U.S. patent application number 17/544618 was filed with the patent office on 2022-05-26 for preparation of (r)-3-hydroxybutyric acid or its salts by one-step fermentation.
This patent application is currently assigned to NNB Nutrition USA, LLC. The applicant listed for this patent is NNB Nutrition USA, LLC. Invention is credited to Wenchao Fan, Kylin Liao.
Application Number | 20220162653 17/544618 |
Document ID | / |
Family ID | 1000006128668 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220162653 |
Kind Code |
A1 |
Liao; Kylin ; et
al. |
May 26, 2022 |
Preparation of (R)-3-Hydroxybutyric Acid or Its Salts by One-Step
Fermentation
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.
Inventors: |
Liao; Kylin; (Nanjing,
CN) ; Fan; Wenchao; (Nanjing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NNB Nutrition USA, LLC |
Frisco |
TX |
US |
|
|
Assignee: |
NNB Nutrition USA, LLC
Frisco
TX
|
Family ID: |
1000006128668 |
Appl. No.: |
17/544618 |
Filed: |
December 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16542044 |
Aug 15, 2019 |
11198890 |
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17544618 |
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15944331 |
Apr 3, 2018 |
10428357 |
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16542044 |
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62481476 |
Apr 4, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/13 20130101; C12R
2001/15 20210501; C12N 1/20 20130101; C12N 9/1029 20130101; C12P
7/42 20130101; C12N 1/205 20210501; C12N 9/0006 20130101; C07C
53/124 20130101 |
International
Class: |
C12P 7/42 20060101
C12P007/42; C12N 1/20 20060101 C12N001/20; C07C 53/124 20060101
C07C053/124; C12N 9/04 20060101 C12N009/04; C12N 9/10 20060101
C12N009/10 |
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
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
priority to U.S. application Ser. No. 16/542,044, filed on Aug. 15,
2019, which is a divisional application of U.S. application Ser.
No. 15/944,331, filed on Apr. 3, 2018, which claims priority to
U.S. application No. 62/481,476, filed on Apr. 4, 2017, the
contents of which are incorporated herein by reference in their
entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The subject invention pertains to the field of
bioengineering, in particular, genetically modified microorganisms
having (R)-3-hydroxybutyrate biosynthesis capability, and a method
for preparing (R)-3-hydroxybutyric acid and its salts by microbial
fermentation.
BACKGROUND OF THE INVENTION
[0003] (R)-3-hydroxybutyrate (R-3HB) is an optically chiral
compound with the CAS No. 625-72-9. Naturally occurring
(R)-3-hydroxybutyric acid is produced by the metabolism of long
chain fatty acids in the liver of mammals. It exists as a major
ketone in plasma and peripheral tissues and can be used as an
energy source in most tissues of the body.
[0004] (R)-3-hydroxybutyric acid has positive effect on treating
many diseases and nutritional functions as well. For example, it
can be used to treat many diseases that arise from elevated levels
of ketone (such as nerve disorders including epilepsy and
myoclonus, and neurodegenerative diseases including Alzheimer's
disease and dementia); it can reduce free radical damage by
oxidizing the coenzyme Q (such as ischemia); it can enhance the
efficiency of metabolism to achieve the treatment of inadequate
support, angina, myocardial infarction, etc. by improving training
efficiency and athletic performance; it can also be used to treat
cancer related diseases such as brain cancer (astrocytoma, etc.).
Further, it has good effects on the treatment of glucose metabolism
disorders (such as type-1 diabetes, type-2 diabetes, hypoglycemia
ketone disease, etc.). It can be used to control osteopenia
(osteopenia), osteoporosis, severe osteoporosis and related
fractures. Based on these functions and therapeutic and nutritional
effects, (R)-3-hydroxybutyric acid and its salts can be used as
food additives and drugs with great health and medicinal
values.
[0005] (R)-3-hydroxybutyric acid has been prepared primarily by
chemical methods. It can be made from direct chemical synthesis or
prepared by enzymatic degradation of poly-3-hydroxybutyrate with
poly-3-hydroxybutyric acid depolymerase. The chemical synthesis of
(R)-3-hydroxybutyric acid requires a high temperature, a high
pressure and expensive chiral metal catalysts. The process of
enzymatic degradation of poly-3-hydroxybutyric acid requires a
large amount of organic solvent, and very pure
poly-3-hydroxybutyrate as starting material. Besides the long
reaction time, it is difficult to control the racemization of
product after reaction. Moreover, this method needs more
optimization in the laboratory to meet even higher requirements for
industrial scale commercialization because of high cost and low
efficiency.
