PREPARATION OF (R)-3-HYDROXYBUTYRIC ACID OR ITS SALTS BY ONE-STEP FERMENTATION

Abstract

The subject invention relates to a process of preparing (R)-3-hydroxybutyric acid or a salt thereof by one-step fermentation with a nonpathogenic microorganism. The fermentation of (R)-3-hydroxybutyric acid was performed by supplying with certain carbon and nitrogen sources. These microorganisms include a Glutamic acid Bacterium HR057 strain or one type of genetically engineered Corynebacterium Glutamicum.

Claims

1. A process for producing (R)-3-hydroxybutyric acid comprising fermenting a fermentation broth with a nonpathogenic microorganism, wherein the fermentation broth comprises carbon and nitrogen sources and an enzyme that is overexpressed by the nonpathogenic microorganism, the carbon and nitrogen sources are directly converted into (R)-3-hydroxybutyric acid by one-step fermentation with the nonpathogenic microorganism, the (R)-3-hydroxybutyric acid was recovered from fermentation broth after it was excreted during fermentation, wherein the nonpathogenic microorganism is selected from a group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum and Brevibacterium breve; and the nonpathogenic microorganism has the following biotransformation capability: converting pyruvic acid and coenzyme A to acetyl-CoA, converting acetyl-CoA into acetoacetyl-CoA, converting acetoacetyl-CoA to acetoacetic acid, and converting acetoacetic acid to (R)-3-hydroxybutyric acid.

2. The process of claim 1, wherein the enzyme overexpressed by the nonpathogenic microorganism comprises a member selected from the group consisting of succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.

3. The process of claim 2, wherein the microorganism overexpresses succinyl-CoA transferase and 3-HB dehydrogenase.

4. The process of claim 3, wherein the nonpathogenic microorganism is Corynebacterium glutamicum, Glutamic acid Bacterium HR057, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum, or Brevibacterium breve.

5. The process of claim 4, wherein the microorganism is Corynebacterium glutamicum or Glutamic acid Bacterium HR057.

6. The process of claim 4, wherein the microorganism is Corynebacterium glutamicum.

7. The process of claim 6, wherein the Corynebacterium glutamicum is as deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 13111.

8. The process of claim 1, wherein the carbon source comprises a member selected from the group consisting of glucose, sucrose, maltose, molasses, starch and glycerol.

9. The process of claim 1, wherein the nitrogen source comprises a member selected from the group consisting of an organic nitrogen source and an inorganic nitrogen source.

10. The process of claim 9, wherein the organic nitrogen source comprises a member selected from the group consisting of corn steep liquor, bran hydrolyzate, soybean cake hydrolyzate, yeast extract, yeast powder, peptone, and urea.

11. The process of claim 9, wherein the inorganic nitrogen source comprises a member selected from the group consisting of ammonium sulfate, ammonium nitrate, and aqueous ammonia.

12. The process of claim 1, wherein the (R)-3-hydroxybutyric acid is free of bacterial endotoxin and has a purity of 95% or more.

13. The process of claim 1, wherein the (R)-3-hydroxybutyric acid is prepared in the form of (R)-3-hydroxybutyrate sodium salt, (R)-3-hydroxybutyrate potassium salt, (R)-3-hydroxybutyrate magnesium salt, or (R)-3-hydroxybutyrate calcium salt.

14. A racemic 3-hydroxybutyric acid prepared by racemization treatment of the (R)-3-hydroxybutyric acid produced in accordance of claim 12.

15. A racemic 3-hydroxybutyric acid prepared by racemization treatment of a (R)-3-hydroxybutyrate salt produced in accordance of claim 13.

16. A nonpathogenic microorganism selected from the group consisting of Corynebacterium glutamicum, Glutamic acid Bacterium HR057, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum, and Brevibacterium breve.

17. The nonpathogenic microorganism of claim 16, wherein the nonpathogenic microorganism is a Corynebacterium glutamicum strain or a Glutamicacid Bacterium HR057 strain.

18. The nonpathogenic microorganism of claim 17, wherein the nonpathogenic microorganism is a Corynebacterium glutamicum strain.

19. The nonpathogenic microorganism of claim 18, wherein the Corynebacterium glutamicum strain is as deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 13111.

20. The nonpathogenic microorganism of claim 16, wherein the microorganism is capable of producing (R)-3-hydroxybutyric acid in a one-step fermentation process as described in claim 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is further described in detail with specific embodiments. The following examples are intended to demonstrate the invention and not to limit the scope of the invention.

