MIXTURES OF GLUCOSE AND XYLOSE FOR THE FERMENTATIVE PREPARATION OF ORTHO-AMINOBENZOIC ACID

Abstract

The present invention relates to the preparation of ortho-aminobenzoic acid by means of microbial fermentation, wherein mixtures of glucose and xylose are used as fermentable substrates.

Claims

1. A method of producing ortho-aminobenzoic acid (oAB) comprising culturing one or more cells of the Corynebacterium genus that are able to convert glucose and xylose to oAB in a culture medium containing a mixture of glucose and xylose with a glucose content between 5% by weight and 86% by weight and a xylose content between 95% by weight and 14% by weight, wherein the proportions of glucose and xylose add up to 100%; wherein oAB is produced; and wherein said cells differ from the wild type at least in the following features: (i) reduced expression of anthranilate phosphoribosyltransferase compared to the wild type, but where there must be residual activity; (ii) elevated activity of shikimate kinase; (iii) elevated activity of 3-phosphoshikimate 1-carboxyvinyltransferase and chorismate synthase; (iv) presence of a feedback-resistant 3-deoxyarabinoheptulosanate-7-phosphate synthase; and (v) elevated activity of xylose isomerase and of xylulokinase.

2. The method as claimed in claim 1, wherein the mixture of glucose and xylose has a glucose content between 16% by weight and 86% by weight, and xylose contributes the proportion lacking from 100% by weight.

3. The method as claimed in claim 2, wherein the carbon yield is at least 0.138 mol of carbon in the form of oAB per mole of carbon consumed in glucose and xylose.

4. A cell of the Corynebacterium genus which is suitable for release of oAB and differs from the wild type at least in the following features: (i) reduced expression of anthranilate phosphoribosyltransferase compared to the wild type, but where there must be residual activity; (ii) elevated activity of shikimate kinase; (iii) elevated activity of 3-phosphoshikimate 1-carboxyvinyltransferase and chorismate synthase; (iv) presence of a feedback-resistant 3-deoxyarabinoheptulosanate-7-phosphate synthase; and (v) elevated activity of xylose isomerase and of xylulokinase.

5. A method of producing OaB comprising microbial fermentation of strain of the Corynebacterium genus as claimed in claim 4 with a mixture of glucose and xylose as energy source and carbon source.

6. The method as claimed in claim 5, wherein the mixture of glucose and xylose has a glucose content between 5% by weight and 86% by weight and a xylose content between 95% by weight and 14% by weight, and wherein the proportions of glucose and xylose add up to 100%.

7. A composition comprising a) microbial cells of the Corynebacterium genus as claimed in claim 4; b) a mixture of glucose and xylose having a glucose content between 5% by weight and 86% by weight and a xylose content between 95% by weight and 14% by weight, where the proportions of glucose and xylose add up to 100%; and c) at least one nitrogen source, at least one phosphorus source, at least one sulfur source and trace elements.

8. The composition as claimed in claim 7, additionally comprising oAB.

9. A method of producing oAB comprising microbial fermentation of a culture medium comprising a mixture of glucose and xylose having a glucose content between 5% by weight and 86% by weight and a xylose content between 95% by weight and 14% by weight, where the proportions of glucose and xylose add up to 100%.

Description

EXAMPLES

[0054] All experiments except that in example 3 were conducted with the strain produced as described below.

[0055] The bacterium Corynebacterium glutamicum ATCC13032 was used as the basis for production of a microbial strain capable of utilizing xylose as carbon source for production of anthranilic acid. For this purpose, the original strain was first adapted by adaptive laboratory evolution (ALE) to rising anthranilic acid concentrations in the culture supernatant. Thereafter, this was made to produce anthranilic acid by directed chromosomal modifications. Subsequently, the strain was provided with genes from what is called the xylose isomerase pathway, since C. glutamicum ATCC13032 does not have a native metabolic pathway for utilization of xylose. All genetic modifications, i.e. chromosomal deletions and integration of genes, were effected by double homologous recombination using corresponding pk19mobsacB derivatives (Schfer et al., 1994: Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pk18 and pk19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69-73. doi: 10.1016/0378-1119 (94) 90324-7).

[0056] The activity of anthranilate phosphoribosyltransferase TrpD was lowered by first deleting the native trpD allele and replacing it with an allele (called trpD5) having a GTG rather than ATG start codon and a ribosome binding site with reduced distance from the start codon (SEQ ID NO. 1).

[0057] The gene (SEQ ID NO. 5) that encodes one or the only phosphoenolpyruvate carboxylase in C. glutamicum (SEQ ID NO. 4) was deleted.

[0058] In order to boost the aromatic biosynthesis pathway, an artificial polycistronic P.sub.tuf-aroLAC operon (SEQ ID NO. 15) consisting of the aroL (b0388) genes from Escherichia coli, and the aroA (cg0873) and aroC (cg1829) genes from C. glutamicum, were integrated downstream of cg2563 under the control of the constitutive promoter of the elongation factor Tuf. In addition, the aroG.sup.new allele, which encodes a feedback-resistant variant of DAHP synthase (SEQ ID NO. 11) from E. coli, was integrated into the genome of the strain downstream of cg3132.

[0059] In order to render the strain capable of feeding xylose into the nonoxidative pentose phosphate pathway, a synthetic construct consisting of a codon-optimized xylose isomerase gene xylA (xcc1758) from Xanthomonas campestris pv. campestris (amino acid sequence of the enzyme according to SEQ ID NO. 13), and the xylulokinase gene xylB (cg0147) from C. glutamicum (amino acid sequence of the enzyme according to SEQ ID NO. 14) was produced, in each case under the control of the Ptuf promoter sequence (SEQ ID NO. 8) and followed by an rrnB terminator from E. coli. The construct, called P.sub.tuf-xylA.sub.Xcc-P.sub.tuf-xylB.sub.Cg-T.sub.mnB (SEQ ID NO. 16), was integrated into the genome of the strain downstream of cg3344.

Example 1

[0060] Comparative cultivations of the above-specified strain for production of ortho-aminobenzoic acid were conducted proceeding from two different sugars in each of four different ratios to one another. The main culture media were produced here such that the two sugars D-glucose and D-xylose were present in amounts as in table 1, based on the total sugar content of 20 g/L.

TABLE-US-00001 TABLE 1 Overview of the compositions of the two different sugars D-glucose and D-xylose in the main culture media. For media 1-4 and hence each ratio of amounts of sugar, four replicates were run in each case, each with an initial cultivation volume of 50 mL. % by wt. of sugar (based on the total sugar content) Medium name D-Glucose D-Xylose Medium 1 100 0 Medium 2 64 36 Medium 3 75 25 Medium 4 88 12

[0061] For each condition and each ratio of amounts of sugar from table 1, four replicates were run. The cultivation conditions are shown below (table 2).

TABLE-US-00002 TABLE 2 Overview of the culture conditions for the preparatory and main cultures. For the incubation of the second preparatory cultures (25 mL each), Erlenmeyer flasks with a maximum capacity of 500 mL each were used, and for the incubation of the main cultures (initially 50 mL each before the first sampling) Erlenmeyer flasks having a maximum capacity of 1000 mL each. The sterile barriers used were cotton plugs. Parameter Setting Comment Shake frequency (rpm) 200 Temperature ( C.) 30 Initial volume per 25 In 500 mL Erlenmeyer flask, replicate (mL) for the preparatory culture Initial volume per 50 In 1000 mL Erlenmeyer flask, replicate (mL) for the main culture

Media Used, and Composition and Production Thereof

[0062] Unless stated otherwise, all media were produced with ddH.sub.2O and autoclaved.

