Preparation of (R)-3-Hydroxybutyric Acid or Its Salts by One-Step Fermentation
20220162653 · 2022-05-26
Assignee
Inventors
Cpc classification
C12N9/1029
CHEMISTRY; METALLURGY
International classification
Abstract
The subject invention relates to a genetically modified microorganism comprising a (R)-3-hydroxybutyric acid pathway and being able of producing (R)-3-hydroxybutyric acid, and a method of preparing (R)-3-hydroxybutyric acid or a salt thereof using the genetically modified microorganism.
Claims
1. A genetically modified microorganism having (R)-3-hydroxybutyrate biosynthesis capability, comprising one or more exogenous nucleic acids encoding at least one (R)-3-hydroxybutyric acid pathway enzyme selected from succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
2. The genetically modified microorganism of claim 1, wherein the genetically modified microorganism is able to produce (R)-3-hydroxybutyric acid in a pathway comprising the following steps: (i) converting pyruvic acid and coenzyme A to acetyl-CoA, (ii) converting acetyl-CoA into acetoacetyl-CoA, (iii) converting acetoacetyl-CoA to acetoacetic acid, and (iv) converting acetoacetic acid to (R)-3-hydroxybutyric acid.
3. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid is codon-optimized gene for expression in the genetically modified microorganism.
4. The genetically modified microorganism of claim 1, wherein the pathway enzyme encoded by exogenous nucleic acids is succinyl-CoA transferase, acetoacetyl-CoA synthase, or 3-HB dehydrogenase.
5. The genetically modified microorganism of claim 1, wherein the pathway enzyme encoded by exogenous nucleic acids are succinyl-CoA transferase, acetoacetyl-CoA synthase and 3-HB dehydrogenase.
6. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding succinyl-CoA transferase is at least 90% identical to the nucleotide sequence of SEQ ID NO:1.
7. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding acetoacetyl-CoA synthase is at least 90% identical to the nucleotide sequence of SEQ ID NO:2.
8. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding 3-HB dehydrogenase is at least 90% identical to the nucleotide sequence of SEQ ID NO:3.
9. The genetically modified microorganism of claim 1, wherein the microorganism is nonpathogenic microorganism selected from the group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum, and Brevibacterium breve.
10. The genetically modified microorganism of claim 9, wherein the microorganism is Corynebacterium glutamicum or Bacillus subtilis.
11. The genetically modified microorganism of claim 9, wherein the microorganism is Corynebacterium glutamicum.
12. The genetically modified microorganism of claim 11, wherein the Corynebacterium glutamicum is the the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 13111.
13. A method for producing (R)-3-hydroxybutyric acid, comprising using the genetically modified microorganism of claim 1.
14. The method of claim 13, wherein the method is a one-step fermentation process comprising fermenting the genetically modified microorganism of claim 1.
15. A recombinant nucleic acid segment encoding succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0045]
[0046]
[0047]
[0048]
DETAILED DESCRIPTION OF THE INVENTION
[0049] 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.
[0050] Mass percentage is referred in the invention such as the added amount, content and concentration of multiple substances unless otherwise provided descried or defined.
[0051] In the embodiments provided under the present invention, room temperature (15-30° C.) is referred to by default unless otherwise provided descried or specified.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] The constant dO.sub.2 is controlled at 15% to 30% during fermentation. Fermentation can be carried out under the following conditions: air flow is about 1-1.3 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).
[0056] 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.
[0057] 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.
[0058] The above term “later stage of fermentation” refers to from exponential stage to stationary stage of microbial growth. For example, the OD.sub.600 nm value is no longer rising and tending to decrease when monitoring cell density with OD.sub.600 nm values.
[0059] The residual sugar is controlled at 1.0%.sup.˜3.0% during fermentation process, more precisely at 1.5%.sup.˜2.5%.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] As used herein, the term “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term “nonpathogenic microorganism” refers to a microorganism that is not capable of causing disease or harmful responses in a host. In preferred embodiments, the microorganism in this invention is nonpathogenic microorganism selected from the group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium breve and Brevibacterium brevica.
[0065] As used herein, the terms “overexpressed”, “overexpression” and the like, refer to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein the polypeptide or protein is either not normally present in the host cell, or wherein the polypeptide or protein is present in the host cell at a higher level than that normally expressed from the endogenous gene encoding the polypeptide or protein.
