Method for producing 3-Oxoadipic acid

Abstract

A method of producing 3-oxoadipic acid from an aliphatic compound easily utilizable by a microorganism, such as a saccharide, by utilization of a metabolic pathway of the microorganism is disclosed. The method of producing 3-oxoadipic acid includes the step of culturing at least one type of microorganism having a capacity to produce 3-oxoadipic acid, selected from the group consisting of, for example, microorganisms belonging to the genus Serratia, microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Hafnia, microorganisms belonging to the genus Bacillus, microorganisms belonging to the genus Escherichia, microorganisms belonging to the genus Pseudomonas, microorganisms belonging to the genus Acinetobacter, microorganisms belonging to the genus Alcaligenes, microorganisms belonging to the genus Shimwellia, microorganisms belonging to the genus Planomicrobium, microorganisms belonging to the genus Nocardioides, microorganisms belonging to the genus Yarrowia, microorganisms belonging to the genus Cupriavidus, microorganisms belonging to the genus Rhodosporidium, microorganisms belonging to the genus Streptomyces, and microorganisms belonging to the genus Microbacterium.

Claims

1. A method of producing 3-oxoadipic acid, said method comprising the steps of: culturing at least one type of microorganism having a capacity to produce 3-oxoadipic acid in a medium through a naturally occurring metabolic pathway, selected from the group consisting of Escherichia coli and Escherichia fergusonii, and recovering the produced 3-oxoadipic acid, wherein the microorganism metabolizes succinic acid into said 3-oxoadipic acid, and wherein said microorganism is cultured in a medium containing at least one inducer selected from the group consisting of ferulic acid and p-coumaric acid.

2. The method according to claim 1, wherein the medium for culturing said microorganism contains at least one carbon source selected from the group consisting of saccharides, succinic acid, 2-oxoglutaric acid, and glycerol.

3. A method of producing 3-oxoadipic acid, said method comprising the step of: culturing at least one type of microorganism having a capacity to produce 3-oxoadipic acid in a medium through a naturally occurring metabolic pathway, selected from the group consisting of Escherichia coli and Escherichia fergusonii, wherein the microorganism metabolizes succinic acid into said 3-oxoadipic acid, and wherein said microorganism is cultured in a medium containing at least one inducer selected from the group consisting of ferulic acid and p-coumaric acid.

Description

EXAMPLES

(1) The present invention is described below concretely by way of Examples. However, the present invention is not limited to these.

Reference Example 1 Providing 3-Oxoadipic Acid

(2) For use in quantitative analysis of 3-oxoadipic acid produced by microorganisms, samples of the substance were provided by chemical synthesis.

(3) First, 1.5 L of super-dehydrated tetrahydrofuran (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 13.2 g (0.1 mol) of succinic acid monomethyl ester (manufactured by Wako Pure Chemical Industries, Ltd.), and 16.2 g (0.1 mol) of carbonyldiimidazole (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto with stirring, followed by stirring the resulting mixture under nitrogen atmosphere for 1 hour at room temperature. To this suspension, 15.6 g (0.1 mol) of malonic acid monomethyl ester potassium salt and 9.5 g (0.1 mol) of magnesium chloride were added. The resulting mixture was stirred under a nitrogen atmosphere for 1 hour at room temperature, and then stirred at 40° C. for 12 hours. After the reaction, 0.05 L of 1 mol/L hydrochloric acid was added to the mixture at room temperature, and extraction with ethyl acetate was carried out. By separation purification by silica gel column chromatography (hexane:ethyl acetate=1:5), 13.1 g of pure 3-oxohexanedicarboxylic acid dimethyl ester was obtained. Yield: 70%.

(4) To 5 g (0.026 mol) of the 3-oxohexanedicarboxylic acid dimethyl ester obtained, 26 mL of methanol (manufactured by Kokusan Chemical Co., Ltd.) was added, and 12 mL of 5 mol/L aqueous sodium hydroxide solution was added to the resulting mixture with stirring, followed by stirring the mixture at room temperature overnight. After completion of the reaction, 12 mL of 5 mol/L hydrochloric acid was added to the reaction product, and extraction with 100 mL of ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was carried out. After concentrating the resulting extract using a rotary evaporator, recrystallization with acetone/petroleum ether was carried out to obtain 2 g of pure 3-oxoadipic acid.

(5) Yield: 47%.

(6) .sup.1H-NMR Spectrum of 3-Oxoadipic Acid:

(7) .sup.1H-NMR (400 MHz, D.sub.2O): δ2.62 (t, 2H), δ2.88 (t, 2H), δ3.73 (s, 1H).