TABLE-US-00003 TABLE 3 Liquid and solid complex media composed of Brain Heart Infusion (BHI) for growing of cells. Medium Composition Addition before/after the autoclave BHI broth 37 g/L BHI broth Before autoclaving BHI agar plates 52 g/L BHI agar Before autoclaving

TABLE-US-00004 TABLE 4 Liquid minimal medium with complex constituents for the growing of cells in the preparatory culture. The amounts weighed out are given for 1 L of complete CGXII preparatory culture medium. Once all the reagents have been supplemented in the medium, the final target concentrations are attained in the complete medium. Corresponding to final concentration in the Addition before/ Medium Composition complete medium after the autoclave CGXII preparatory 1 g KH.sub.2PO.sub.4 1 g/L Everything hereafter culture medium 1 g K.sub.2HPO.sub.4 1 g/L added before autoclaving. 10 g (NH.sub.4).sub.2SO.sub.4 10 g/L 5 g urea 5 g/L 62 g MOPS 62 g/L 5 g yeast extract 5 g/L 1 mL CaCl.sub.2 stock solution 1 mL/L Dissolve in 800 mL of ddH.sub.2O, adjust pH to pH = 7.0-7.5 with KOH pellets, make up to 940 mL with ddH.sub.2O and autoclave. 1.25 mL MgSO.sub.4 stock solution 1.25 mL/L Everything hereafter added after autoclaving: 1 mL trace element stock solution 1 mL/L 1 mL biotin stock solution 1 mL/L 33 mL D-glucose stock solution OR 20 g/L D-glucose OR 33 mL D-glucose and 16.5 mL D- 20 g/L D-glucose and xylose stock solution 10 g/L D-xylose 23.75 mL water OR 7.25 mL water

TABLE-US-00005 TABLE 5 Liquid minimal medium for the main culture. The amounts weighed out are given for 1 L of complete CGXII main culture medium. Once all the reagents have been supplemented in the medium, the final target concentrations are attained in the complete medium. Corresponding to final concentration in the Addition before/ Medium Composition complete medium after the autoclave CGXII main culture 1 g KH.sub.2PO.sub.4 1 g/L Everything hereafter medium 1 g K.sub.2HPO.sub.4 1 g/L added before autoclaving: 10 g (NH.sub.4).sub.2SO.sub.4 10 g/L 62 g MOPS 62 g/L 1 mL CaCl.sub.2 stock solution 1 mL/L Dissolve in 800 mL of ddH.sub.2O, adjust pH to pH = 7.0-7.5 with KOH pellets, make up to 940 mL with ddH.sub.2O and autoclave. 1.25 mL MgSO.sub.4 stock solution 1.25 mL/L Everything hereafter added after autoclaving: 1 mL trace element stock solution 1 mL/L 1 mL biotin stock solution 1 mL/L 33 mL D-glucose stock solution OR 20 g/L D-glucose OR 33 mL D-xylose stock solution 20 g/L D-xylose 23.75 mL water

TABLE-US-00006 TABLE 6 Overview of production of the 2 g/L biotin stock solution. Rather than being autoclaved, the solution is sterile-filtered (0.2 m). The solution can be stored at 4 C. for 1 month. Medium Composition Note Biotin 2 g/L Solution is sterile-filtered. stock biotin During the dissolving, the pH is measured solution while stirring and adjusted to pH = 7.2 with 1M KOH.

TABLE-US-00007 TABLE 7 Overview of production of the 600 g/L D-glucose stock solution. Medium Composition Note D-glucose 660 g D- Substance is dissolved in 554 g of hot stock glucose ddH.sub.2O. Total weight of 1 L of the complete solution monohydrate solution: 1214 g. Boil briefly for complete dissolution prior to autoclaving.

TABLE-US-00008 TABLE 8 Overview of production of the 600 g/L D-xylose stock solution. Medium Composition Note D-xylose 600 g D- Substance is dissolved in 595 g of hot ddH.sub.2O. stock xylose Total weight of 1 L of the complete solution: solution 1195 g. Boil briefly for complete dissolution prior to autoclaving.

TABLE-US-00009 TABLE 9 Overview of production of the 10 g/L CaCl.sub.2 stock solution. Medium Composition Note CaCl.sub.2 10 g/L CaCl.sub.2 Solution need not be autoclaved since it is stock 2 H.sub.2O supplemented before the autoclaving solution operation of the CGXII media.

TABLE-US-00010 TABLE 10 Overview of production of the 200 g/L MgSO.sub.4 stock solution. Medium Composition Note MgSO.sub.4 stock 200 g/L MgSO.sub.4 7 H.sub.2O Solution is sterile-filtered. solution

TABLE-US-00011 TABLE 11 Overview of production of the 1000x trace element solution. Rather than being autoclaved, the solution is sterile-filtered (0.2 m). This solution has a shelf life of 6 months at 4 C. Because of the small weights, it is advisable to produce a 1 L batch. Medium Composition Note Trace 10 g/L MnSO.sub.4 H.sub.2O Solution is sterile-filtered. element 10 g/L FeSO.sub.4 7 H.sub.2O solution 1 g/L ZnSO.sub.4 7 H.sub.2O 0.2 g/L CuSO.sub.4 5 H.sub.2O 0.02 g/L NiCl.sub.2 6 H.sub.2O Dissolve in ddH.sub.2O and adjust pH with HCl to pH = 1.

TABLE-US-00012 TABLE 12 Overview of production of 1x phosphate buffer (PBS). 10x PBS (article number BP399-1) from Fisher Scientific GmbH was used. Medium Composition Note 1x phosphate buffer 10x PBS Dilute 1:10 with ddH.sub.2O, autoclave.

Instruments Used

TABLE-US-00013 TABLE 13 Overview of the parameters examined in this study and instruments and methods for examination thereof. Parameter Instrument Description Glucose concentration Cedex Bio Analyzer, Roche Cedex Bio Glucose assay with article number RD- 06343732001, used according to manufacturer's instructions NH.sub.3 concentration Cedex Bio Analyzer, Roche Cedex Bio NH3 assay with article number RD- 06343775001, used according to manufacturer's instructions ortho-Aminobenzoic acid HPLC, Agilent For separation and concentration G7104C 1260 flexible pump quantification of ortho- G7167A 1260 multisampler aminobenzoic acid in the G7116A 1260 multicolumn sterile-filtered supernatant of thermostat (MCT) the samples, an Agilent Eclipse G7117C 1260 DAD HS Plus C18 column (4.6 150 mm, G7162A 1260 RID 5 m) and a Zorbax Eclipse Plus C18 precolumn cartridge (4.6 12 mm, 5 m, 5 mm) were used. The ortho-aminobenzoic acid was detected by means of the diode array detector (DAD). D-Xylose concentration HPLC, ROA For separation and HPLC, Agilent quantification of D-xylose in the G7104C 1260 flexible pump sterile-filtered supernatant of G7167A 1260 multisampler the samples, a Phenomenex G7116A 1260 multicolumn Rezex ROA-Organic Acid H + 8% thermostat (MCT) column (300 7.8 mm) and a G7117C 1260 DAD HS SecurityGuard Cartridge Carbo- G7162A 1260 RID H precolumn cartridge (4 3.0 mm) were used. The D- xylose was detected by means of the refractive index detector (RID). Incubation of the cultures ISF1-X shaker incubator, Kuhner Shaker GmbH pH SevenCompact S210 pH meter, Mettler Toledo InLab Expert Pro-ISM pH electrode, Mettler Toledo Optical density (OD.sub.600) CO8000 cell density meter, WPA biowave Weight of dry biomass SARTORIUS CUBIS 225S Semi- Micro Balance, Sartorius

Procedure

[0063] The starter cultures were generated by taking cell mass from dormant forms in glycerol of the microbial cultures used for the culturing, and these were used to inoculate a BHI agar plate. The BHI agar plates were incubated at 30 C. for 72 h.

[0064] The agar plate culture set up in this way was used to inoculate a BHI liquid culture. For this purpose, a little cell mass was taken from the respective BHI agar plate cultures and 4 mL of liquid BHI medium in each case was inoculated in a round-bottom tube. The liquid cultures were incubated at 30 C. and 200 rpm for about 7.5 h (preparatory culture I).

[0065] For the second preparatory culture, two media with different carbon sources were used. For this purpose, CGXII preparatory culture medium was produced with either 20 g/L D-glucose or with 20 g/L D-glucose and additionally 10 g/L D-xylose. For this purpose, 23 mL in each case of the corresponding preparatory culture medium was transferred into a 500 mL Erlenmeyer flask, 2 mL of the BHI liquid preparatory culture I was added to each batch, and these two shaken flask preparatory cultures were incubated at 200 rpm and 30 C. for 17 h.

[0066] The preparatory culture with D-glucose as the sole energy source and carbon source was used for the inoculation of the main cultures with medium 1. Proceeding from the preparatory culture with 20 g/L D-glucose and additionally 10 g/L D-xylose, three different main cultures (media 2-4) were inoculated.

[0067] On completion of incubation of the second preparatory culture in the shaken flask, the optical density of the cultures was measured. Depending on the cell density achieved, a proportion of these second preparatory cultures was taken, the cells were pelletized by centrifugation and washed in sterile phosphate buffer (singly concentrated), and resuspended in 50 mL in each case of the corresponding main culture medium (media 1-4).