[0066] As used herein, the term “modified” or “engineered” refers to any manipulation of a microorganism, wherein the manipulation includes but not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism. The term “genetically modified microorganism” refers to a microorganism having at least one genetic alteration not normally found in the wild type strain of the reference species, for example, involving rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired product.
[0067] As used herein, the term “pathway” is “biosynthetic pathway”, which refers to a set of biochemical reactions for converting one chemical species into another.
[0068] As used herein, the term “native” or “endogenous” as used herein with reference to molecules, in particular nucleic acids, enzymes and polynucleotides, which are originated or present in the organism. It is understood that expression of native enzymes or polynucleotides may be modified in genetically modified microorganisms.
[0069] On the contrary, the term “exogenous” refers to molecules, in particular nucleic acids, enzymes and polynucleotides, which are not originated, but introduced into the host organism. For example, the molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid.
[0070] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLE 1
Genetically Modification of Corynebacterium glutamicum
[0071] The genetically modified Corynebacterium glutamicum comprises an exemplary (R)-3-hydroxybutyric acid synthesis pathway.
[0072] Plasmid Construction
[0073] Codon optimized genes for t3, OXCT1 and BDH1, encoding acetoacetyl-CoA synthase, succinyl-CoA transferase and 3-HB dehydrogenase respectively, were cloned into plasmid pUC57 to give a cloning vector PUC57-t3-OXCT1-BDH1. The codon optimized nucleic acid sequences are listed in SEQ ID NOs: 1-3 respectively. Table 1 below lists the exemplary pathway enzymes.
TABLE-US-00001 TABLE 1 Exemplary pathway enzymes for production of (R)-3-hydroxybutyric acid Gene Name Enzyme Name GenBank ID t3 acetoacetyl-CoA synthase WP_007111820 OXCT1 Succinyl-CoA transferase AAH09001.1 BDH1 3-HB dehydrogenase EAW53609.1
[0074] Next, the gene fragment t3-OXCT1-BDH1 was digested from the cloning vector PUC57-t3-OXCT1-BDH1 using restriction endonuclease enzymes EcoR1 and Hind3, and inserted into the same endonuclease digested plasmid pXMJ19 to construct recombinant vectors. For confirming the insertion, the constructed recombinant vectors were transformed into competent bacterial strain DH5a for clone selection. The presence of the inserted fragment t3-OXCT1-BDH1 was confirmed by colony-PCR using specific primers, restriction enzyme digestion and sequencing. The confirmed vector was the expression vector named as pXMJ19-t3-OXCT1-BDH1.
[0075] Generation of Genetically Modified Corynebacterium glutamicum
[0076] The Corynebacterium glutamicum ATCC 13032 cells were made electro-competent. Then, the expression vector pXMJ19-t3-OXCT1-BDH1 was transformed into this competent cell and used for producing (R)-3-hydroxybutyric acid. The transformed bacteria were plated on 2% agar plate with CM medium (beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, and 0.5 g/L D-alanine) containing 25 mg/L chloramphenicol for clone selection.
[0077] This modified Corynebacterium glutamicum harboring plasmid pXMJ19-t3-OXCT1-BDH1 is the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC 13111.
EXAMPLE 2
Fermentation of the Genetically Modified Corynebacterium glutamicum
[0078] The genetically modified C. glutamicum strain CGMCC No. 13111 was take out from a glycerol stock stored at −80° C. and was used to inoculate 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 μg/L of biotin, pH 7.0). The cells were cultured at 30° C. for 18 hours, at which time the absorbance at 600 nm (known as OD.sub.600) reaches 0.4-0.5.
[0079] Next, the 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters fermentation medium, which was the same as the seed medium described above. The pH of the medium was controlled at 6.4.sup.˜6.7 after autoclaving. The fermentation began with an agitation speed of 600 rpm and aeration rate of 1 vvm. The tank pressure was maintained at 0.05 Mpa, and the cultivation temperature was controlled at 30° C. IPTG was added with a final concentration of 0.1 mmol/L after 8-10 h.
[0080] At the later stage of the fermentation, the pH was controlled at 6.7 and the temperature was raised to 35° C. The dissolved oxygen concentration (dO.sub.2) was controlled at 15.sup.˜25% by adjusting aeration rate and agitation speed. To maintain the residual sugar level, sugar was fed slowly while the concentration of the original sugar dropped to about 3.0%. The residual sugar level was maintained at 1.5%.sup.˜2.0%.
[0081] After 72 h, production of (R)-3-hydroxybutyric acid was accumulated to 11.8 g/L.