Example 1 3-Oxoadipic Acid Production Test Using Aliphatic Compound

(8) [Microbial Culture]

(9) The 3-oxoadipic acid productivities of the microorganisms shown in the following Table 1 (all microorganisms were purchased from microorganism-distributing agencies; the distributors are described in the strain names) were investigated. To 5 mL of a medium prepared such that it contains 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride, and, as inducers, 2.5 mM each of benzoic acid, cis,cis-muconic acid, terephthalic acid, protocatechuic acid, catechol, adipic acid, phenylalanine, and phenethylamine, wherein the pH was adjusted to 7, a loopful of each microorganism was inoculated. Shake culture was then carried out at 30° C. until the microorganism was sufficiently suspended (preculture). To the culture liquid, 10 mL of 0.9% sodium chloride was added, and the microbial cells were centrifuged, followed by completely removing the supernatant, thereby washing the microbial cells. After carrying out this operation three times, the microbial cells were suspended in 1 mL of 0.9% sodium chloride. To 5 mL of the medium having the following composition containing aliphatic compounds as carbon sources, 0.5 mL of the resulting suspension was added, and shake culture was performed at 30° C. for 20 hours (main culture). The main culture liquid was subjected to centrifugation to separate microbial cells, and the resulting supernatant was analyzed by LC-MS/MS.

(10) Medium Composition for the Main Culture

(11) 10 g/L succinic acid 10 g/L glucose 10 g/L glycerol 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 2.5 g/L Bacto tryptone 1.25 g/L yeast extract pH 6.5.
[Quantitative Analysis of 3-Oxoadipic Acid]

(12) Quantitative analysis of 3-oxoadipic acid by LC-MS/MS was carried out under the following conditions. HPLC: 1290 Infinity (manufactured by Agilent Technologies) Column: Synergi hydro-RP (manufactured by Phenomenex); length, 100 mm; inner diameter, 3 mm; particle size, 2.5 μm Mobile phase: 0.1% aqueous formic acid solution/methanol=70/30 Flow rate: 0.3 mL/minute Column temperature: 40° C. LC detector: DAD (210 nm) MS/MS: Triple-Quad LC/MS (manufactured by Agilent Technologies) Ionization method: ESI negative mode.

(13) The concentration of 3-oxoadipic acid accumulated in the culture supernatant was as shown in Table 1. It was able to be confirmed that all microorganisms have a capacity to produce 3-oxoadipic acid.

(14) TABLE-US-00001 TABLE 1 3-Oxoadipic acid Test microorganism (mg/L) Serratia plymuthica NBRC102599 350 Serratia grimesii NBRC13537 250 Serratia ficaria NBRC102596 230 Corynebacterium glutamicum ATCC13826 190 Corynebacterium glutamicum ATCC21492 180 Corynebacterium glutamicum ATCC13032 116 Corynebacterium acetoacidophilum ATCC 21270 45 Pseudomonas putida NBRC3738 43 Corynebacterium acetoglutamicum ATCC 15806 27 Pseudomonas fragi NBRC3458 25 Bacillus megaterium ATCC10778 22 Corynebacterium ammoniagenes ATCC 6871 13 Hafnia alvei NBRC3731 10 Acinetobacter radioresistens NBRC102413 8.0 Pseudomonas reptilivora ATCC14039 7.4 Bacillus badius ATCC 14574 6.8 Escherichia fergusonii NBRC102419 6.3 Alcaligenes faecalis NBRC13111 6.2 Shimwellia blattae NBRC105725 5.8 Pseudomonas fluorescens NBRC3925 5.0 Planomicrobium okeanokoites NBRC12536 3.9 Nocardioides albus NBRC13917 3.4 Yarrowia lipolytica NBRC0717 3.1 Escherichia coli NBRC12713 2.4 Pseudomonas azotoformans NBRC12693 2.4 Cupriavidus necator NBRC102504 2.1 Rhodosporidium toruloides ATCC 10788 1.2 Streptomyces olivaceus NBRC3049 1.0

Example 2 3-Oxoadipic Acid Production Test Using Single Aliphatic Compound

(15) Each of S. plymuthica NBRC102599, C. glutamicum ATCC13826, and P. fragi NBRC3458 was cultured under the same conditions as in Example 1 except that 10 g/L of one of succinic acid, glucose, and glycerol was included as a sole carbon source in the main culture. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results for the three strains of microorganisms are shown in Tables 2 to 4, respectively.

(16) TABLE-US-00002 TABLE 2 Carbon source 3-Oxoadipic acid (mg/L) Succinic acid 16 Glucose 16 Glycerol 11

(17) TABLE-US-00003 TABLE 3 Carbon source 3-Oxoadipic acid (mg/L) Succinic acid 10 Glucose 10 Glycerol 5.3

(18) TABLE-US-00004 TABLE 4 Carbon source 3-Oxoadipic acid (mg/L) Succinic acid 3.2 Glucose 2.6 Glycerol 1.1

Reference Example 2 Growth Test Using Single Aliphatic Compound

(19) A loopful of each of S. plymuthica NBRC102599, C. glutamicum ATCC13826, and P. fragi NBRC3458 was inoculated to a growth test medium containing 10 g/L of one of the aliphatic compounds shown in Tables 5 to 7 as a sole carbon source in the culture, and shake culture was carried out at 30° C. Two days after the beginning of the culture, the turbidity (McFarland units) of the culture liquid was measured using a densitometer DEN-1B (Wakenbtech Co., Ltd.). At the same time, culture was carried out to provide a control without addition of a carbon source. The difference, from the control, in the turbidity was calculated. The results for the three strains of microorganisms are shown in Tables 5 to 7, respectively.