TABLE-US-00014 TABLE 14 Measurement of optical density (OD.sub.600) on completion of incubation of the second preparatory culture in the shaken flask. This was used to calculate what volume of the second preparatory culture is needed to inoculate 50 mL in each case of main culture with an initial OD.sub.600 of 1. For each medium, four replicates were run. Since three xylose-containing media (media 2-4) were to be tested, a total of 12 aliquots were taken for the inoculation from the preparatory culture with 20 g/L D-glucose and 10 g/L D-xylose. OD.sub.600 () Volume required with 20 g/L D-glucose 30 4 1.7 mL for medium 1 with 20 g/L D-glucose and 32 4 1.6 mL for medium 2 10 g/L D-xylose 4 1.6 mL for medium 3 4 1.6 mL for medium 4

[0068] The main cultures produced in this way were transferred to a Kuhner incubation shaker (table 13) and incubated. For the sampling over the period of cultivation, the shaken flasks were taken from the incubation shaker and weighed in order to ascertain evaporation effects. Samples were taken under sterile conditions for determination of glucose, xylose, ortho-aminobenzoic acid and dry biomass.

Results

D-Xylose Concentration

TABLE-US-00015 TABLE 15 Overview of the D-xylose concentrations over time for four replicates in each case with medium 1 and medium 2. Condition (% by wt. of sugars based on Medium 1 Medium 2 total sugar) (100% by wt. of D-glucose, (64% by wt. of D-glucose, Replicate 0% by wt. of D-xylose) 36% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 0 0 0 0 7.53 7.28 7.36 7.46 19.00 0 0 0 0 7.20 7.05 7.09 7.24 26.00 0 0 0 0 7.22 7.15 7.18 7.38 42.83 0 0 0 0 5.37 5.20 5.25 5.43 50.00 0 0 0 0 4.19 4.06 4.09 4.21 66.50 0 0 0 0 2.41 2.22 2.26 2.51

D-Xylose Concentration

TABLE-US-00016 TABLE 16 Overview of the D-xylose concentrations over time for four replicates in each case with medium 3 and medium 4. Condition (% by wt. of sugars based on Medium 3 Medium 4 total sugar) (75% by wt. of D-glucose, (88% by wt. of D-glucose, Replicate 25% by wt. of D-xylose) 12% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 5.15 5.11 5.10 5.11 2.51 2.51 2.54 2.51 19.00 4.95 4.94 4.94 4.96 2.34 2.38 2.37 2.39 26.00 4.88 4.89 4.89 4.90 2.24 2.28 2.26 2.28 42.83 3.31 3.32 3.30 3.18 1.08 1.16 1.07 1.20 50.00 2.15 2.16 2.15 2.03 0.46 0.48 0.44 0.49 66.50 0.83 0.84 0.81 0.75 0.10 0.10 0.08 0.10

D-Glucose Concentration

TABLE-US-00017 TABLE 17 Overview of the D-glucose concentrations over time for four replicates in each case with medium 1 and medium 2. Condition (% by wt. of sugars based on Medium 1 Medium 2 total sugar) (100% by wt. of D-glucose, (64% by wt. of D-glucose, Replicate 0% by wt. of D-xylose) 36% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 21.46 21.46 21.46 21.46 13.19 13.19 13.19 13.19 19.00 16.08 16.51 16.23 16.62 9.18 8.90 9.03 9.37 26.00 12.38 12.56 12.40 12.89 6.21 5.95 6.15 6.44 42.83 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 50.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 66.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

D-Glucose Concentration

TABLE-US-00018 TABLE 18 Overview of the D-glucose concentrations over time for four replicates in each case with medium 3 and medium 4. Condition (% by wt. of sugars based on Medium 3 Medium 4 total sugar) (75% by wt. of D-glucose, (88% by wt. of D-glucose, Replicate 25% by wt. of D-xylose) 12% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 15.78 15.78 15.78 15.78 18.27 18.27 18.27 18.27 19.00 11.64 11.65 11.62 11.57 13.80 13.99 13.68 14.024 26.00 8.47 8.49 8.48 8.38 10.26 10.53 10.21 10.61937 42.83 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0 50.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0 66.50 0.00 0.00 0.00 0.00 0.00 0.00 #NV 0

Dry Biomass Concentration (DBM)

TABLE-US-00019 TABLE 19 Final dry biomass concentrations in double determination for four replicates in each case with medium 1 and medium 2. Condition (% by wt. of sugars based on Medium 1 Medium 2 total sugar) (100% by wt. of D-glucose, (64% by wt. of D-glucose, Replicate 0% by wt. of D-xylose) 36% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture DBM DBM DBM DBM DBM DBM DBM DBM time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 66.50 4.91 5.21 4.85 5.14 4.22 4.25 3.93 4.02 66.50 5.01 5.26 4.84 4.97 3.72 4.12 4.01 4.16

Dry Biomass Concentration (DBM)

TABLE-US-00020 TABLE 20 Final dry biomass concentrations in double determination for four replicates in each case with medium 3 and medium 4. Condition (% by wt. of sugars based on Medium 3 Medium 4 total sugar) (75% by wt. of D-glucose, (88% by wt. of D-glucose, Replicate 25% by wt. of D-xylose) 12% by wt. of D-xylose) () 1 2 3 4 1 2 3 4 Culture DBM DBM DBM DBM DBM DBM DBM DBM time (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 66.50 4.61 4.39 4.46 4.48 4.76 4.4 4.7 4.7 66.50 4.49 4.34 4.41 4.56 4.67 4.62 4.81 4.88
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00021 TABLE 21 Overview of the ortho-aminobenzoic acid concentration over time for four replicates in each case with medium 1 and medium 2. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (64% by wt. of D-glucose, 0% by wt. of D-xylose) 36% by wt. of D-xylose) Replicate () 1 2 3 4 1 2 3 4 Culture time oAB oAB oAB oAB oAB oAB oAB oAB (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 0.27 0.27 0.26 0.26 0.26 0.25 0.25 0.24 19.00 0.34 0.33 0.32 0.34 0.18 0.30 0.28 0.26 26.00 0.62 0.62 0.61 0.60 0.53 0.52 0.51 0.51 42.83 2.15 2.21 2.15 2.18 1.68 1.68 1.74 1.73 50.00 2.56 2.55 2.41 2.47 2.11 2.08 2.13 2.09 66.50 2.31 2.35 2.30 2.37 2.40 2.38 2.36 2.42
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00022 TABLE 22 Overview of the ortho-aminobenzoic acid concentration over time for four replicates in each case with medium 3 and medium 4. Condition (% by wt. of sugars based on total sugar) Medium 3 (75% by wt. of D-glucose, Medium 4 (88% by wt. of D-glucose, 25% by wt. of D-xylose) 12% by wt. of D-xylose) Replicate () 1 2 3 4 1 2 3 4 Culture time oAB oAB oAB oAB oAB oAB oAB oAB (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 0.25 0.26 0.27 0.27 0.27 0.27 0.28 0.26 19.00 0.28 0.27 0.28 0.28 0.28 0.28 0.29 0.27 26.00 0.53 0.53 0.53 0.54 0.56 0.56 0.58 0.55 42.83 1.80 1.81 1.81 1.94 2.07 2.02 2.13 1.97 50.00 2.39 2.36 2.39 2.41 2.41 2.43 2.48 2.52 66.50 2.51 2.50 2.51 2.62 2.46 2.48 2.46 2.43

Final Product Yield

TABLE-US-00023 TABLE 23 Overview of the ortho-aminobenzoic acid yields after 66.50 h for four replicates with medium 1 and medium 2. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (64% by wt. of D-glucose, 0% by wt. of D-xylose) 36% by wt. of D-xylose) Replicate () 1 2 3 4 1 2 3 4 Yield after 0.0857 0.0881 0.0860 0.0900 0.1054 0.1044 0.1035 0.1090 cultivation for 66.50 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.0819 0.0841 0.0822 0.0858 0.0890 0.0890 0.0880 0.0914 cultivation for 66.50 h (g.sub.oAB/ g.sub.C source initially charged) Yield after 0.1126 0.1157 0.1130 0.1132 0.1312 0.1300 0.1288 0.1359 cultivation for 66.50 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.1075 0.1105 0.1079 0.1128 0.1090 0.1091 0.1079 0.1120 cultivation for 66.50 h (mol.sub.oAB/ mol.sub.C source initially charged)