EXAMPLE 3
Genetically Modification of Bacillus subtilis
[0082] The genetically modified Bacillus subtilis comprises an exemplary (R)-3-hydroxybutyric acid synthesis pathway as shown in
[0083] Plasmid Construction
[0084] Similar to the modification of Corynebacterium glutamicum, the target gene fragment t3-OXCT1-BDH1 was digested from the cloning vector PUC57-t3-OXCT1-BDH1, and then inserted into the digested and purified plasmid pMA5 to construct recombinant vectors. For confirming the insertion, the constructed recombinant vectors were transformed into competent bacterial strain DH5a for clone selection. The presence of the inserted fragment t3-OXCT1-BDH1 was confirmed by colony-PCR using specific primers, restriction enzyme digestion and sequencing. The confirmed vector was the expression vector named as pMA5-t3-OXCT1-BDH1.
[0085] Generation of Genetically Modified Bacillus subtilis
[0086] The Bacillus subtilis WB600 cells were made competent. Then, the expression vector pMA5-t3-OXCT1-BDH1 was transformed into this competent cell and used for producing (R)-3-hydroxybutyric acid. The transformed bacteria were plated on LB agar plate containing 100 mg/L ampicillin for clone selection.
[0087] This modified Bacillus subtilis harboring plasmid pMA5-t3-OXCT1-BDH1 was named as Bacillus subtilis NNB01.
EXAMPLE 4
Fermentation of the Genetically Modified Bacillus subtilis
[0088] The genetically modified Bacillus subtilis NNB01 was taken out from a glycerol stock stored at −80° C. and used to inoculate a 5000 mL flask containing 500 mL seed medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L Yeast extract, Ammonium sulfate 0.4 g/L, Potassium dihydrogen phosphate 0.19 g/L, Magnesium sulfate 0.16 g/L). The cells were cultured at 37° C. for 20 hours, at which time the absorbance at 600 nm (known as OD.sub.600) reaches 0.4-0.5.
[0089] Next, the 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters fermentation medium, which is the same as the seed medium described above. The pH of the medium was controlled at 7.2.sup.˜7.5 after autoclaving. The tank pressure was maintained at 0.05 Mpa, and the cultivation temperature was controlled at 37° C. The fermentation began with an agitation speed of 700 rpm and an aeration rate of 1.3 vvm.
[0090] At the later stage of the fermentation, the pH of the medium was controlled at 7.2. The dissolved oxygen concentration (dO.sub.2) was controlled at 25.sup.˜30% by adjusting aeration rate and agitation 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%.
[0091] After 60 hours, the production of (R)-3-hydroxybutyric acid was accumulated to 10.2 g/L.
EXAMPLE 5
Isolation of Fermentation Broth and Extraction of (R)-3-hydroxybutyric Acid
[0092] The fermentation broth was harvested from the fermenter and centrifuged at 4500 rpm. After centrifugation, cell pellets were discarded, and the supernatant was collected and mixed with 1% diatomaceous earth. After 30 min of mixing, clear supernatant was recovered from the mixture.
[0093] Next, the clear supernatant was concentrated using a nanofiltration membrane, and further purified by passing through a 732 cation exchange resin. The pass-through containing (R)-3-hydroxybutyric acid was collected, giving 56.8 g of (R)-3-hydroxybutyric acid with a yield of 92.5%.
[0094] The purity of (R)-3-hydroxybutyric acid was determined by high performance liquid chromatography. The chromatographic column was Shim-pack Vp-ODSC18 column (150 L×4.6). The mobile phase consisted of acetonitrile: water (v/v)=15:85, UV detection wavelength was 210 nm, injection volume was 20 μL, 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 [α] D20=−25° (C=6%, H.sub.2O).
[0095]
EXAMPLE 6
Preparation of Sodium (R)-3-hydroxybutyrate
[0096] 5 g of (R)-3-hydroxybutyric acid was neutralized with an equivalent amount of sodium hydroxide at 25° C. The obtained neutralized product was concentrated, filtered and dried, giving sodium (R)-3-hydroxybutyrate powder with a yield of 81%. The obtained salt has a melting point of 152° C. and a specific optical value [α].sub.D.sup.20 of −14.1° (C=10%, H2O).
[0097] In summary, the present invention disclosed a genetically modified microorganism comprising a (R)-3-hydroxybutyric acid pathway and being able of producing (R)-3-hydroxybutyric acid, and a method for producing (R)-3-hydroxybutyric acid at a high yield using the modified microorganism, 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.