedback resistant versions of the TrpEG protein have been reported based on trpEG sequences from Salmonella typhimurium ( Ikeda M, Appl Microbiol Biotechnol, 2006, 69: 615 ). The most favorable version of trpEG, based on the S. typhimurium genes, trpEGS40F (SEQ ID NO: 53), was expressed in P. putida strains. The feedback inhibition-resistant version of the trpEG gene was cloned into the P. putida vector pSEVA234 to allow plasmid-based expression in P. putida KT2440, P. putida ΔtrpDC, and P. putida ΔtrpDCΔpheA strains (with control of the lacIq-Ptrc system, which encodes a kanamycin resistance and a pBBR1 origin of replication) (Silva-Rocha et al , Nucleic Acids Research, 2013, D666-75). The trpEGS40F gene was restricted from a donor plasmid using EcoRI and BamHI restriction enzymes and standard conditions as described above (Example 3), resulting in a fragment of 2200 bp. The pSEVA234 vector was restricted with BamHI, resulting in a 4550 bp fragment. The fragments were purified using a High Pure PCR Product Purification Kit (Roche). Ligation of fragments to generate pSEVA234-trpEGS40F (Table 1) was performed with a T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer's instructions. The ligation mix was introduced into electro-competent Escherichia coli DH5α cells (ElectroMAX™ DH5α-E™ Competent Cells; Life Technologies, Darmstadt). After electrotransformation (conditions: ~20 ms pulse with exponential decay, 2.5 kV/cm, 25 M, 200 Q) in 0.2 cm gap electroporation crucibles (BioRad, Hercules, CA) using a Gene Pulser Xcell system (BioRad, Hercules, CA), 800 μl of LB regeneration medium (Luria-Bertani; Roth, Karl sruhe) and the suspension was transferred to a 1.5 ml microcentrifuge tube. After 1 h at 37 °C, a cell suspension was spread onto LB-agar plates (Abcr GmbH & Co. Kg, Karlsruhe), supplemented with appropriate antibiotics and incubated at 37 °C overnight. Clones were collected and correct transformation was confirmed by restriction analysis and sequencing. For plasmid preparation, strains were cultivated in 15 ml tubes for 14-16 h, with constant stirring at 200 rpm, in 3 ml of LB medium and 50 mg/l of kanamycin at 37 °C (Kuhner Shaker ISF-4-W; Adolf Kühner AG, Basel (Switzerland)). Plasmid DNA purification was performed using the NucleoSpin® Plasmid Pure kit (Macherey & Nagel, Düren), according to the manufacturer's instructions. Electrocompetent P. putida KT2440, P. putida ΔtrpDC and P. putida ΔtrpDCΔpheA cells were obtained according to Choi et al ( Choi et al, J Microbiol Methods, 2005, 64(3):391 ). Electroporation was performed using a Biorad Gene Pulser Xcell electroporator (2.5 kV, 200 ohms, 25 μF) and the cell suspension was seeded on LB-agar plates supplemented with 50 mg/l of kanamycin. This resulted in the generation of P. putida KT2440/pSEVA234-trpEGS40F, P. putida ΔtrpDC/pSEVA234-trpEGS40F and P. putida ΔtrpDCΔpheA/pSEVA234-trpEGS40F strains (Table 2).