Final Product Yield

TABLE-US-00024 TABLE 24 Overview of the ortho-aminobenzoic acid yields after 66.50 h for four replicates with medium 3 and medium 4. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 3 (75% by wt. of D-glucose, Medium 4 (88% by wt. of D-glucose, 25% by wt. of D-xylose) 12% by wt. of D-xylose) Replicate () 1 2 3 4 1 2 3 4 Yield after 0.1020 0.0999 0.1017 0.1055 0.0949 0.0967 0.0943 0.0949 cultivation for 66.50 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.0933 0.0927 0.0930 0.0968 0.0901 0.0917 0.0865 0.0900 cultivation for 66.50 h (g.sub.oAB/ g.sub.C source initially charged) Yield after 0.1286 0.1253 0.1282 0.1329 0.1219 0.1242 0.1210 0.1219 cultivation for 66.50 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.1168 0.1161 0.1165 0.1212 0.1155 0.1176 0.1147 0.1154 cultivation for 66.50 h (mol.sub.oAB/ mol.sub.C source initially charged)

Averages of the Final Product Yields and Standard Deviations

TABLE-US-00025 TABLE 25 Overview of the averages and standard deviations of the ortho-aminobenzoic acid yields from four replicates in each case after 66.50 h. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Medium No. () Medium 1 Medium 2 Medium 3 Medium 4 Condition (% by wt. 100% by wt. of 64% by wt. of 75% by wt. of 88% by wt. of of sugars based on D-glucose, 0% D-glucose, 36% D-glucose, 25% D-glucose, 12% total sugar) by wt. of D- by wt. of D- by wt. of D- by wt. of D- xylose xylose xylose xylose Average yield after 0.087 0.002 0.106 0.002 0.102 0.002 0.095 0.001 cultivation for 66.50 h (g.sub.oAB/g.sub.C source consumed) Average yield after 0.083 0.002 0.089 0.001 0.094 0.002 0.090 0.001 cultivation for 66.50 h (g.sub.oAB/g.sub.C source initially charged) Average yield after 0.114 0.001 0.131 0.003 0.129 0.003 0.122 0.001 cultivation for 66.50 h (mol.sub.oAB/mol.sub.C source consumed) Average yield after 0.110 0.002 0.110 0.002 0.118 0.002 0.116 0.001 cultivation for 66.50 h (mol.sub.oAB/mol.sub.C source initially charged)

SUMMARY

[0069] In the shaken flask culturing described here of a xylose-metabolizing ortho-aminobenzoic acid producer based on C. glutamicum, the effect of various mixing ratios of glucose and xylose on the yield of ortho-aminobenzoic acid was examined.

[0070] By the time the cultures had ended, the xylose was in most cases still not yet fully consumed, and therefore a distinction was made in respect of the final product yield between the substrate yield based on the mass or molar amount of substrate consumed (g.sub.oAB/g.sub.C source consumed or mol.sub.oAB/mol.sub.C source consumed) and the process yield based on the mass or molar amount of the substrate initially charged (g.sub.oAB/g.sub.C source initially charged or mol.sub.oAB/mol.sub.C source initially charged).

[0071] It was found that the substrate yield was at its highest with use of medium 2 at 0.106 g.sub.oAB/g.sub.C source consumed or 0.129 mol.sub.oAB/mol.sub.C source consumed. The dry biomass concentration attained decreased with increasing xylose content. In respect of process yield, a maximum of 0.094 g.sub.oAB/g.sub.C source initially charged or 0.118 mol.sub.oAB/mol.sub.C source initially charged was achieved with medium 3, in which the energy source and carbon source used was composed to an extent of 76% by weight of glucose and 24% by weight of xylose.

Example 2

[0072] Comparative cultivations of the above-described strain for production of ortho-aminobenzoic acid were conducted proceeding from two different sugars in eight different ratios. The main culture media were produced here such that the two sugars D-glucose and D-xylose were present in amounts as in table 26, based on the total sugar content of 20 g/L.

TABLE-US-00026 TABLE 26 Overview of the compositions of the different sugars in the main cultivation media used. % by wt. of sugar (based on the total sugar content) Medium name D-Glucose D-Xylose Medium 1 100 0 Medium 2 86 14 Medium 3 76 24 Medium 4 63 37 Medium 5 50 50 Medium 6 39 61 Medium 7 27 73 Medium 8 14 86

[0073] For each condition and each ratio of amounts of sugar, four replicates were run. The cultivation conditions are shown below.

TABLE-US-00027 TABLE 27 Settings on the BioLector Pro for the cultivation of the 48-well microtiter plate. Parameter Setting Temperature ( C.) 30 Shake frequency (rpm) 1000 Culture time (h) 70.79 Air humidity (%) 85 Time between measurement cycles (min) 5 Oxygen in cultivation chamber (% by wt.) 21

TABLE-US-00028 TABLE 28 Layout of the culture plate of the M2P-MTP-48-BOH2 type. The same volume of 1 mL of the main culture was used at each position. 1 2 3 4 5 6 7 8 A Medium 1 Medium 1 Medium 1 Medium 1 Medium 1 Medium 1 Medium 2 Medium 2 B Medium 2 Medium 2 Medium 2 Medium 2 Medium 3 Medium 3 Medium 3 Medium 3 C Medium 3 Medium 3 Medium 4 Medium 4 Medium 4 Medium 4 Medium 4 Medium 4 D Medium 5 Medium 5 Medium 5 Medium 5 Medium 5 Medium 5 Medium 6 Medium 6 E Medium 6 Medium 6 Medium 6 Medium 6 Medium 7 Medium 7 Medium 7 Medium 7 F Medium 7 Medium 7 Medium 8 Medium 8 Medium 8 Medium 8 Medium 8 Medium 8

Media Used, and Composition and Production Thereof

[0074] Unless stated otherwise, all media were produced with ddH.sub.2O and autoclaved.

TABLE-US-00029 TABLE 29 Liquid and solid complex media composed of Brain Heart Infusion (BHI) for growing of cells. Addition before/ Medium Composition after the autoclave BHI broth 37 g/L BHI broth Before autoclaving BHI agar plates 52 g/L BHI agar Before autoclaving

TABLE-US-00030 TABLE 30 Liquid minimal medium with complex constituents for the growing of cells in the preparatory culture. The amounts weighed out are given for 1 L of complete CGXII preparatory culture medium. Once all the reagents have been supplemented in the medium, the final target concentrations are attained in the complete medium. Corresponding to final concentration in the Addition before/ Medium Composition complete medium after the autoclave CGXII preparatory 1 g KH.sub.2PO.sub.4 1 g/L Everything hereafter culture medium 1 g K.sub.2HPO.sub.4 1 g/L added before autoclaving. 10 g (NH.sub.4).sub.2SO.sub.4 10 g/L 5 g urea 5 g/L 62 g MOPS 62 g/L 5 g yeast extract 5 g/L 1 mL CaCl.sub.2 stock solution 1 mL/L Dissolve in 800 mL of ddH.sub.2O, adjust pH to pH = 7.0-7.5 with KOH pellets, make up to 940 mL with ddH.sub.2O and autoclave. 1.25 mL MgSO.sub.4 stock solution 1.25 ml/L Everything hereafter added after autoclaving: 1 mL trace element stock solution 1 mL/L 1 mL biotin stock solution 1 mL/L 33 mL D-glucose stock solution OR 20 g/L D-glucose OR 33 mL D-glucose and 20 g/L D-glucose and 16.5 mL D-xylose stock solution 10 g/L D-xylose 23.75 mL water OR 7.25 mL water

TABLE-US-00031 TABLE 31 Liquid minimal medium for the main culture. The amounts weighed out are given for 1 L of complete CGXII main culture medium. Once all the reagents have been supplemented in the medium, the final target concentrations are attained in the complete medium. Corresponding to final concentration in the Addition before/ Medium Composition complete medium after the autoclave CGXII main culture 1 g KH.sub.2PO.sub.4 1 g/L Everything hereafter medium 1 g K.sub.2HPO.sub.4 1 g/L added before autoclaving: 10 g (NH.sub.4).sub.2SO.sub.4 10 g/L 62 g MOPS 62 g/L 1 mL CaCl.sub.2 stock solution 1 mL/L Dissolve in 800 ml of ddH.sub.2O, adjust pH to pH = 7.0-7.5 with KOH pellets, make up to 940 mL with ddH.sub.2O and autoclave. 1.25 mL MgSO.sub.4 stock solution 1.25 mL/L Everything hereafter 1 mL trace element stock solution 1 mL/L added after autoclaving: 1 mL biotin stock solution 1 mL/L 33 mL D-glucose stock solution OR 20 g/L D-glucose OR 33 mL D-xylose stock solution 20 g/L D-xylose 23.75 mL water

TABLE-US-00032 TABLE 32 Overview of production of the 2 g/L biotin stock solution. Rather than being autoclaved, the solution is sterile-filtered (0.2 m). The solution can be stored at 4 C. for 1 month. Medium Composition Note Biotin stock solution 2 g/L biotin Solution is sterile-filtered. During the dissolving, the pH is measured while stirring and adjusted to pH = 7.2 with 1M KOH.