(20) Growth Test Medium Composition (S. plymuthica and P. fragi)

(21) 10 g/L carbon source 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride pH 6.5.
Growth Test Medium Composition (C. glutamicum) 10 g/L carbon source 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 0.03 mg/L biotin 1 mg/L thiamine hydrochloride 1 mg/L protocatechuic acid pH 6.5.

(22) TABLE-US-00005 TABLE 5 McFarland units Carbon source (Difference from control) Succinic acid >6 Glucose 5.8 Xylose >6 Glycerol 1.9 Acetic acid 0.9 Galactitol −0.5 Ethylene glycol −0.3 D(−)-Tartaric acid −0.2

(23) TABLE-US-00006 TABLE 6 McFarland units Carbon source (Difference from control) Succinic acid 4.7 Glucose 5.4 Xylose 0.0 Glycerol 3.7 Acetic acid 5.9 Galactitol −0.4 Ethylene glycol −0.4 D(−)-Tartaric acid −0.4

(24) TABLE-US-00007 TABLE 7 McFarland units Carbon source (Difference from control) Succinic acid 3.0 Glucose 1.2 Xylose 1.0 Glycerol 0.9 Acetic acid 1.1 Galactitol 0.0 Ethylene glycol −0.2 D(-)-Tartaric acid −0.3

Example 3 3-Oxoadipic Acid Production Test Using Two Kinds of Aliphatic Compounds

(25) Each of S. plymuthica NBRC102599, C. glutamicum ATCC13826, and P. fragi NBRC3458 was cultured under the same conditions as in Example 1 except that 10 g/L each of two kinds of aliphatic compounds shown in Tables 8 to 10 were contained as carbon sources in the main culture. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. At the same time, culture was carried out to provide a control using only succinic acid as a carbon source. The difference, from the control, in the concentration of 3-oxoadipic acid accumulated was calculated. The results for the three strains of microorganisms are shown in Tables 8 to 10, respectively.

(26) TABLE-US-00008 TABLE 8 3-Oxoadipic acid (mg/L) Carbon source (Difference from control) Glucose Glycerol 5.0 Succinic acid Glucose 150 Succinic acid Xylose 120 Succinic acid Glycerol 79 Succinic acid Acetic acid 13 Succinic acid Galactitol −4.6 Succinic acid Ethylene glycol −5.2 Succinic acid D(-)-Tartaric acid −4.5

(27) TABLE-US-00009 TABLE 9 3-Oxoadipic acid (mg/L) Carbon source (Difference from control) Glucose Glycerol 2.3 Succinic acid Glucose 180 Succinic acid Xylose −1.2 Succinic acid Glycerol 26 Succinic acid Acetic acid 13 Succinic acid Galactitol −1.2 Succinic acid Ethylene glycol −1.2 Succinic acid D(-)-Tartaric acid −1.2

(28) TABLE-US-00010 TABLE 10 3-Oxoadipic acid (mg/L) Carbon source (Difference from control) Glucose Glycerol 1.5 Succinic acid Glucose 33 Succinic acid Xylose 17 Succinic acid Glycerol 11 Succinic acid Acetic acid 6.1 Succinic acid Galactitol 0.0 Succinic acid Ethylene glycol −0.7 Succinic acid D(-)-Tartaric acid −0.1

Example 4 3-Oxoadipic Acid Production Test Using Single Inducer

(29) Each of S. plymuthica NBRC102599, C. glutamicum ATCC13826, and P. fragi NBRC3458 was precultured using media containing 2.5 mM of a compound shown in Tables 11 to 13 as an inducer, or a medium containing no inducer. Culture was performed under the same conditions as in Example 1 except that a medium containing 10 g/L each of succinic acid and glucose as carbon sources was used in the main culture. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results for the three strains of microorganisms are shown in Tables 11 to 13, respectively.

(30) TABLE-US-00011 TABLE 11 Inducer 3-Oxoadipic acid (mg/L) No addition 16 Benzoic acid 53 Catechol 35 Protocatechuic acid 90 Adipic acid 55

(31) TABLE-US-00012 TABLE 12 Inducer 3-Oxoadipic acid (mg/L) No addition 49 Benzoic acid 120 Catechol 110 Protocatechuic acid 52 Adipic acid 82

(32) TABLE-US-00013 TABLE 13 Inducer 3-Oxoadipic acid (mg/L) No addition 9.2 Benzoic acid 9.6 Catechol 15 Protocatechuic acid 15 Adipic acid 20