[194] All resulting P. putida strains (P. putida KT2440, P. putida ΔtrpDC/pSEVA234, P. putida ΔtrpDCΔpheA/pSEVA234, P. putida/pSEVA234-aroGD146N, P. putida/pSEVA234-trpEGS40F, P. putida ΔtrpDC/pSE VA234-aroGD146N, P. putida ΔtrpDC/pSEVA234-trpEGS40F, P. putida ΔtrpDCΔpheA/pSEVA234-aroGD146N and P. putida ΔtrpDCΔpheA/pSEVA234-trpEGS40F) (Table 2) were characterized with respect to their oAB-producing properties in shake culture flasks. The strains were grown in minimal medium, according to Wierckx et al. (Wierckx et al., AEM, 2005, 71:8221) at 30 °C and stirring at 250 rpm. The composition of the minimal medium was: was: 2 g/l of (NH4)2SO4, 1.63 g/l of NaH2PO4, 3.88 g/l of K2HPO4, 0.1 g/l of MgSO4^7H2O, 1.0 mg/l of CaCl2, 10 mg/l of EDTA, 5.0 mg/l of FeSO4^7H2O, 1.0 mg/l of MnCl ^O, 2.0 mg/l ZnSOv7H2O, 0.2 mg/l CuSO4^5H2O, 0.4 mg/l CoCl2^6H2O, 0.2 mg/l Na2MoO4 • 2H2O, and 5 g/l glucose. The pH was adjusted to 7 with 5 mol/l sodium hydroxide solution. The strains were pre-cultured on minimal medium and inoculated into 50 ml main cultures in 500 ml shake flasks with an OD600 of approximately 0.1. Cultures of plasmid-bearing strains were supplemented with 50 mg/l of kanamycin. Cultures of strains with a trpDC gene disruption were additionally supplemented with 0.1 mM L-tryptophan and cultures of strains with a pheA gene disruption were additionally supplemented with 1 mM L-phenylalanine. The lacIq system was induced at the beginning of the exponential-average phase by addition of 1 mM IPTG to allow production of oAB. Characteristics of the strains as oAB producers were measured as described in Example 3. During culture, OD600, glucose concentration, and anthranilate concentrations were measured off-line from 1.5 ml culture samples. A 1 ml aliquot of each sample was centrifuged for 5 min at 16000 xg (5415R; Eppendorf, Hamburg, Germany) in a reaction tube, after measuring the OD600 using a photometer. The supernatant was analyzed by HPLC-DAD (1100; Agilent Technologies, Santa Clara (USA)) and YSI (YSI-Select 2700; Kreienbaum Neoscience GmBH, Langenfeld). The data collected from Example 3 and Example 4 are presented in Table 4. Table 1 - Vectors and plasmids used and/or generated in the present invention Table 2 - Bacterial strains used and/or generated in the present invention Table 3 - Primers used in the present invention Table 4 - Characteristics in relation to the production of oAB of bacterial strains used and/or generated in the present invention (CDW: dry weight of cells; Y: yield; μmax: maximum growth rate; STY: space-time yield).

Example 5 - Cultivation of C. glutamicum strains in continuous fermenters with cell retention

[195] Continuous fermentations have been used for anthranilate production to achieve space-time optimized yield. A cell retention system was applied during the continuous fermentation, to increase the biomass concentration without increasing the glucose concentration of the feed solution. Two different systems were used for laboratory scale cell retention experiments: a hollow fiber filtration module provided by JM Water Separations (WaterSep Technology Corporation, Marlborough, MA, USA), with a cut-off value of 750 kDa, and a disposable centrifuge (Centritech Lab III) provided by Pneumatic Scale Angelus (Allen Road, Stow, OH, USA). Both systems were connected to 1 L laboratory scale bioreactors (OmniFerm, HiTec Zang, Herzogenrath, Germany). The filtration module integration is shown in Figure 30 and the centrifuge integration is shown in Figure 31.

Continuous fermentation of the C. glutamicum ΔtrpD strain

[196] A preculture of C. glutamicum ΔtrpD (see Table 2) was prepared using a sterile 250 ml Erlenmeyer flask filled with 50 ml of sterile BHIS medium containing 37 g/l Brains-Heart Infusion (BHI) and 91 g/l sorbitol. Incubation for 24 h at 28°C with a shaking frequency of 140 rpm resulted in an OD600 of 6.0. A 9 ml culture volume was transferred to 91 ml of freshly derived CGXII-MOPS medium containing 42 g/l MOPS buffer, 20 g/l (NH4)2SO4, 5 g/l urea, 3.7 g/l Brain-Heart Infusion, 9.1 g/l sorbitol, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g/l l of MgSO4^7H2O, 0.01 g/l of CaCl2 and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l of biotin, 0.1 mM tryptophan, 0.01 g/l of MnSO4^H2O, 0.01 g/l of FeSO4^7H2O, 1 mg/l of ZnSO4^7H2O, 0.2 mg/l of CuSO4^5H2O, 0.02 mg/l of NiCl2^6H2O and 0.03 g/l of 3,4-dihydroxybenzoic acid. Incubation of a 100 ml culture volume for 24 h at 28°C with a shaking frequency of 140 rpm resulted in an OD600 of 18.7. A 27 ml culture volume was centrifuged, the supernatant discarded and the pellet resuspended in 25 ml of sterile isotonic PBS buffer. The suspension was injected into a fermenter with 1 l of sterile derived CGXII medium containing 20 g/l of (NH4)2SO4, 5 g/l of urea, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.25 g/l of MgSθ4'7H2θ, 0.01 g/l of CaCl2, 1 μl/l of polypropylene glycol ol and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l Biotin, 0.1 mM tryptophan, 0.01 g/l MnSO4^H2O, 0.01 g/l FeSO4-7H2O, 1 mg/l ZnSθ4-7H2θ, 0.2 mg/l CuSO4^5H2O, 0.02 mg/l NiCl2^6H2 0 and 0.03 g/l of 3,4-dihydroxybenzoic acid. The same medium was used for feeding during continuous operation. The dissolved oxygen concentration was controlled at 30% by adjusting the stirring speed between 200 and 1200 rpm. A constant gas flow of 0.2 l/h of air was adjusted to allow for aeration and the pH was controlled at pH 7 with 1 M NH4OH. The fermentation results are shown in Figure 32. During the batch phase, in the first 24 h, glucose was consumed and biomass and oAB were produced. The fermenter was switched to continuous operation after a 24 h culture time, by feeding 50 ml/h of sterile medium and purging 50 ml/h of fermentation broth, including biomass and oAB. A constant production of oAB was obtained during continuous culture. After a culture time of 100 h, cell retention was initiated using a cross-flow ultrafiltration, as specified in Figure 30. An increase in the dry weight of cells was achieved by cell retention, as shown in Figure 32.