TABLE-US-00033 TABLE 33 Overview of production of the 600 g/L D-glucose stock solution. Medium Composition Note D-glucose stock 660 g D-glucose Substance is dissolved in 554 g of hot ddH.sub.2O. Total solution monohydrate weight of 1 L of the complete solution: 1214 g. Boil briefly for complete dissolution prior to autoclaving.

TABLE-US-00034 TABLE 34 Overview of production of the 600 g/L D-xylose stock solution. Medium Composition Note D-xylose 600 g Substance is dissolved in 595 g of hot ddH.sub.2O. stock D-xylose Total weight of 1 L of the complete solution: solution 1195 g. Boil briefly for complete dissolution prior to autoclaving.

TABLE-US-00035 TABLE 35 Overview of production of the 10 g/L CaCl.sub.2 stock solution. Medium Composition Note CaCl.sub.2 stock 10 g/L CaCl.sub.2 Solution need not be autoclaved since it solution 2 H.sub.2O is supplemented before the autoclaving operation of the CGXII media.

TABLE-US-00036 TABLE 36 Overview of production of the 200 g/L MgSO.sub.4 stock solution. Medium Composition Note MgSO.sub.4 stock 200 g/L MgSO.sub.4 7 H.sub.2O Solution is sterile-filtered. solution

TABLE-US-00037 TABLE 37 Overview of production of the 1000x trace element solution. Rather than being autoclaved, the solution is sterile-filtered (0.2 m). This solution has a shelf life of 6 months at 4 C. Because of the small weights, it is advisable to produce a 1 L batch. Medium Composition Note Trace element 10 g/L MnSO.sub.4 H.sub.2O Solution is sterile-filtered. solution 10 g/L FeSO.sub.4 7 H.sub.2O 1 g/L ZnSO.sub.4 7 H.sub.2O 0.2 g/L CuSO.sub.4 5 H.sub.2O 0.02 g/L NiCl.sub.2 6 H.sub.2O Dissolve in ddH.sub.2O and adjust pH with HCl to pH = 1.

TABLE-US-00038 TABLE 38 Overview of production of 1x phosphate buffer (PBS). 10x PBS (article number BP399-1) from Fisher Scientific GmbH was used. Medium Composition Note 1x phosphate buffer 10x PBS Dilute 1:10 with ddH.sub.2O, autoclave.

Instruments Used

TABLE-US-00039 TABLE 39 Overview of the parameters examined in this study and instruments and methods for examination thereof. Parameter Instrument Description Glucose concentration Cedex Bio Analyzer, Roche Cedex Bio Glucose assay with article number RD- 06343732001, used according to manufacturer's instructions NH.sub.3 concentration Cedex Bio Analyzer, Roche Cedex Bio NH3 assay with article number RD- 06343775001, used according to manufacturer's instructions ortho-Aminobenzoic acid HPLC, Agilent For separation and concentration G7104C 1260 flexible pump quantification of ortho- G7167A 1260 multisampler aminobenzoic acid in the G7116A 1260 multicolumn sterile-filtered supernatant of thermostat (MCT) the samples, an Agilent Eclipse G7117C 1260 DAD HS Plus C18 column (4.6 G7162A 1260 RID 150 mm, 5 m) and a Zorbax Eclipse Plus C18 precolumn cartridge (4.6 12 mm, 5 m, 5 mm) were used. The ortho- aminobenzoic acid was detected by means of the diode array detector (DAD). D-Xylose concentration HPLC, ROA For separation and HPLC, Agilent quantification of D-xylose in the G7104C 1260 flexible pump sterile-filtered supernatant of G7167A 1260 multisampler the samples, a Phenomenex G7116A 1260 multicolumn Rezex ROA-Organic Acid H+ 8 thermostat (MCT) column (300 7.8 mm, S/N G7117C 1260 DAD HS H20-094694) and a G7162A 1260 RID SecurityGuard Cartridge Carbo- H precolumn cartridge (4 3.0 mm) were used. The D- xylose was detected by means of the refractive index detector (RID). pH SevenCompact S210 pH meter, Mettler Toledo InLab Expert Pro-ISM pH electrode, Mettler Toledo Weight of dry biomass SARTORIUS CUBIS 225S Semi-Micro Balance, Sartorius

TABLE-US-00040 TABLE 40 Overview of the products from Beckman Coulter GmbH used for the culturing of the main cultures. Product Description Properties G-BLMF100 BioLector Pro system Integrated microfluidic system for active pH control and substrate feeding in the microtiter plate, 6 filters for the following parameters: biomass, pH, DO (dissolved oxygen), riboflavin (Ex 436 nm/Em 540 nm), LG1 (pH) and RF (DO); incubation chamber is equipped with sensors for moisture and temperature; moistening function for the incubation chamber (>75% by weight) E-O2-100 O2 enrichment module Module for enrichment of Rev10.3 oxygen (up to 35% by weight), including O2 sensor and pressure reducer E-OP-498 Configurable LED module BL II and Pro Configurable filter module for BioLector II and Pro: The wavelengths for emission and excitation may be between 365 and 800 nm (bandpass filter: 10 nm). M2P-MTP-48-BOH2 FlowerPlate MTP, pH/DO type 2 48-well FlowerPlate microtiter (LG1/RF) plate, transparent base, pH and DO optode type 2 (LG1/RF). M2P-F-GPR48-10 Sealing foil, Gas-permeable, Reduced Self-adhesive, gas-permeable evaporation sealing film, serves as sterile barrier and reduces evaporation

Procedure

[0075] A starter culture was generated by taking cell mass from dormant forms in glycerol of the microbial cultures used for the culturing, and these were used to inoculate a BHI agar plate. The BHI agar plate was incubated at 30 C. for 48 h.

[0076] The agar plate culture set up in this way was used to inoculate a BHI liquid culture. For this purpose, a little cell mass was taken from the BHI agar plate culture and 4 mL of liquid BHI medium was inoculated in a round-bottom tube. The liquid culture was incubated at 30 C. and 200 rpm for about 7.5 h (preparatory culture I).

[0077] For the second preparatory culture, two media with different carbon sources were used. For this purpose, CGXII preparatory culture medium was produced with either 20 g/L D-glucose or with 20 g/L D-glucose and additionally 10 g/L D-xylose, and 50 mL in each case was transferred into a 1 L Erlenmeyer flask. 2 mL of the liquid BHI preparatory culture was added to each of the two batches, and these two shaken flask preparatory cultures were incubated at 200 rpm and 30 C. for 17 h.

[0078] On completion of incubation of the second preparatory culture in the shaken flask, the optical density of the cultures, which either had only glucose available or had a mixture of glucose and xylose available as the carbon source, was measured. Depending on the cell density achieved, a proportion of these second preparatory cultures was taken, the cells were pelletized by centrifugation and washed in sterile phosphate buffer (singly concentrated), and resuspended in appropriate main culture medium (media 1-8).

TABLE-US-00041 TABLE 41 Measurement of optical density (OD.sub.600) on completion of incubation of the second preparatory culture in the shaken flask. The optical density measured was used to calculate what volume of the second preparatory culture is needed to inoculate 10 mL of main culture with an initial OD.sub.600 of 1. Since xylose-containing media with seven different xylose concentrations were to be tested, seven aliquots were taken for the inoculation from the preparatory culture with 20 g/L D-glucose and 10 g/L D-xylose. OD.sub.600 () Volume required 20 g/L D-glucose 23.24 1 430 L for medium 1 20 g/L D-glucose and 18.17 7 550 for media 2-8 10 g/L D-xylose

[0079] The preparatory culture with glucose as the sole carbon source and energy source was used for the production of the main culture with medium 1. By means of the preparatory culture with glucose and xylose, the main cultures were inoculated with media 2-8. In each case 61 mL (6 replicates for each ratio of amounts of sugar) of the main cultures produced in this way were pipetted into different positions of a culture plate, and the culture plate was sealed with a film and transferred into the BioLector Pro.