Example 5 Production Example of 3-Oxoadipic Acid

(33) To 5 mL of LB medium, a loopful of Serratia plymuthica NBRC102599, which was able to be confirmed to be a microorganism having a capacity to produce 3-oxoadipic acid in Example 1, was inoculated, and shake culture was carried out at 30° C. until the microorganism was sufficiently suspended (pre-preculture). To 100 mL of a medium containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride, 2.5 mM benzoic acid, 2.5 mM catechol, 2.5 mM cis,cis-muconic acid, 2.5 mM terephthalic acid, 2.5 mM protocatechuic acid, 2.5 mM adipic acid, 2.5 mM phenylalanine, and 2.5 mM phenethylamine, at pH 7, 2 mL of the pre-preculture liquid was added, and shake culture was carried out at 30° C. until the microorganism was sufficiently suspended (preculture). The preculture liquid was subjected to three times of washing with 200 mL of 0.9% sodium chloride in the same manner as in Example 1, and the microbial cells were suspended in 10 mL of 0.9% sodium chloride. To 100 mL of the same main culture medium as in Example 1, 10 mL of the resulting suspension was added, and shake culture was performed at 30° C. for 20 hours (main culture). The main culture liquid was centrifuged to separate microbial cells, and the resulting supernatant was analyzed by LC-MS/MS in the same manner as in Example 1. As a result, the concentration of 3-oxoadipic acid accumulated in the culture supernatant was found to be 260 mg/L.

(34) Subsequently, the supernatant from the main culture was concentrated under reduced pressure, to obtain 12 mL of a concentrate having a 3-oxoadipic acid concentration of 2200 mg/L. The concentrate was injected into HPLC to which a fraction collection device was connected, and a fraction having the same elution time as a 3-oxoadipic acid sample was collected. This operation was carried out ten times for removal of impurities in the culture liquid, to obtain an aqueous 3-oxoadipic acid solution. The preparative HPLC used for the collection of 3-oxoadipic acid was carried out under the following conditions. HPLC: SHIMADZU 20A (manufactured by Shimadzu Corporation) Column: Synergi hydro-RP (manufactured by Phenomenex); length, 250 mm; inner diameter, 10 mm; particle size, 4 μm Mobile phase: 5 mM aqueous formic acid solution/acetonitrile=98/2 Flow rate: 4 mL/minute Injection volume: 1 mL Column temperature: 45° C. Detector: UV-VIS (210 nm) Fraction collection device: FC204 (manufactured by Gilson)

(35) Subsequently, the aqueous 3-oxoadipic acid solution was concentrated under reduced pressure, to obtain 22 mg of crystals. As a result of analysis of the crystals by .sup.1H-NMR, it was able to confirm that the obtained crystals were 3-oxoadipic acid.

Comparative Example 1 Microorganism Having No Capacity to Produce 3-Oxoadipic Acid

(36) In order to investigate the 3-oxoadipic acid productivity of the microorganism shown in Table 14, microbial culture was carried out under the same conditions as in Example 1, and quantitative analysis of 3-oxoadipic acid was carried out. As a result, no 3-oxoadipic acid was detected in the culture supernatant.

(37) TABLE-US-00014 TABLE 14 Microbial strain 3-Oxoadipic acid (mg/L) Zymomonas mobilis NBRC13756 N.D.

Comparative Example 2 Culture without Addition of Carbon Source

(38) The microorganisms shown in Table 1 were cultured under the same conditions as in Example 1 except that a medium having a composition containing no aliphatic compound (succinic acid, glucose, or glycerol) as a carbon source was used. As a result of quantitative analysis of 3-oxoadipic acid, no 3-oxoadipic acid was detected in the culture supernatant. From this result, it was confirmed that the 3-oxoadipic acid that was able to be quantified in Example 1 was a product resulted by metabolism of aliphatic compounds by the microorganisms.

Example 6 3-Oxoadipic Acid Production Test Using Various Microorganisms

(39) The microorganisms shown in Table 15 (all microorganisms were purchased from microorganism-distributing agencies; the distributors are described in the strain names) were subjected to preculture and microbial cell washing under the same conditions as in Example 1 except that each of ferulic acid, p-coumaric acid, benzoic acid, cis,cis-muconic acid, protocatechuic acid, and catechol was added to 2.5 mM as an inducer to the preculture medium. To 5 mL of the medium having the composition shown below, 0.5 mL of the suspension after the washing was added, and shake culture was performed at 30° C. for 48 hours. 10 g/L succinic acid 10 g/L glucose 10 g/L glycerol 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 2.5 g/L Bacto tryptone 1.25 g/L yeast extract pH 6.5.

(40) The results of quantitative analysis of 3-oxoadipic acid accumulated in the culture supernatant are shown in Table 15. From these results, it was confirmed that all microorganisms have a capacity to produce 3-oxoadipic acid.