Continuous fermentation of the C. glutamicum ΔtrpD::trpD5Δgpi strain

[197] A preculture of C. glutamicum ΔtrpD::trpD5Δgpi (see Table 2) was prepared using a sterile 250 ml Erlenmeyer flask filled with 50 ml of sterile BHIS medium containing 37 g/l Brains-Heart Infusion (BHI) and 91 g/l sorbitol. Incubation for 24 h at 28°C with a shaking frequency of 140 rpm resulted in an OD600 of 8.2. A 10 ml culture volume was transferred to 90 ml of freshly derived CGXII-MOPS medium containing 42 g/l MOPS buffer, 20 g/l (NH4)2SO4, 5 g/l urea, 3.7 g/l Brain-Heart Infusion, 9.1 g/l sorbitol, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g /l of MgSO4^7H2O, 0.01 g/l of CaCl2 and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l Biotin, 0.01 g/l MnSO4^H2O, 0.01 g/l FeSO4^7H2O, 1 mg/l ZnSO4^7H2O, 0.2 mg/l CuSO4^5H2O, 0.02 mg/l NiCl2^6H2O and 0.03 g/l acid 3 ,4-dihydroxybenzoic acid. Incubation of a 100 ml culture volume in two 250 ml Erlenmeyer flasks for 24 h at 30°C with a shaking frequency of 300 rpm resulted in an OD600 of 20.5. A volume of 55 ml of culture broth was injected into a fermenter with 1 l of derived CGXII medium containing 20 g/l of (NH4)2SO4, 5 g/l of urea, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.25 g/l of MgSO4^7H2O, 0.01 g/l of CaCl2, 1 μl/l of polypropylene englycol and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l Biotin, 0.01 g/l MnSO4^H2O, 0.01 g/l FeSO4^7H2O, 1 mg/l ZnSO4^7H2O, 0.2 mg/l CuSO4^5H2O, 0.02 mg/l NiCl2^6H2O and 0.03 g/l acid 3 ,4-dihydroxybenzoic acid. A high concentration medium was used for the feed containing 20 g/l of (NH4)2SO4, 10 g/l of KH2PO4, 10 g/l of K2HPO4, 2.5 g/l of MgSO4^7H2O, 0.1 g/l of CaCl2, 2 ml/l of polypropylene glycol and 100 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 20 mg/l Biotin, 0.1 g/l MnSO4^H2O, 0.1 g/l FeSO4^7H2O, 10 mg/l ZnSO4^7H2O, 2 mg/l CuSO4^5H2O, 0.2 mg/l NiCl2^6H2O and 0.3 g/l 3,4-di acid - hydroxybenzoic. The dissolved oxygen concentration was controlled at 30% by adjusting the stirring speed between 200 and 1400 rpm. A constant gas flow rate of 0.2 l/h of air was adjusted to allow for aeration and the pH was controlled at pH 7 with 1 M NH4OH and 1 M NaCl. The fermentation results are shown in Figure 33. During the batch phase, in the first 40 h, glucose was consumed and biomass and oAB were produced. The fermenter was changed to continuous operation after a culture time of 40 h, by feeding 10 ml/h of sterile medium and purging 10 ml/h of fermentation broth, including biomass and oAB. At the start of continuous operation, the biomass concentration was not high enough to completely consume the added glucose in the feed solution, resulting in an accumulation of glucose in the reactor, as shown in Figure 33. After 136 h, the cell concentration was high enough to completely consume the added glucose. Cell retention was started after 184 h using a centrifuge as shown in Figure 31. An increase in the dry weight of cells was achieved by cell retention as shown in Figure 33. A constant production of oAB was obtained during 400 h of continuous fermentation.