Results

D-Xylose Concentration

TABLE-US-00042 TABLE 42 Overview of the D-xylose concentrations for the six replicates with medium 1 and medium 2 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (86% by wt. of D-glucose, 0% by wt. of D-xylose) 14% by wt. of D-xylose) Position in MTP A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 D-Xylose after 0 0 0 0 0 0 2.983 2.983 2.983 2.983 2.983 2.983 cultivation for 0.00 h (g/L) D-Xylose after 0 0 0 0 0 0 0.041 0.044 0.051 0.049 0.044 0.200 cultivation for 70.79 h (g/L)

D-Xylose Concentration

TABLE-US-00043 TABLE 43 Overview of the D-xylose concentrations for the six replicates with medium 3 and medium 4 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 3 (76% by wt. of D-glucose, Medium 4 (63% by wt. of D-glucose, 24% by wt. of D-xylose) 37% by wt. of D-xylose) Position in MTP B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D-Xylose after 4.884 4.884 4.884 4.884 4.884 4.884 7.567 7.567 7.567 7.567 7.567 7.567 cultivation for 0.00 h (g/L) D-Xylose after 0.398 0.567 0.549 0.522 0.410 0.740 1.809 1.694 2.164 2.160 2.086 2.440 cultivation for 70.79 h (g/L)

D-Xylose Concentration

TABLE-US-00044 TABLE 44 Overview of the D-xylose concentrations for the six replicates with medium 1 and medium 2 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 5 (50% by wt. of D-glucose, Medium 6 (39% by wt. of D-glucose, 50% by wt. of D-xylose) 61% by wt. of D-xylose) Position in MTP D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 D-Xylose after 10.39 10.39 10.39 10.39 10.39 10.39 12.29 12.29 12.29 12.29 12.29 12.29 cultivation for 0.00 h (g/L) D-Xylose after 4.46 4.53 4.45 4.30 4.56 4.68 7.23 7.34 7.3 7.41 7.52 7.85 cultivation for 70.79 h (g/L)

D-Xylose Concentration

TABLE-US-00045 TABLE 45 Overview of the D-xylose concentrations for the six replicates with medium 7 and medium 8 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 7 (27% by wt. of D-glucose, Medium 8 (14% by wt. of D-glucose, 73% by wt. of D-xylose) 86% by wt. of D-xylose) Position in MTP E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 D-Xylose after 14.42 14.42 14.42 14.42 14.42 14.42 16.76 16.76 16.76 16.76 16.76 16.76 cultivation for 0.00 h (g/L) D-Xylose after 10.79 10.66 10.68 10.77 10.73 10.19 14.33 14.73 14.51 14.65 14.68 15.47 cultivation for 70.79 h (g/L)

D-Glucose Concentration

TABLE-US-00046 TABLE 46 Overview of the D-glucose concentrations for the six replicates with medium 1 and medium 2 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (86% by wt. of D-glucose, 0% by wt. of D-xylose) 14% by wt. of D-xylose) Position in MTP A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 D-Glucose 21.05 21.05 21.05 21.05 21.05 21.05 18.31 18.31 18.31 18.31 18.31 18.31 after cultivation for 0.00 h (g/L) D-Glucose 0 0 0 0 0 0 0 0 0 0 0 0 after cultivation for 70.79 h (g/L)

D-Glucose Concentration

TABLE-US-00047 TABLE 47 Overview of the D-glucose concentrations for the six replicates with medium 3 and medium 4 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 3 (76% by wt. of D-glucose, Medium 4 (63% by wt. of D-glucose, 24% by wt. of D-xylose) 37% by wt. of D-xylose) Position in MTP B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D-Glucose 15.74 15.74 15.74 15.74 15.74 15.74 13.06 13.06 13.06 13.06 13.06 13.06 after cultivation for 0.00 h (g/L) D-Glucose 0 0 0 0 0 0 0 0 0 0 0 0 after cultivation for 70.79 h (g/L)

D-Glucose Concentration

TABLE-US-00048 TABLE 48 Overview of the D-glucose concentrations for the six replicates with medium 5 and medium 6 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 5 (50% by wt. of D-glucose, Medium 6 (39% by wt. of D-glucose, 50% by wt. of D-xylose) 61% by wt. of D-xylose) Position in MTP D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 D-Glucose after 10.46 10.46 10.46 10.46 10.46 10.46 7.81 7.81 7.81 7.81 7.81 7.81 cultivation for 0.00 h (g/L) D-Glucose after 0 0 0 0 0 0 0 0 0 0 0 0 cultivation for 70.79 h (g/L)

D-Glucose Concentration

TABLE-US-00049 TABLE 49 Overview of the D-glucose concentrations for the six replicates with medium 7 and medium 8 at the start of cultivation and after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 7 (27% by wt. of D-glucose, Medium 8 (14% by wt. of D-glucose, 73% by wt. of D-xylose) 86% by wt. of D-xylose) Position in MTP E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 D-Glucose after 5.25 5.25 5.25 5.25 5.25 5.25 2.63 2.63 2.63 2.63 2.63 2.63 cultivation for 0.00 h (g/L) D-Glucose after 0 0 0 0 0 0 0 0 0 0 0 0 cultivation for 70.79 h (g/L)

Dry Biomass Concentration (DBM)

TABLE-US-00050 TABLE 50 Overview of the dry biomass concentrations for the six replicates with medium 1 and medium 2 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (86% by wt. of D-glucose, 0% by wt. of D-xylose) 14% by wt. of D-xylose) Position in MTP A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 DBM after 6.71 5.72 5.63 5.69 6.12 5.96 5.05 5.09 4.56 5.20 5.49 4.76 cultivation for 70.79 h (g/L)

Dry Biomass Concentration (DBM)

TABLE-US-00051 TABLE 51 Overview of the dry biomass concentrations for the six replicates with medium 3 and medium 4 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 3 (76% by wt. of D-glucose, Medium 4 (63% by wt. of D-glucose, 24% by wt. of D-xylose) 37% by wt. of D-xylose) Position in MTP B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 DBM after 5.37 4.95 5.36 4.55 5.37 6.17 5.11 4.75 4.64 4.79 5.56 4.11 cultivation for 70.79 h (g/L)

Dry Biomass Concentration (DBM)

TABLE-US-00052 TABLE 52 Overview of the dry biomass concentrations for the six replicates with medium 5 and medium 6 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 5 (50% by wt. of D-glucose, Medium 6 (39% by wt. of D-glucose, 50% by wt. of D-xylose) 61% by wt. of D-xylose) Position in MTP D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 DBM after 5.33 4.43 4.01 4.27 4.19 4.73 3.88 2.09 3.52 2.63 3.57 3.19 cultivation for 70.79 h (g/L)

Dry Biomass Concentration (DBM)

TABLE-US-00053 TABLE 53 Overview of the dry biomass concentrations for the six replicates with medium 7 and medium 8 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 7 (27% by wt. of D-glucose, Medium 8 (14% by wt. of D-glucose, 73% by wt. of D-xylose) 86% by wt. of D-xylose) Position in MTP E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 DBM after 2.68 2.37 2.37 2.29 2.84 3.68 #NV 1.49 2.63 1.17 1.56 1.49 cultivation for 70.79 h (g/L)
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00054 TABLE 54 Overview of the ortho-aminobenzoic acid concentration for the six replicates with medium 1 and medium 2 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (86% by wt. of D-glucose, 0% by wt. of D-xylose) 14% by wt. of D-xylose) Position in MTP A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 oAB after 2.258 2.295 2.341 2.245 2.345 2.322 2.464 2.450 2.427 2.409 2.450 2.432 cultivation for 70.79 h (g/L)
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00055 TABLE 55 Overview of the ortho-aminobenzoic acid concentration for the six replicates with medium 3 and medium 4 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 3 (76% by wt. of D-glucose, Medium 4 (63% by wt. of D-glucose, 24% by wt. of D-xylose) 37% by wt. of D-xylose) Position in MTP B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 oAB after 2.425 2.469 2.430 2.448 2.441 2.400 2.341 2.336 2.343 2.315 2.357 2.345 cultivation for 70.79 h (g/L)
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00056 TABLE 56 Overview of the ortho-aminobenzoic acid concentration for the six replicates with medium 5 and medium 6 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 5 (50% by wt. of D-glucose, Medium 6 (39% by wt. of D-glucose, 50% by wt. of D-xylose) 61% by wt. of D-xylose) Position in MTP D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 oAB after 2.119 2.103 2.098 2.085 2.082 2.137 1.783 1.758 1.778 1.758 1.717 1.719 cultivation for 70.79 h (g/L)
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00057 TABLE 57 Overview of the ortho-aminobenzoic acid concentration for the six replicates with medium 7 and medium 8 after 70.79 h. Condition (% by wt. of sugars based on total sugar) Medium 7 (27% by wt. of D-glucose, Medium 8 (14% by wt. of D-glucose, 73% by wt. of D-xylose) 86% by wt. of D-xylose) Position in MTP E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 oAB after 1.305 1.323 1.327 1.320 1.375 1.397 0.876 0.790 0.821 0.801 0.771 0.773 cultivation for 70.79 h (g/L)