(41) TABLE-US-00015 TABLE 15 Test microorganism 3-Oxoadipic acid (mg/L) Serratia entomophila DSM12358 24 Serratia nematodiphila DS M21420 6.2 Serratia fonticola DSM9663 6.1 Serratia fonticola NBRC 102597 6.1 Serratia odorifera NBRC102598 2.0 Corynebacterium glutamicum ATCC 14020 58 Corynebacterium glutamicum ATCC13059 131 Corynebacterium glutamicum ATCC13060 137 Corynebacterium glutamicum ATCC13287 63 Corynebacterium glutamicum ATCC14067 26 Corynebacterium glutamicum ATCC21086 51 Corynebacterium glutamicum ATCC21127 32 Corynebacterium glutamicum ATCC21128 42 Corynebacterium glutamicum ATCC21129 36 Corynebacterium glutamicum ATCC21300 34 Corynebacterium glutamicum ATCC21474 55 Corynebacterium glutamicum ATCC21526 57 Corynebacterium glutamicum ATCC21527 57 Corynebacterium glutamicum ATCC21650 117 Corynebacterium glutamicum ATCC21651 60 Corynebacterium ammoniagenes NBRC12071 22 Corynebacterium ammoniagenes NBRC12072 14 Microbacterium ammoniaphilum ATCC15354 42 Bacillus megaterium ATCC10778 22 Pseudomonas putida ATCC8209 7.2 Pseudomonas putida NBRC12653 6.2 Pseudomonas putida NBRC12996 3.4 Pseudomonas sp. NBRC12691 3.0 Pseudomonas putida ATCC12633 3.0 Pseudomonas putida ATCC17642 2.5 Pseudomonas sp. ATCC13867 1.9 Pseudomonas sp. ATCC14718 1.3 Hafnia alvei ATCC 9760 7.0 Cupriavidus necator DSM545 1.6 Saccharomyces cerevisiae NBRC0206 1.6 Yersinia ruckeri NBRC102019 1.4

Example 7 3-Oxoadipic Acid Production Test without Addition of Inducers

(42) The microorganisms shown in Table 16 were subjected to preculture and microbial cell washing under the same conditions as in Example 6 except that the inducers used in Example 6 were not added. To 5 mL of the medium having the composition shown below, 0.5 mL of the suspension after the washing was added, and shake culture was performed at 30° C. for 48 hours. 10 g/L succinic acid 10 g/L glucose 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 2.5 g/L Bacto tryptone 1.25 g/L yeast extract pH 6.5.

(43) The results of quantitative analysis of 3-oxoadipic acid in the culture supernatant are shown in Table 16.

(44) From these results, it was confirmed that the microorganisms shown in Table 16 have a capacity to produce 3-oxoadipic acid even in cases where preculture is carried out without addition of inducers.

(45) TABLE-US-00016 TABLE 16 3-Oxoadipic acid Test microorganism production (mg/L) Serratia grimesii NBRC13537 2.5 Serratia ficaria NBRC102596 3.2 Serratia plymuthica NBRC102599 12.4 Serratia fonticola NBRC102597 2.0 Serratia odorifera NBRC102598 1.6 Serratia entomophila DSM12358 3.2 Serratia nematodiphila DSM21420 1.0 Corynebacterium glutamicum ATCC13032 14.6 Corynebacterium glutamicum ATCC21492 5.7 Corynebacterium glutamicum ATCC13826 9.1

Example 8 3-Oxoadipic Acid Production Test Using p-Coumaric Acid or Ferulic Acid as Inducer

(46) The microorganisms shown in Table 17 were subjected to preculture and microbial cell washing under the same conditions as in Example 6 except that p-coumaric acid or ferulic acid, among the substances added as inducers to the preculture medium in Example 6, was added to 0.5 mM. To 5 mL of the medium having the composition shown in Example 7, 0.5 mL of the suspension after the washing was added, and shake culture was performed at 30° C. for 48 hours. The results of quantitative analysis of 3-oxoadipic acid in the culture supernatant are shown in Table 17. From these results, it was found that the productivity of 3-oxoadipic acid can be increased even by addition of p-coumaric acid or ferulic acid alone as an inducer to the preculture medium compared to cases where neither of these is added.

(47) TABLE-US-00017 TABLE 17 3-Oxoadipic acid production (mg/L) No p-Coumaric acid Ferulic acid Test microorganism addition added added Serratia plymuthica 12.4 18.0 16.6 NBRC102599 Serratia grimesii NBRC13537 2.5 6.0 5.7 Serratia ficaria NERC102596 3.2 5.0 4.8 Corynebacterium glutamicum 9.1 15.6 13.2 ATCC13826 Corynebacterium glutamicum 5.7 14.0 9.6 ATCC21492 Corynebacterium glutamicum 14.6 32.2 28.7 ATCC13032 Corynebacterium 12.5 44.5 36.0 acetoglutamicum ATCC 15806 Bacillus badius ATCC 14574 6.3 33.6 9.8 Cupriavidus necator DSM545 1.4 3.4 3.2

Example 9 3-Oxoadipic Acid Production Test Using Ferulic Acid as Inducer at Various Concentrations

(48) The microorganisms shown in Table 18 were subjected to preculture in which, among the substances added as inducers to the preculture medium in Example 6, ferulic acid was added to the preculture medium in Example 7 to the concentrations shown in Table 18. Main culture was carried out under the same conditions as in Example 7, and quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results are shown in Table 18. From these results, it was found that the productivity of 3-oxoadipic acid can be increased even by addition of ferulic acid alone as an inducer to the preculture medium.