Continuous fermentation of C. glutamicum ΔtrpD strain with complex carbon sources

[198] A preculture of C. glutamicum ΔtrpD (Table 2) was prepared using a sterile 250 ml Erlenmeyer flask filled with 50 ml of sterile BHIS medium containing 37 g/l Brains-Heart infusion and 91 g/l sorbitol. Incubation for 24 h at 28°C with a shaking frequency of 140 rpm resulted in an OD600 of 7.53. A 7 ml culture volume was transferred to 93 ml of freshly derived CGXII-MOPS medium containing 42 g/l MOPS buffer, 20 g/l (NH4)2SO4, 5 g/l urea, 3.7 g/l Brain-Heart Infusion, 9.1 g/l sorbitol, 1 g/l KH2PO4, 1 g/l K2HPO4, 0.25 g/l l of MgSO4^7H2O, 0.01 g/l of CaCl2 and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l of biotin, 0.1 mM tryptophan, 0.01 g/l of MnSO4^H2O, 0.01 g/l of FeSO4^7H2O, 1 mg/l of ZnSO4^7H2O, 0.2 mg/l of CuSO4^5H2O, 0.02 mg/l of NiCl2^6H2O and 0.03 g/l of 3,4-dihydroxybenzoic acid. Incubation of a 100 ml culture volume for 24 h at 28°C with a shaking frequency of 140 rpm resulted in an OD600 of 21.3. A 25 ml culture volume was centrifuged, the supernatant discarded and the pellet resuspended in 25 ml of sterile isotonic PBS buffer. The suspension was injected into a fermenter with 1 l of sterile derived CGXII medium containing 20 g/l of (NH4)2SO4, 5 g/l of urea, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 0.25 g/l of MgSO4^7H2O, 0.01 g/l of CaCl2, 1 μl/l of polypropylene glycol and 10 g/l of glucose (autoclaved separately). The following components were added after sterile filtration: 2 mg/l of biotin, 0.1 mM tryptophan, 0.01 g/l of MnSO4^H2O, 0.01 g/l of FeSO4^7H2O, 1 mg/l of ZnSO4^7H2O, 0.2 mg/l of CuSO4^5H2O, 0.02 mg/l of NiCl2^6H2O and 0.03 g/l of 3,4-dihydroxybenzoic acid. The same medium was used for feeding during the first 772 hours of continuous operation (feeding medium 1). After 772 hours, the feed composition was changed to feed medium 2, 40 g/l (NH4)2SO4, 0.5 g/l KH2PO4, 5 ml/l maizecin (Sigma Aldrich, C4648, lot no.: MKBP5720V), 1 ml/l polypropylene glycol, 267 g/l glucose syrup (Cargill C*Sweet D027 67, lot no.: 03285882), autoclaved separately to obtain a final concentration of glucose in the feed solution of 200 g/l and 0.1 mM L-tryptophan (filtered under sterile conditions and added after autoclave sterilization). The dissolved oxygen concentration was controlled at 30% by adjusting the stirring speed between 200 and 1200 rpm. A constant gas flow rate of 0.2 l/hr of air was set to allow for aeration and the pH was controlled to pH 7 with 1 M NH 4 OH. The results of continuous fermentation with feed medium 2 are shown in Figure 34. The preceding 772 h of continuous fermentation with feed medium 1 are not shown. Continuous cell retention was achieved by cross-flow ultrafiltration as specified in Figure 30 and continuous production of anthranilate was obtained during culture as shown in Figure 34. Continuous operation was temporarily stopped, as indicated by a stop phase in Figure 34, due to an accumulation of glucose. Feeding and collection were resumed after the glucose level had decreased.

Example 6 - Anthranilic acid (AA) adsorption/desorption

[199] To obtain anthranilic acid (AA) in a solution with a higher concentration, the AA was transferred from the aqueous solution to an organic solvent by means of an adsorption-desorption operation. To do this, the adsorption capacity of AA was tested on different types of adsorbents.

[200] Zeolite Y (Zeolyst International, catalog no. CBV600) and ZSM5 (Süd-Chemie/Clariant, catalog no. H-MFI-27) were the selected zeolites, which function as molecular sieves for different molecules. Hydroxyapatite (Calo(PO4)e(OH)2) (Sigma-Aldrich Catalog No. 289396) was tested for its ability to adsorb AA and other similar compounds in different solvents.

[201] Adsorption test: The adsorbents had been calcined at 300°C for 3 h to eliminate any remaining moisture. A solution of AA (0.5% by weight) in water was prepared. 20 ml of this solution was transferred to a 50 ml vial containing 0.2 g of adsorbent. After a certain period of time with stirring, the concentration of AA in the water was analyzed by HPLC. Decreased AA concentration in water was considered to be adsorbed AA.