Final Product Yield

TABLE-US-00058 TABLE 58 Overview of the ortho-aminobenzoic acid yields after 70.79 h for six replicates with medium 1 and medium 2. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 1 (100% by wt. of D-glucose, Medium 2 (86% by wt. of D-glucose, 0% by wt. of D-xylose) 14% by wt. of D-xylose) Position in MTP A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 Yield after 0.107 0.109 0.111 0.107 0.111 0.110 0.116 0.115 0.114 0.113 0.115 0.115 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.107 0.109 0.111 0.107 0.111 0.110 0.116 0.115 0.114 0.113 0.115 0.114 cultivation for 70.79 h (go.sub.AB/ g.sub.C source initially charged) Yield after 0.141 0.143 0.146 0.140 0.146 0.145 0.148 0.147 0.146 0.145 0.147 0.148 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.141 0.143 0.146 0.140 0.146 0.145 0.148 0.147 0.146 0.145 0.147 0.146 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source initially charged)

Final Product Yield

TABLE-US-00059 TABLE 59 Overview of the ortho-aminobenzoic acid yields after 70.79 h for six replicates with medium 3 and medium 4. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 3 (76% by wt. of D-glucose, Medium 4 (63% by wt. of D-glucose, 24% by wt. of D-xylose) 37% by wt. of D-xylose) Position in MTP B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 Yield after 0.120 0.123 0.121 0.122 0.121 0.121 0.124 0.123 0.127 0.125 0.127 0.129 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.118 0.120 0.118 0.119 0.118 0.116 0.113 0.113 0.114 0.112 0.114 0.114 cultivation for 70.79 h (go.sub.AB/ g.sub.C source initially charged) Yield after 0.151 0.155 0.152 0.153 0.152 0.152 0.154 0.153 0.157 0.156 0.158 0.160 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.147 0.150 0.148 0.149 0.148 0.146 0.139 0.139 0.139 0.137 0.140 0.139 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source initially charged)

Final Product Yield

TABLE-US-00060 TABLE 60 Overview of the ortho-aminobenzoic acid yields after 70.79 h for six replicates with medium 5 and medium 6. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 5 (50% by wt. of D-glucose, Medium 6 (39% by wt. of D-glucose, 50% by wt. of D-xylose) 61% by wt. of D-xylose) Position in MTP D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 Yield after 0.129 0.129 0.128 0.126 0.128 0.132 0.139 0.138 0.139 0.139 0.136 0.140 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.102 0.101 0.101 0.100 0.100 0.103 0.089 0.087 0.088 0.087 0.085 0.086 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source initially charged) Yield after 0.158 0.158 0.157 0.154 0.157 0.162 0.169 0.168 0.170 0.169 0.167 0.172 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.121 0.120 0.120 0.119 0.119 0.122 0.104 0.102 0.104 0.102 0.100 0.100 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source initially charged)

Final Product Yield

TABLE-US-00061 TABLE 61 Overview of the ortho-aminobenzoic acid yields after 70.79 h for six replicates with medium 7 and medium 8. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Condition (% by wt. of sugars based on total sugar) Medium 7 (27% by wt. of D-glucose, Medium 8 (14% by wt. of D-glucose, 73% by wt. of D-xylose) 86% by wt. of D-xylose) Position in MTP E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 Yield after 0.147 0.147 0.148 0.148 0.154 0.147 0.173 0.170 0.168 0.169 0.164 0.198 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.066 0.067 0.067 0.067 0.070 0.071 0.045 0.041 0.042 0.041 0.040 0.040 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source initially charged) Yield after 0.178 0.178 0.179 0.180 0.187 0.178 0.208 0.205 0.203 0.204 0.198 0.244 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.076 0.077 0.077 0.077 0.080 0.081 0.051 0.046 0.047 0.046 0.045 0.045 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source initially charged)

Averages of the Final Product Yields and Standard Deviations

TABLE-US-00062 TABLE 62 Overview of the averages and standard deviations of the ortho-aminobenzoic acid yields from six replicates in each case after 70.79 h. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Medium No. () Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 6 Medium 7 Medium 8 Condition (% by wt. of sugars based on total sugar) 100% by 86% by 76% by 63% by 50% by 39% by 27% by 14% by wt. of wt. of wt. of wt. of wt. of wt. of wt. of wt. of D-glucose, D-glucose, D-glucose, D-glucose, D-glucose, D-glucose, D-glucose, D-glucose, 0% by wt. 14% by wt. 24% by wt. 37% by wt. 50% by wt. 61% by wt. 73% by wt. 86% by wt. of D-xylose of D-xylose of D-xylose of D-xylose of D-xylose of D-xylose of D-xylose of D-xylose Average yield 0.109 0.115 0.121 0.126 0.129 0.139 0.149 0.174 after 0.002 0.001 0.001 0.002 0.002 0.001 0.003 0.012 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source consumed) Average yield 0.109 0.115 0.118 0.113 0.101 0.087 0.068 0.042 after 0.002 0.001 0.001 0.001 0.001 0.001 0.002 0.002 cultivation for 70.79 h (g.sub.oAB/ g.sub.C source initially charged) Average yield 0.144 0.147 0.153 0.156 0.158 0.169 0.180 0.210 after 0.003 0.001 0.001 0.003 0.003 0.002 0.003 0.017 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source consumed) Average yield 0.144 0.146 0.148 0.139 0.121 0.102 0.078 0.047 after 0.003 0.001 0.001 0.001 0.001 0.002 0.002 0.002 cultivation for 70.79 h (mol.sub.oAB/ mol.sub.C source initially charged)

SUMMARY

[0080] In the culturing described here of a xylose-metabolizing ortho-aminobenzoic acid producer based on C. glutamicum in microtiter plate format, the effect of various mixing ratios of glucose and xylose on the yield of ortho-aminobenzoic acid was examined over an extended mixing range.

[0081] By the time the cultures had ended, the xylose was in most cases still not yet fully consumed, and therefore a distinction was made in respect of the final product yield between the substrate yield based on the mass or molar amount of substrate consumed (g.sub.oAB/g.sub.C source consumed or mol.sub.oAB/mol.sub.C source consumed) and the process yield based on the mass or molar amount of the substrate initially charged (g.sub.oAB/g.sub.C source initially charged or mol.sub.oAB/mol.sub.C source initially charged).

[0082] It was found that metabolic yield rose with increasing proportion of xylose. In respect of process yield, a maximum of 0.153 g.sub.oAB/g.sub.C source initially charged or 0.148 mol.sub.oAB/mol.sub.C source initially charged was achieved with medium 3, in which the energy source and carbon source used was composed to an extent of 76% by weight of glucose and 24% by weight of xylose.

[0083] In this study, it was thus found that, surprisingly, the combination of D-glucose and D-xylose in mixtures having a glucose content of 86% to 14% by weight and a xylose content between 14% and 86% by weight leads to more efficient ortho-aminobenzoic acid product formation than the pure use of D-glucose as carbon source and energy source for production thereof.

Example 3

[0084] In this example, it was examined whether the use of a longer (SEQ ID NO. 13) or shorter (SEQ ID NO. 12) variant of xylose isomerase xylA (xcc1758) from Xanthomonas campestris pv. campestris leads to the same results. For this purpose, variants of the above-describe strain were used, which differ solely in the variants of xylose isomerase present. The strain containing the extended variant of the enzyme as also used in examples 1 and 2 is referred to hereinafter as Gizmo, and the strain containing the shorter variant instead as Geronimo.

Media

[0085] The media used in this example 3 can be found in tables 3 to 12 or tables 29 to 38 and table 63.

TABLE-US-00063 TABLE 63 Overview of production of SY medium. The medium is made up with ddH.sub.2O and autoclaved. Addition before/ Medium Composition after the autoclave SY medium 16 g/L soy peptone Before autoclaving 5 g/L sodium chloride Before autoclaving 10 g/L yeast extract Before autoclaving pH with KOH to pH = 7.2 Before autoclaving Glucose (final After autoclaving concentration: 16 g/L)

[0086] The cultivation parameters corresponded to those from table 27, except that the cultivation time in example 3 was 70.95 h.