(49) TABLE-US-00018 TABLE 18 3-Oxoadipic acid production (mg/L) Test Concentration of ferulic acid added (mM) microorganism 0.00 0.05 0.10 0.25 0.50 1.00 2.50 S. grimesii 1.4 1.5 1.5 1.6 1.8 1.8 2.5 NBRC13537 S. ficaria 2.2 2.5 2.5 2.8 2.9 3.1 3.4 NBRC102596 S. plymuthica 17.1 17.7 17.8 17.8 18.6 20.5 24.7 NBRC102599

Example 10 3-Oxoadipic Acid Production Test Using p-Coumaric Acid as Inducer at Various Concentrations

(50) The microorganism shown in Table 19 was subjected to preculture in which, among the substances added as inducers to the preculture medium in Example 6, p-coumaric acid was added to the preculture medium in Example 7 to the concentrations shown in Table 19. Main culture was carried out under the same conditions as in Example 7, and quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results are shown in Table 19. From these results, it was found that the productivity of 3-oxoadipic acid can be increased even by addition of p-coumaric acid alone as an inducer to the preculture medium.

(51) TABLE-US-00019 TABLE 19 Test 3-Oxoadipic acid production (mg/L) micro- Concentration of p-coumaric acid added (mM) organism 0.00 0.05 0.10 0.25 0.50 1.00 2.50 S. grimesii 1.4  1.7  1.8  2.0  2.3  3.0  6.5  NBRC13537

Reference Example 3 Method of Preparing Filamentous Fungus-Derived Cellulase (Culture Liquid)

(52) A filamentous fungus-derived cellulase (culture liquid) was prepared by the following method.

(53) [Preculture]

(54) The mixture of 5% (w/vol) corn steep liquor (CSL), 2% (w/vol) glucose, 0.37% (w/vol) ammonium tartrate, 0.14 (w/vol) ammonium sulfate, 0.2% (w/vol) potassium dihydrogen phosphate, 0.03% (w/vol) calcium chloride dihydrate, 0.03% (w/vol) magnesium sulfate heptahydrate, 0.02% (w/vol) zinc chloride, 0.01% (w/vol) iron (III) chloride hexahydrate, 0.004% (w/vol) copper (II) sulfate pentahydrate, 0.0008% (w/vol) manganese chloride tetrahydrate, 0.0006% (w/vol) boric acid, and 0.0026% (w/vol) hexaammonium heptamolybdate tetrahydrate in distilled water was prepared, and 100 mL of this mixture was placed in a baffled 500-mL Erlenmeyer flask, followed by sterilization by autoclaving at a temperature of 121° C. for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each of which was sterilized by autoclaving at a temperature of 121° C. for 15 minutes separately from the mixture, were added thereto to 0.01% (w/vol) each. To this preculture medium, Trichoderma reesei ATCC66589 was inoculated at 1×10.sup.5 cells/mL, and the cells were cultured at a temperature of 28° C. for 72 hours with shaking at 180 rpm to perform preculture (shaker: BIO-SHAKER BR-40LF, manufactured by TAITEC CORPORATION).

(55) [Main Culture]

(56) The mixture of 5% (w/vol) corn steep liquor (CSL), 2% (w/vol) glucose, 10% (w/vol) cellulose (manufactured by Asahi Kasei Chemicals Corporation; trade name, Avicel), 0.37% (w/vol) ammonium tartrate, 0.14% (w/vol) ammonium sulfate, 0.2% (w/vol) potassium dihydrogen phosphate, 0.03% (w/vol) calcium chloride dihydrate, 0.03% (w/vol) magnesium sulfate heptahydrate, 0.02% (w/vol) zinc chloride, 0.01% (w/vol) iron (III) chloride hexahydrate, 0.004% (w/vol) copper (II) sulfate pentahydrate, 0.0008% (w/vol) manganese chloride tetrahydrate, 0.0006% (w/vol) boric acid, and 0.0026% (w/vol) hexaammonium heptamolybdate tetrahydrate in distilled water was prepared, and 2.5 L of this mixture was placed in a 5-L stiffing jar (manufactured by ABLE, DPC-2A), followed by sterilization by autoclaving at a temperature of 121° C. for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each of which was sterilized by autoclaving at a temperature of 121° C. for 15 minutes separately from the mixture, were added thereto to 0.1% each. To the resulting mixture, 250 mL of the preculture of Trichoderma reesei ATCC66589 preliminarily prepared with a liquid medium by the method described above was inoculated. Shake culture was then carried out at a temperature of 28° C. for 87 hours at 300 rpm at an aeration rate of 1 vvm. After centrifugation, the supernatant was subjected to membrane filtration (“Stericup-GV”, manufactured by Millipore, material: PVDF). The culture liquid prepared under the above conditions was used as a filamentous fungus-derived cellulase in the following Reference Examples.

Reference Example 4 Measurement of Cellulase Concentration

(57) The cellulase concentration in the aqueous solution was evaluated using as a standard the value of the protein concentration (mg/mL) in an enzyme liquid as measured by the Bradford method. The protein concentration was measured using an assay kit based on the Bradford method (Quick Start Bradford Protein Assay, manufactured by Bio-Rad).