[0041] Mass percentage is referred in the invention such as the added amount, content and concentration of multiple substances unless otherwise provided descried or defined.

[0042] In the embodiments provided under the present invention, room temperature (15-30 C.) is referred to by default unless otherwise provided descried or specified.

[0043] In the present invention, a microorganism strain capable of producing (R)-3-hydroxybutyric acid by fermentation is exemplified by but not limited to Corynebacterium glutamicum, Corynebacterium glutamicum strain CGMCC No. 13111, or Glutamic acid Bacterium HR057.

[0044] Fermentation of (R)-3-hydroxybutyric acid by microorganism was investigated in the present invention in order to supply the consumer and pharmaceutical and food manufacturers with the naturalized or biogenic sources of (R)-3-hydroxybutyric acid and its salts, 3-hydroxybutyric acid and its salts.

[0045] The inventors screened and selected species for the construction of genetic engineering strains. It is not considered that general strain with potentially pathogenic feature such as Escherichia coli. Nonpathogenic microorganism was chosen and genetically engineered as fermentation strains such as Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium breve and Brevibacterium brevica. Several strains were obtained through screening which are able to produce (R)-3-hydroxybutyric acid by fermentation. No endotoxin was produced at all during fermentation, which may cause harm to most people. So, it is considered as non-toxic and harmless design.

[0046] It is necessary to adjust and control some parameters such as dissolved oxygen, temperature, pH etc., to have a higher yield of (R)-3-hydroxybutyric acid during fermentation.

[0047] The constant dO2 is controlled at 15% to 25% during fermentation. Fermentation can be carried out under the following conditions: air flow is about 1 vvm, where vvm is the ratio of the amount of ventilation per minute to the actual volume of liquid in the tank (for example, 1 vvm is equal to 30 L/min for a fermenter containing 30 liters of fermentation broth, and 1 vvm is equal to 5 L/min as for a fermentation tank containing 5 liters of fermentation broth,).

[0048] Preferably, the temperature is first controlled at 30.sup.32 C. during the initial stage of fermentation and then increased to 34.sup.37 C. at the later stage of the fermentation which facilitates the synthesis and excretion of (R)-3-hydroxybutyric acid by the microorganism.

[0049] The pH is generally controlled at pH 6.0.sup.8.0, preferably at pH 6.5.sup.7.0, during the initial stage of fermentation. It can then be adjusted to 6.8.sup.7.0 in the later stages of fermentation to facilitate the synthesis and drainage of (R)-3-hydroxybutyric acid from the fermentation broth.

[0050] The above term later stage of fermentation refers to from exponential stage to stationary stage of microbial growth. For example, the OD600 nm value is no longer rising and tending to decrease when monitoring cell density with OD600 nm values.

[0051] The residual sugar is controlled at 1.0%.sup.3.0% during fermentation process, more precisely at 1.5%.sup.2.5%.

[0052] After the fermentation is completed, the fermentation broth needs to be recovered and (R)-3-hydroxybutyric acid is extracted therefrom. For example, the supernatant of the fermentation broth is obtained by centrifugation. The supernatant is concentrated if necessary; (R)-3-hydroxybutyric acid is separated by a post-treatment such as purification and drying.

[0053] Cells and macromolecules in the fermentation medium can be removed by filtration (including ultrafiltration, nanofiltration, etc.). Concentrated filtration, and other post-processing means such as drying, purification and other methods may be applied if necessary to isolate (R)-3-hydroxybutyric acid. Alternatively, (R)-3-hydroxybutyric acid can be isolated by centrifugation which obtains supernatant of fermentation broth, then go through ultrafiltration, nanofiltration and other methods to remove macromolecules, or through concentrated filtration if necessary, then by drying, purification and other post-processing means.

[0054] To prepare (R)-3-hydroxybutyrate such as sodium salt, potassium salt, magnesium salt, calcium salt, an equivalent amount of (R)-3-hydroxybutyric acid is reacted with the corresponding base or metal oxide such as sodium hydroxide. The reaction temperature is controlled to be 30 C. or lower, preferably at 25 C. or lower, and more preferably at 20 C. or less, where the racemic reaction could be avoided as much as possible.

[0055] As the whole preparation process does not require or involve an organic solvent, chemical odor like bitterness was not detected from the product (R)-3-hydroxybutyric acid which could be directly used to manufacture pharmaceuticals and health care products.