[202] Synthesis of metal-substituted zeolite: given the better adsorption capacity of the zeolite with incorporated Ca (according to W. H. Goodman, US 4910336, 1990) Ca-substituted zeolites were prepared by ion exchange, to be tested in the adsorption of AA (S. M. Seo, S. Y. Coi, J. M. Suh, K. J. Jung, N. H. He o and W. T. Lim, Bull. Korean Chem. Soc. 2009, 30:1703). 3 g of powdered zeolite were added to a solution of Ca(NO3)2.4H2O (0.5 M). The suspension was stirred for 4 h and then the solution was replaced with fresh solution and this procedure was repeated two more times. Finally, the solids were separated by centrifugation, dried at 80 °C and calcined at 300 °C for 3 h. Four other samples of metal substituted zeolite Y using K, Na, Mg and Fe were prepared with the same method explained above. The samples were then labeled as K-Y, Na-Y, Y-Ca, Mg-Y and Fe-Y. The pore size of the H-ZSM5 zeolite can be in the range of 0.4-0.6 nm, preferably 0.5 nm. The pore size of the H-Y zeolite can be in the range of 0.6-0.9 nm, preferably 0.7-0.9 nm (eg as supplied by Zeolyst International, catalog no. CBV600).

[203] The AA adsorption assay was performed using the adsorbents mentioned above. The results of the analysis are presented below, in Table 6: Table 6: Adsorption of AA at 0.5%

[204] Zeolite Y showed the highest AA adsorption capacity from the aqueous solution. Ca-substituted zeolite Y improved AA adsorption by about 50%. Fe-substituted zeolite Y improved AA adsorption almost twice as much. The tests were carried out with the AA solution in distilled water and also in a buffer solution containing ((NH4)2SO4 (20 g/l), Na2HPO4 (1 g/l), KH2PO4 (1 g/l) and MgSO4 (0.25 g/l). Later, MgSO4 was eliminated from the buffer solution, because it caused the formation of insoluble magnesium anthranilate salt. Among the metal-substituted zeolites above, Fe-Y showed the highest AA adsorption capacity (greater than 50 g of AA/kg of Fe-Y). Fortunately, the contents of the buffer solution did not influence the adsorption of AA and the adsorption capacities were similar to those in distilled water. The IR spectrum of the Fe-Y sample after the adsorption test showed some characteristic peaks of AA, which may be evidence of the existence of AA on the surface of Fe-Y, probably chelated with Fe. Table 7: Capacities of adsorption of AA from zeolite Y replaced by metal in distilled water and buffered aqueous solution after 10 and 60 minutes of contact. ag of AA/kg of adsorbent

[205] The Fe-Y zeolite after the adsorption assay and a sample of the fresh Fe-Y zeolite were analyzed by ICP-MS. This analysis showed that the Fe/Al ratio in both samples were 1.25 and 1.3, respectively, which are values very close to each other. Therefore, the amount of Fe leached was insignificant. In the case of Ca-Y, 64% Ca leaching was detected after the adsorption test.

[206] The decomposition of desorbed AA to Fe-Y at temperatures up to 550 °C was studied using a thermal gravimetric analyzer equipped with a mass analyzer (TGA-MS). As can be seen in Figure 7, there was no formation of ANL at temperatures below 400°C. At temperatures greater than 400 °C, AA was directly decomposed into polyaniline, which was observed as dark materials on the sample holder. A small amount of ANL was detected at temperatures above 400 °C. Therefore, direct thermal conversion of AA adsorbed on the adsorbent to ANL was not successful.

AA desorption from the adsorbent to the liquid phase

[207] The desorption assay of anthranilic acid from Fe-Y zeolite into water showed that the adsorption of anthranilic acid on metal-substituted zeolite in aqueous solution is reversible. Subsequently, the desorption of AA into an organic solvent was tested. 1-dodecanol was selected as the organic solvent because of the high solubility of anthranilic acid in it and also because of its very low miscibility in water (0.004 g/l). Furthermore, its boiling point (259 °C) is much higher than that of aniline (183 °C) and water, so that its separation from the final mixture was more convenient. The desorption of AA adsorbed on the Fe-Y zeolite to 1-dodecanol was carried out by suspending 0.2 g of Fe-Y containing 10.8 mg of AA in 2 ml of 1-dodecanol. The suspension was stirred for 0.5 h in the temperature range 25-120 °C. The desorption results are shown in Figure 8, where a maximum of 27.8% AA can be desorbed to 1-dodecanol at 120 °C.

Example 7 - kinetics of the decarboxylation reaction of anthranilic acid in aniline, by thermal decarboxylation in an organic solvent Decarboxylation of AA dissolved in 1-dodecanol

[208] The decarboxylation of 3 wt% AA dissolved in 1-dodecanol at 160 °C was tested. Catalysts with different characteristics were selected. The results can be seen in Figure 9. The blank test, without any catalyst, showed only 1.4% conversion, while in the presence of zeolite Y (CBV600, "GO257") it resulted in more than 70% conversion to ANL in 1h.