Reference Example 5 Preparation of Cellulose-containing-biomass-derived Saccharified Liquid

(58) To bagasse with a dry weight of 1 kg, 30 g of caustic soda was added at a biomass feeding amount of 5%. The resulting mixture was reacted at 90° C. for 3 hours to prepare alkali-treated bagasse. The alkali-treated bagasse was subjected to solid-liquid separation by screw pressing to obtain a solid-liquid-separated solid having a moisture content of 60%.

(59) The solid-liquid-separated solid was resuspended at a solid concentration of 5%, and hydrolysis was carried out with the filamentous fungus-derived cellulase prepared in Reference Example 3 at a protein amount of 8 mg/g-bagasse according to the measurement described in Reference Example 4, to obtain a saccharified liquid. The hydrolysis was carried out at 40° C. at pH 7.0 for a reaction time 24 hours. The solid component was removed from the obtained saccharified liquid using a screw decanter, and the whole amount of the recovered saccharified liquid was filtered through a microfiltration membrane having a pore size of 0.22 μm, followed by subjecting the obtained permeate to filtration treatment through an ultrafiltration membrane. As the ultrafiltration membrane, TMUS10k (manufactured by Toray Membrane USA; material: polyvinylidene fluoride; molecular weight cutoff: 10,000) was used. In the ultrafiltration, filtration treatment was carried out using a flat membrane filtration unit “SEPA-II” (GE Osmonics) at a membrane surface linear velocity of 20 cm/sec. and a filtration pressure of 3 MPa until the volume of the liquid collected from the feed side reached 0.6 L, to obtain a saccharified liquid in the permeate side.

(60) Filtration treatment of the obtained saccharified liquid was carried out using a separation membrane (manufactured by Synder; NFW (material: piperazine polyamide; molecular weight cutoff, 300 to 500)). The filtration treatment was carried out at a membrane surface linear velocity of 20 cm/sec. and a filtration pressure of 3 MPa until the concentration rate in the feed side reached 12-fold, to obtain a saccharified liquid in the permeate side.

(61) The obtained saccharified liquid was concentrated using an evaporator to prepare a saccharified liquid containing 100 g/L glucose, 22.3 g/L xylose, 0.5 g/L coumaric acid, and 0.06 g/L ferulic acid. The final pH of the saccharified liquid was adjusted to 7 using 6 N sodium hydroxide.

Example 11 3-Oxoadipic Acid Production Test Using Cellulose-Containing-Biomass-Derived Saccharified Liquid

(62) For the microorganisms shown in Table 20, a 3-oxoadipic acid production test was carried out using as a carbon source the cellulose-containing-biomass-derived saccharified liquid prepared in Reference Example 5.

(63) In 5 mL of a preculture medium prepared to have the following composition using the cellulose-containing-biomass-derived saccharified liquid, each microorganism was cultured with shaking at 30° C. until the microorganism was sufficiently suspended (preculture). Subsequently, the microbial cells were washed under the same conditions as in Example 6. To 5 mL of a main culture medium prepared to have the following composition using the cellulose-containing-biomass-derived saccharified liquid, 0.5 mL of the suspension after the washing was added, and culture was performed with shaking at 30° C. for 48 hours. For comparison, culture was performed under the same conditions as described above except that carbon sources containing neither p-coumaric acid nor ferulic acid were used for the preculture and the main culture. The results of quantitative analysis of 3-oxoadipic acid in the culture supernatant are shown in Table 20. From these results, it was found that the productivity of 3-oxoadipic acid can be increased also in cases where culture is carried out using a cellulose-containing-biomass-derived saccharified liquid containing p-coumaric acid and ferulic acid, relative to cases where carbon sources containing neither p-coumaric acid nor ferulic acid are used.

(64) Medium Composition for the Preculture

(65) 5 g/L glucose 1.1 g/L xylose 25 mg/L p-coumaric acid 3 mg/L ferulic acid 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 2.5 g/L Bacto tryptone 1.25 g/L yeast extract pH 6.5.
Medium Composition for the Main Culture 50 g/L glucose 11 g/L xylose 250 mg/L p-coumaric acid 30 mg/L ferulic acid 10 g/L succinic acid 1 g/L ammonium sulfate 50 mM potassium phosphate 0.025 g/L magnesium sulfate 0.0625 mg/L iron sulfate 2.7 mg/L manganese sulfate 0.33 mg/L calcium chloride 1.25 g/L sodium chloride 2.5 g/L Bacto tryptone 1.25 g/L yeast extract pH 6.5.

(66) TABLE-US-00020 TABLE 20 3-Oxoadipic acid production (mg/L) Reagent Test microorganism saccharides Sugar liquid Serratia plymuthica NBRC102599 8.0 11.3 Serratia grimesii NBRC13537 1.3 5.1 Serratia ficaria NBRC102596 3.5 5.3 Corynebacterium glutamicum 14.1 28.3 ATCC13826 Corynebacterium glutamicum 9.8 24.1 ATCC21492 Corynebacterium glutamicum 18.7 26.4 ATCC13032

Example 12 3-Oxoadipic Acid Production Test Using Two Kinds of Carbon Sources

(67) The microorganisms shown in Table 21 and Table 22 were precultured using the same medium as in Example 6, and then cultured in media containing 10 g/L each of the compounds shown in Table 21 and Table 22 as carbon sources under the same conditions as in Example 6. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results are shown in Table 21 and Table 22. From these results, it was found that 3-oxoadipic acid can be efficiently produced also by culture using two kinds of carbon sources.