Example 1: Pre-Culture and Fermentation

[0056] A glycerol stock CGMCC No. 13111 stored at 80 C. was thawed and inoculated to a 5000 mL flask containing 500 mL seed medium (75 g/L of glucose, 25-30 g/L of corn steep liquor, 20 g/L of (NH.sub.4).sub.2SO.sub.4, 1.5 g/L of KH.sub.2PO.sub.4, 0.5 g/L of MgSO.sub.4.7H.sub.2O, 1.0 g/L of urea, 30 mg/L of histidine, 25 g/L of molasses, 100 g/L of biotin, pH 7.0), and cultured at 30 C. for 18 hours. The seed culture cultivation was completed when OD=0.4-0.5.

[0057] 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters medium. The composition of fermentation medium was the same as the seed medium described above, and the pH was controlled at 6.4.sup.6.7 after sterilization. Feed medium contains 500 g/L ammonium sulfate and 650 g/L glucose. The fermentation temperature was set at 30 C., the tank pressure was kept at 0.05 Mpa, and the initial ventilation ratio was 1 vvm. Stirring speed was 600 rpm. The pH of the fermentation was about pH 6.5.

[0058] The pH was controlled at 6.7 and the temperature was raised to 35 C. at the later phase of fermentation. The dissolved oxygen constant (dO2) was controlled at 15.sup.25% by adjusting ventilation and stirring speed. To control residual sugar level, sugar was fed slowly while the concentration of the original sugar dropped to about 3.0% and the residual sugar was controlled at 1.5%.sup.2.0%. (R)-3-hydroxybutyric acid was accumulated to 11.8 g/L after 72 hours.

Example 2: Isolation of Fermentation Broth and Extraction of (R)-3-Hydroxybutyric Acid

[0059] 5.2 liters of the fermentation broth obtained in Example 1 was centrifuged at 4500 rpm and the cells were discarded. The supernatant was filtered with 1% diatomaceous earth. Clear filtrate was recovered after stirring for 30 minutes mixed with 1% activated carbon.

[0060] The filtrate was concentrated through nanofiltration membrane, and the resulting filtrate was passed through a 732 cation exchange resin to get a 1000 g/L concentrated filtrate. The concentrated collection was oily and collected while hot to give 56.8 g of (R)-3-hydroxybutyric acid with a yield of 92.5%. The purity of (R)-3-hydroxybutyric acid was determined by high performance liquid chromatography. The chromatographic column was Shim-pack Vp-ODSC18 column (150 L4.6). The mobile phase consisted of acetonitrile:water (v/v)=15:85, UV detection wavelength was 210 nm, injection volume was 20 L, flow rate was 1 mL/min, column temperature was 10 C. The purity of (R)-3-hydroxybutyric acid was 98% and the specific optical value was [] D20=25 (C=6%, H.sub.2O).

Example 3: Preparation of sodium (R)-3-hydroxybutyrate

[0061] A neutralization reaction was performed by mixing 5 g of (R)-3-hydroxybutyric acid obtained in Example 2 with an equivalent amount of a 2.0 N sodium hydroxide solution at 25 C. or lower. 4.9 g of sodium (R)-3-hydroxybutyrate powder was finally obtained with an 81% yield by rotating concentration, standing for 2 hours, and being collected by suction filtration and dried at 60 C. The salt has a melting point of 152 C. and a specific optical value [] D20 of 14.1 (C=10%, H2O).

Example 4: Racemization of (R)-3-hydroxybutyric acid

[0062] 10 g of (R)-3-hydroxybutyric acid was slowly added to 100 mL of a 2.0 N sodium hydroxide solution, and the mixture was heated to 60 C. for 4 hours. The optical value of product was 0 at room temperature. A neutralization reaction was carried out by adding hydrochloric acid to adjust the pH of the mixture to 7.0. 10.3 g of sodium 3-hydroxybutyrate powder was obtained with a yield of 85% by rotary evaporation until a solid was observed, allowing to stand for 2 hours, and then filtration. The results showed that racemic 3-hydroxybutyrate was obtained.

[0063] In summary, the present invention disclosed that the engineered Corynebacterium glutaricum by genetic modification could ferment (R)-3-hydroxybutyric acid in a one-step process at a high yield, which was proved to be safe and non-toxic food grade. The engineered strain and manufacturing process that were disclosed in this invention has broad industrial application prospects.