[209] The high conversion of AA over zeolite Y (CBV600, "GO257") may be due to its highly acidic character and also due to pore size (0.7-0.8 nm). ZSM5 (MFI-27), despite having an acid character, has a smaller pore size (0.5 nm), so that AA molecules cannot penetrate these and, consequently, do not have access to active sites. Hydrotalcite (HTC, Mg6Al2(CO3)(OH)16.4H2O) has a basic character and the low conversion of AA indicates that the basic sites are not as active as the acidic sites in the decarboxylation of AA. The addition of Na to the H-Y zeolite ("GO257") decreases its acidic character and, therefore, a low AA conversion was obtained. The ammoniacal form (CBV500, "GO55") also has a lower number of acidic sites than the H-Y (CBV600, "GO257").

[210] As can be seen in Figure 10, the decarboxylation profile of AA in 1-dodecanol at 160 °C and 180 °C showed that AA conversion and ANL formation follow zero-order kinetics. The models simulated for the reaction at 160 °C and 180 °C could calculate rate coefficients (k) as 0.235 mol L-1.h-1 and 0.522 mol.L-1.h-1, respectively. The selectivity of these reactions for ANL was 100% and a mass balance of 95-110% (with a mean of 100.4%) was observed.

Decarboxylation of AA dissolved in Aniline

[211] Aniline is a much better solvent for AA than dodecanol and, being also the decarboxylation product, its use has great advantages compared to 1-dodecanol. It was found that up to 30% of AA could be dissolved in aniline at room temperature (20°C), up to 40% of AA could be dissolved in aniline at 50°C, and up to 50% of AA could be dissolved at 90°C.

[212] The decarboxylation of 40% by weight of AA dissolved in aniline at 160, 180 and 200 °C was tested. The blank test without any catalyst showed, as before, negligible conversion, while in the presence of zeolite Y (CBV600, "GO257") resulted in more than 99% conversion to ANL in 2 h. The results are shown in Figure 35. We were also able to fit the data according to a simple kinetic model:

[213] Where "r" is the reaction rate in g of aniline/g of catalyst per hour, "K0" is the kinetic constant also in g of aniline/g of catalyst per hour, "Ea" is the activation energy of the reaction in kJ/mol, "R" is the ideal gas constant equal to 8.31 J/Kmol, "t" is the temperature in K, "AA" is the concentration of AA and "n" is the order of reaction. Figure 35 also shows the adjustments of the present reaction model, at different temperatures. The results of the adjustments can be summarized as follows: k0 = 55492 g AN/g CAT h Ea = 37.4 kJ/mol (n = 0.16)

[214] Figure 35 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) in aniline with a catalyst. The decarboxylation kinetics of anthranilic acid (AA) dissolved 40% by weight in aniline in the presence of zeolite Y at 160 °C, 180 °C and 200 °C is shown.

[215] Anthranilic acid (40% by weight) was dissolved in aniline. At this concentration the acid was perfectly soluble in the organic solvent once heated to about 50°C. 80 ml of the above solution is then transferred to a 160 ml autoclave, 1.5 g of zeolite catalyst is added and the mixture is heated to 160°C or 180°C or 200°C. Samples were collected at different time intervals and analyzed by HPLC methods to determine the rate of aniline formation.

Decarboxylation of AA dissolved in Aniline in the presence of water

[216] As the anthranilic acid separated in the crystallization process may have some residual moisture (approximately 10%), we decided to analyze the reaction kinetics in the presence of water. Experiments analogous to the previous ones were carried out, with the exception that 10% by weight of water was deliberately added to the reaction mixture. We found that the overall kinetic and reaction profile remain unchanged. The results for the reaction temperature of 200 °C are shown in Figure 36.

[217] Figure 36 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) in aniline with a catalyst. The decarboxylation kinetics of anthranilic acid (AA) dissolved at 40% by weight in aniline and in 10% water-90% aniline in the presence of zeolite Y at 200°C is shown.

[218] Anthranilic acid (40% by weight) was dissolved in aniline. At this concentration the acid was perfectly soluble in the organic solvent once heated to about 50°C. 80 ml of the above solution was then transferred to a 160 ml autoclave, then 10% water (8 g), 1.5 g of zeolite catalyst was added and the mixture was heated to 160°C or 180°C or 200°C. Samples were collected at different time intervals and analyzed by HPLC methods to determine the rate of aniline formation.

Decarboxylation of AA dissolved in Aniline from different organic sources

[219] According to the present invention, anthranilic acid can be produced biologically by fermentation of different sugar-containing media. It is to be expected that differences in trace elements present in different media (ie hydrolyzed corn starch, sugar cane juice, or glucose) could affect anthranilic acid, which must be further decarboxylated to aniline. To verify if these differences could interfere with the catalyst and with the decarboxylation reaction, different anthranilic acids, separated by crystallization from different growth media, were tested. The comparison was always made in relation to chemically pure anthranilic acid, purchased from the supplier laboratory Sigma Aldrich.