(68) TABLE-US-00021 TABLE 21 3-Oxoadipic acid production (mg/L) (with addition of inducers) Carbon source Glucose Glycerol Xylose Arabinose Glucose Glycerol Xylose Arabinose Test Glucose Succinic Succinic Succinic Succinic 2-Oxoglutaric 2-Oxoglutaric 2-Oxoglutaric 2-Oxoglutaric microorganism Glycerol acid acid acid acid acid acid acid acid S.grimesti 17.5 136.9 185.3 113.2 169.4 40.5 115.0 151.3 123.0 NBRC13537 S.ficaria 15.3 68.6 42.6 19.0 84.1 45.1 75.1 73.7 61.0 NBRC102596 S.plyinuthica 26.3 182.3 84.8 64.7 172.4 35.1 94.1 103.4 69.5 NBRC102599

(69) TABLE-US-00022 TABLE 22 3-Oxoadipic acid production (mg/L) (with addition of inducers) Carbon source Test Glucose Glucose Glycerol microorganism Glycerol Succinic acid Succinic acid C. glutamicum 16.3 115.5 16.4 ATCC13032 C. glutamicum 13.4 250.3 14.2 ATCC21492 C. glutamicum 13.7 188.8 16.3 ATCC13826

Example 13 3-Oxoadipic Acid Production Test Using Two Kinds of Carbon Sources at Various Concentrations

(70) The microorganisms shown in Table 23 and Table 24 were precultured using the same medium as in Example 6, and then cultured in media containing as carbon sources the compounds shown in Table 23 and Table 24 at the concentrations shown in the Tables, under the same conditions as in Example 6 for 48 to 120 hours. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results are shown in Table 23 and Table 24. From these results, it was found that 3-oxoadipic acid can be produced also in cases where the ratios of carbon sources are changed.

(71) TABLE-US-00023 TABLE 23 3-Oxoadipic acid production (mg/L) (with addition of inducers) Carbon source Glucose 25 g/L Glucose 50 g/L Xylose 25 g/L Xylose 50 g/L Glucose 10 g/L Glucose 100 g/L Xylose 50 g/L Test Succinic acid Succinic acid Succinic acid Succinic acid Succinic acid Succinic acid Succinic Acid microorganism 10 g/L 10 g/L 10 g/L 10 g/L 20 g/L 20 g/L 20 g/L S. grimesii 114.8 120.9 90.8 123.2 242.7 241.5 382.9 NBRC13537 S. ficaria 13.5 18.1 35.3 37.4 65.9 165.4 103.6 NBRC102596 S. plymuthica 81.6 71.9 197.6 213.6 268.2 222.9 247.3 NBRC102599

Example 14 3-Oxoadipic Acid Production Test Using Single Carbon Source

(72) The microorganisms shown in Table 24 and Table 25 were precultured without addition of an inducer using the same medium as in Example 7, and then cultured in media containing 10 g/L of one of succinic acid, glucose, and glycerol as a carbon source, under the same conditions as in Example 7. Thereafter, quantitative analysis of 3-oxoadipic acid in the culture supernatant was carried out. The results are shown in Table 24. The same experiment was carried out under the same conditions as in Example 6, wherein inducers were added only to the preculture medium. The amounts of 3-oxoadipic acid produced are shown in Table 25. From these results, it was found that 3-oxoadipic acid can be produced even in cases where a sole carbon source is used, and that the amount of 3-oxoadipic acid produced can be increased by adding inducers to the preculture medium even in cases where a sole carbon source is used.

(73) TABLE-US-00024 TABLE 24 3-Oxoadipic acid production (mg/L) (without addition of Inducers) Carbon source Test Succinic microorganism acid Glucose Glycerol Xylose Arabinose S. grimesii 1.2 1.0 1.5 1.8 1.7 NBRC13537 S. ficaria 1.0 1.3 3.0 2.0 1.5 NBRC102596 S. plymuthica 2.0 2.0 4.7 4.0 3.3 NBRC102599

(74) TABLE-US-00025 TABLE 25 3-Oxoadipic acid production (mg/L) (with addition of inducers) Carbon source Test Succinic microorganism acid Glucose Glycerol Xylose Arabinose S. grimesii 4.7 8.3 54.9 14.3 13.8 NBRC13537 S. ficaria 2.1 3.2 20.3 14.8 12.0 NBRC102596 S. plymuthica 15.8 16.3 10.6 11.1 10.9 NBRC102599

INDUSTRIAL APPLICABILITY

(75) By the present invention, 3-oxoadipic acid can be produced using a microorganism. The obtained 3-oxoadipic acid can be used as a raw material for polymers.