Instruments Used

[0087] Apart from the measurement of dry biomass, in example 3, the parameters from table 39 were analyzed with the aid of the instruments from table 39, and the instruments in table 40 were used for the culturing.

Procedure

[0088] A starter culture was generated by taking cell mass from dormant forms in glycerol of the microbial cultures used for the culturing, and these were used to inoculate a BHI agar plate. The BHI agar plate was incubated at 30 C. for 48 h and stored if necessary at 4 C. until further use.

[0089] The agar plate culture set up in this way was used to inoculate an SY liquid culture. For this purpose, a little cell mass was taken from the BHI agar plate culture and 4 mL of liquid SY medium was inoculated in a round-bottom tube. The liquid culture was incubated at 30 C. and 200 rpm for about 7.5 h (preparatory culture I).

[0090] For the second preparatory culture, CGXII preparatory culture medium with 15 g/L D-glucose and 5 g/L D-xylose was produced, and 24 mL was transferred into each of two 500 mL Erlenmeyer flasks. 1 ml of the liquid SY preparatory culture was added to each of the two batches, and these two shaken flask preparatory cultures were incubated at 200 rpm and 30 C. for 17 h.

[0091] On completion of incubation of the second preparatory cultures in the shaken flask, the optical density of the cultures, which had a mixture of glucose and xylose available as the carbon source, was measured. Depending on the cell density achieved, a proportion of these second preparatory cultures was taken, the cells were pelletized by centrifugation and washed in sterile phosphate buffer (singly concentrated), and resuspended in appropriate main culture medium.

TABLE-US-00064 TABLE 64 Measurement of optical density (OD.sub.600) in double determination on completion of incubation of the second preparatory cultures in the shaken flask. The averages of the optical density measured were used to calculate what volume of the second preparatory culture is needed in each case to inoculate 25 mL of main culture of C. glutamicum Gizmo and C. glutamicum Geronimo with an initial OD.sub.600 of 1. Preparatory OD.sub.600 OD.sub.600 Average Volume culture II () () OD .sub.600 () required (mL) C. glutamicum Gizmo 17 16 16.5 1.52 C. glutamicum 16 17 16.5 1.52 Geronimo

[0092] The main cultures of C. glutamicum Gizmo and C. glutamicum Geronimo that had been produced in this way had an identical initial sugar ratio of 76% by weight of D-glucose and 24% by weight of D-xylose based on total sugar. Aliquots of these main cultures were pipetted into different positions of a culture plate, and the culture plate was sealed with a film and transferred into the BioLector pro.

Results

D-Xylose Concentration

TABLE-US-00065 TABLE 65 Overview of the D-xylose concentrations at the start and at the end of culturing for three replicates in each case of C. glutamicum Gizmo and C. glutamicum Geronimo. The medium contained initial sugar contents of 76% by weight of D-glucose and 24% by weight of D-xylose based on total sugar. The initial D-xylose concentration was determined in uninoculated main culture medium. Name of culture, condition (% by wt. of sugars based on total sugar) C. glutamicum Gizmo, C. glutamicum Geronimo, 76% by wt. of D-glucose, 76% by wt. of D-glucose, 24% by wt. of D-xylose 24% by wt. of D-xylose Replicate () 1 2 3 1 2 3 Culture time D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose D-Xylose (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 4.93 4.93 4.93 4.93 4.93 4.93 70.95 0.64 0.70 0.81 0.34 0.18 0.19

D-Glucose Concentration

TABLE-US-00066 TABLE 66 Overview of the D-glucose concentrations at the start and at the end of culturing for three replicates in each case of C. glutamicum Gizmo and C. glutamicum Geronimo. The medium contained initial sugar contents of 76% by weight of D-glucose and 24% by weight of D-xylose based on total sugar. The initial D-glucose concentration was determined in uninoculated main culture medium. Name of culture, condition (% by wt. of sugars based on total sugar) C. glutamicum Gizmo, C. glutamicum Geronimo, 76% by wt. of D-glucose, 76% by wt. of D-glucose, 24% by wt. of D-xylose 24% by wt. of D-xylose Replicate () 1 2 3 1 2 3 Culture time D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose D-Glucose (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 15.63 15.63 15.63 15.63 15.63 15.63 70.95 0.00 0.00 0.00 0.00 0.00 0.00
Ortho-Aminobenzoic Acid Concentration (oAB)

TABLE-US-00067 TABLE 67 Overview of the ortho-aminobenzoic acid concentrations (oAB) at the start and at the end of culturing for three replicates in each case of C. glutamicum Gizmo and C. glutamicum Geronimo. The medium contained initial sugar contents of 76% by weight of D-glucose and 24% by weight of D-xylose based on total sugar. Name of culture, condition (% by wt. of sugars based on total sugar) C. glutamicum Gizmo, C. glutamicum Geronimo, 76% by wt. of D-glucose, 76% by wt. of D-glucose, 24% by wt. of D-xylose 24% by wt. of D-xylose Replicate () 1 2 3 1 2 3 Culture time oAB oAB oAB oAB oAB oAB (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) 0.00 0 0 0 0 0 0 70.95 2.46 2.49 2.48 2.45 2.42 2.43

Final Product Yield

TABLE-US-00068 TABLE 68 Overview of the ortho-aminobenzoic acid yields after 70.95 h for three replicates in each case of C. glutamicum Gizmo and C. glutamicum Geronimo in medium with initial sugar contents of 74% by weight of D-glucose and 26% by weight of D- xylose based on total sugar. These are reported as the mass-based (g.sub.oAB/g.sub.C source) and molar amount-based quotient (mol.sub.oAB/mol.sub.C source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. Name of culture, condition (% by wt. of sugars based on total sugar) C. glutamicum Gizmo, C. glutamicum Geronimo, 76% by wt. of D-glucose, 76% by wt. of D-glucose, 24% by wt. of D-xylose 24% by wt. of D-xylose Replicate () 1 2 3 1 2 3 Yield after 0.124 0.125 0.126 0.121 0.119 0.119 cultivation for 70.95 h (g.sub.oAB/ g.sub.C source consumed) Yield after 0.120 0.121 0.121 0.119 0.118 0.118 cultivation for 70.95 h (g.sub.oAB/ g.sub.C source initially charged) Yield after 0.156 0.158 0.158 0.152 0.149 0.150 cultivation for 70.95 h (mol.sub.oAB/ mol.sub.C source consumed) Yield after 0.150 0.152 0.151 0.149 0.147 0.148 cultivation for 70.95 h (mol.sub.oAB/ mol.sub.C source initially charged)

Averages of the Final Product Yields and Standard Deviations

TABLE-US-00069 TABLE 69 Overview of the averages and standard deviations of the ortho-aminobenzoic acid yields from three replicates in each case of C. glutamicum Gizmo and C. glutamicum Geronimo in medium with initial sugar contents of 74% by weight of D-glucose and 26% by weight of D-xylose based on total sugar after 70.95 h. These are reported as the mass-based (goAB/gc source) and molar amount-based quotient (moloAB/molc source), including firstly the metabolized carbon source and secondly the carbon source included in the initial charge of medium in the calculation. C. glutamicum C. glutamicum Name of culture, Gizmo, Geronimo, condition (% by wt. of 76% by wt. of D- 76% by wt. of D- sugars based on total glucose, 24% by wt. glucose, 24% by wt. sugar) of D-xylose of D-xylose Average yield after 0.125 0.001 0.120 0.001 cultivation for 70.95 h (goAB/gC source consumed) Average yield after 0.120 0.001 0.118 0.001 cultivation for 70.95 h (goAB/ gC source initially charged) Average yield after 0.157 0.001 0.150 0.002 cultivation for 70.95 h (moloAB/molc source consumed) Average yield after 0.151 0.001 0.148 0.000 cultivation for 70.95 h (moloAB/molc source initially charged)

SUMMARY

[0093] In the culturing described here of two xylose-metabolizing ortho-aminobenzoic acid producers based on C. glutamicum in microtiter plate format, the effect of various xylose isomerase variants on the yield of ortho-aminobenzoic acid was examined.

[0094] Example 3 shows that the use of the two different variants of xylose isomerase does not lead to relevant differences in the performance of the strains. Both variants are thus equivalent in the context of the present invention.