[220] The different AA were tested under the same conditions as the previous tests, starting with a 40% solution of AA in aniline at 180°C. The results are summarized in Figure 37. All AA sources were fully converted to aniline in less than 90 minutes. The fastest conversion is shown by the chemically pure material (Aldrich). This is comparable to material produced by fermenting chemically pure glucose (VN35). The hydrolyzed corn starch isolate material is slightly slower (VN32 and 33), followed by the sugarcane juice isolate material (VN 34). This sequence follows the degree of refinement of the media used. The purer sugar source resulted in faster decomposition of the AA isolated from it. This can be explained by the presence of trace elements, such as ions or mineral salts, which could interfere with the zeolite catalyst. However, the effect is so limited that it would not interfere with the decarboxylation reaction, which proceeds very rapidly to completion, regardless of the AA source.

[221] Figure 37 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) in aniline with a catalyst. The decarboxylation kinetics of anthranilic acid (AA) dissolved at 40% by weight in aniline in the presence of zeolite Y at 180°C is shown.

[222] Anthranilic acid was obtained by crystallization from different sugar-containing media, such as chemically pure glucose, hydrolyzed corn starch, and sugar cane juice. The resulting isolated anthranilic acid (40% by weight) was dissolved in aniline. At this concentration the acid was perfectly soluble in the organic solvent once heated to about 50 °C. 80 ml of the above solutions were then transferred to a 160 ml autoclave, 1.5 g of zeolite catalyst was added and the mixture was heated to 180 °C. Samples were collected at different time intervals and analyzed by HPLC methods to determine the rate of aniline formation.

Stability of the catalyst for the decarboxylation of AA dissolved in aniline from hydrolyzed corn starch

[223] Considering the effect of trace elements observed before, the stability of the catalyst was tested over several batches. This was done by recycling the catalyst and using it in the next reaction without any washing step, following the reaction procedure described above. The results are shown in Figure 38. The difference in activity between the freshly prepared and reused catalyst is minimal. The reused catalyst appears to be even faster or better than the fresh system. This showed that there is no inhibition or poisoning effect of the trace elements on the catalyst, which remains active in the decarboxylation reaction. For further confirmation, the catalyst was analyzed post mortem and compared to freshly prepared. The IR spectrum of the two catalysts is illustrated in Figure 39. Apart from some absorbed aniline species, only a small decrease in the OH signal is present. This OH can be abruptly cooled after exchange with other ions (trace elements), however, it is probably not the active site for catalysis, since the strong acidity of zeolites derives from Al-O-Si bridges.

[224] Figure 38 shows the decomposition of 2-aminobenzoic acid (anthranilic acid, AA) in aniline with a catalyst. The decarboxylation kinetics of anthranilic acid (AA) dissolved at 40% by weight in aniline in the presence of zeolite Y at 180°C is shown.

[225] Anthranilic acid was obtained by crystallization from hydrolyzed corn starch. The resulting isolated anthranilic acid (40% by weight) was dissolved in aniline. At this concentration the acid was perfectly soluble in the organic solvent once heated to about 50 °C. 80 ml of the above solution are then transferred to a 160 m autoclave, 1.5 g of zeolite catalyst are added and the mixture is heated to 180 °C. The catalyst was isolated and recycled for a subsequent reaction without further treatment or washing. 80 ml of the above solution was then transferred to a 160 ml autoclave, 1.5 g of zeolite catalyst was added and the mixture heated to 180°C. The catalyst was isolated and recycled to the subsequent reaction without further treatment or washing. Samples were collected at different time intervals and analyzed by HPLC methods to determine the rate of aniline formation.

[226] Figure 39 shows the IR spectrum of a freshly prepared H-Y catalyst and one after the reaction as above using AA isolated from hydrolyzed corn starch. Typical bands for aniline are marked. Indeed, absorption of this highly basic species takes place. The OH region is also highlighted. The reduction of the OH signal is compatible with some partial ion exchange that could occur with present trace elements, however the OH groups do not seem to be active in the catalysis, but the AL-O-Si bridge is probably responsible.

Example 8 - dissolution and crystallization of oAB from fermentation broth

[227] An industrial sugar source was used for fermentation with Corynebacterium glutamicum. 100 ml of broth with a content of 21.5 g/l of glucose, 3.7 g/l of lactate was mixed with 10 g of anthranilic acid in a 250 ml laboratory reactor. The suspension had a pH value of 4.7. The solubility of oAB was about 4.5 g/l at room temperature and pH 4.5. Then 9.12 g of NaOH (32%) were added in 4 portions until a solution was formed. The solution had a pH value of 8.3. Then 7 g of HCl (37%) was added in 1 hour. By dosage of HCl, the first nucleation occurred from a pH value of 5.8. The number of crystals increased during dosing. At the end of dosing the suspension had a pH value of 3.6. Then the suspension was stirred for 1 hour. Finally the suspension was filtered. Filtration:

[228] Filtration was performed according to the VDI guideline “VDI 2762”, at 0.2 bar and a filter area of 12.6 cm2. 125 g of suspension was filtered and washed with 25 g of water. 91 g of mother liquor and 23.1 g of wet solid were measured. The resistance of the filter cake was determined according to the "VDI 2762" standard and presented a value of 5 x 1010 l/m2. The filter cake was dried in a drying oven. After drying, 9.1 g of dry solid were weighed, which corresponds to a yield of 91%.

[229] The anthranilic acid fraction in the mother liquor was 0.72%. The ash content of the solid was determined as an indication of the anthranilic acid salt content. The ash content was determined to be 0.55%.

Example 9 - Solubility of oAB in 1-dodecanol

[230] The solubility of oAB in 1-dodecanol was tested with increasing temperature. SLE = solid-liquid balance

[231] The solubility of oAB in 1-dodecanol increased with increasing temperature.

Claims (14)

1. Recombinant microbial host cell characterized by being capable of biologically converting a raw material comprising a fermentable carbon substrate into o-aminobenzoate (oAB), wherein the recombinant microbial host cell is Corynebacterium glutamicum, wherein said fermentable carbon substrate is selected from the group consisting of C-5 monosaccharides, C-6 monosaccharides, disaccharides and trisaccharides, and wherein said microbial host cell recombinant has a genetic modification of the trpD gene, said modification being in accordance with a genetic sequence selected from the group consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, which is capable of decreasing the expression of the trpD gene with respect to a wild type. 2. Recombinant microbial host cell, according to claim 1, characterized in that the microbial host cell Corynebacterium glutamicum additionally comprises a deletion in the hpr gene selected from SEQ ID NO: 17 or SEQ ID NO: 18 or in the ptsG gene selected from SEQ ID NO: 19 or SEQ ID NO: 20, both encoding phosphoenolpyruvate-phosphotransferase. 3. Recombinant microbial host cell, according to claim 1, characterized in that said raw material is selected from the group consisting of sugar beet, sugar cane, plants containing starch, lignocellulose, glycerol and C1 compounds. 4. Method for the production of aniline, characterized in that it comprises the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising a fermentable carbon substrate using the recombinant microbial host cell, as defined in claim 1, wherein said o-aminobenzoate comprises anthranilate anion, b) converting said o-aminobenzoate from said anthranilate anion into anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolution in an organic solvent, and d) converting said anthranilic acid into aniline by thermal decarboxylation in an organic solvent. 5. Method according to claim 4, characterized in that said fermentation of step a) is a batch fermentation, a fed-batch fermentation or a continuous fermentation. 6. Method according to claim 4 or 5, characterized in that step a) and step b) are performed continuously. 7. Method according to any one of claims 4 to 6, characterized in that said recombinant microbial host is removed before carrying out step b) of converting said o-aminobenzoate from said anthranilate anion into anthranilic acid. 8. Method according to any one of claims 4 to 7, characterized in that said acid protonation of step b) is carried out by adding HCl. 9. Method according to any one of claims 4 to 8, characterized in that in step c) said recovery of said anthranilic acid by precipitation comprises filtration, thus generating a suspension comprising said recovered anthranilic acid. 10. Method according to any one of claims 4 to 9, characterized in that in step c) said recovery of anthranilic acid by dissolving said anthranilic acid in an organic solvent comprises adding said organic solvent or a mixture thereof to said anthranilic acid, such that said anthranilic acid is recovered as a solute in the organic solvent. 11. Method according to any one of claims 4 to 10, characterized in that step c) is followed by washing and drying the recovered anthranilic acid precipitate, before carrying out the thermal decarboxylation of step d). 12. Method according to any one of claims 4 to 11, characterized in that step d) is carried out in the presence of a catalyst. 13. Method according to any one of claims 4 to 12, characterized in that, after step c), the residual anthranilate anion is recovered by adsorption on an ion exchange resin, or on an active carbon material, or on a zeolite, followed by desorption of the recovered anthranilate anion. 14. Method according to claim 13, characterized by the fact that, after recovering the residual anthranilate anion by adsorption following step c), a stream of water free of adsorbed anthranilate anion is partially or completely fed back to the fermentation of step a).