[0006] At present, most of the commercially available
3-hydroxybutyric acid is a racemic mixture, with the ratio of
(R)-3-hydroxybutyric acid to (S)-3-hydroxybutyric acid being about
one to one. Although study showed that (S)-3-hydroxybutyric acid is
not physiologically active, racemic 3-hydroxybutyric acid and its
salts, especially sodium salts, are still the main commodity form
and accepted by most consumers. It is expected that a single
optical (R)-3-hydroxybutyric acid and its salts will become popular
in future, replacing the racemic 3-hydroxybutyric acid and its
salts.
[0007] Generally, it is believed that naturally or biologically
produced compounds are safer (and non-toxic) than chemically
produced compounds. People prefer the "natural" or "biological"
feature of the source of pharmaceutical, food and cosmetic
ingredients.
BRIEF SUMMARY OF THE INVENTION
[0008] In order to produce "safe and nontoxic" (R)-3-hydroxybutyric
acid by a biosynthetic method, the inventors of this invention have
developed genetically modified nonpathogenic microorganisms and
unexpectedly found that fermentation with such genetically modified
nonpathogenic microorganisms in an unusually efficient one-step
process can produce (R)-3-hydroxybutyric acid with high yield and
high purity. Optimally engineered organisms have been screened and
selected by genetic engineering technology to enhance the
expression of genes associated with (R)-3-hydroxybutyric acid main
metabolic pathway and weaken the branched metabolic pathway. After
that, (R)-3-hydroxybutyric acid can be effectively accumulated in
the fermentation process, which has broad application prospect in
industrial scale.
[0009] Accordingly, in one aspect, the invention provides 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 the group consisting of succinyl-CoA
transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
[0010] The genetically modified microorganism is able to produce
(R)-3-hydroxybutyric acid by a novel (R)-3-hydroxybutyric acid
pathway which includes the following steps: [0011] (i) converting
pyruvic acid and coenzyme A to acetyl-CoA, [0012] (ii) converting
acetyl-CoA into acetoacetyl-CoA, [0013] (iii) converting
acetoacetyl-CoA to acetoacetic acid, and [0014] (iv) converting
acetoacetic acid to (R)-3-hydroxybutyric acid. The genetically
modified microorganism comprises one or more exogenous nucleic acid
encoding at least one (R)-3-hydroxybutyric acid pathway enzyme
expressed in a sufficient amount to produce (R)-3-hydroxybutyric
acid. The overexpressed pathway enzyme is selected from
succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB
dehydrogenase.
[0015] In some embodiments, the exogenous nucleic acid is a
codon-optimized gene for better expression in the genetically
modified microorganism.
[0016] In some embodiments, the overexpressed pathway enzyme is
selected from the group consisting of succinyl-CoA transferase,
acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
[0017] In some embodiments, the exogenous nucleic acid encoding
succinyl-CoA transferase is at least 90% identical to the
nucleotide sequence of SEQ ID NO:1, the exogenous nucleic acid
encoding acetoacetyl-CoA synthase is at least 90% identical to the
nucleotide sequence of SEQ ID NO:2, and the exogenous nucleic acid
encoding 3-HB dehydrogenase is at least 90% identical to the
nucleotide sequence of SEQ ID NO:3.
[0018] In some embodiments, the genetically modified microorganism
is a nonpathogenic microorganism. Examples of suitable
nonpathogenic microorganism includes, but not limited to,
Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium
lactofermentum, Brevibacterium difficile, Brevibacterium flavum, or
Brevibacterium breve.
[0019] In some preferred embodiments, the nonpathogenic
microorganism is Corynebacterium glutamicum and Bacillus subtilis.
In some more preferred embodiments, the microorganism is
Corynebacterium glutamicum (such as the strain deposited at the
China General Microbiological Culture Collection Center under the
accession number CGMCC No. 13111).
[0020] In another aspect, this invention provides a biological
method for efficiently producing (R)-3-hydroxybutyric acid using
the genetically modified microorganism. In some embodiments, the
method is a one-step fermentation process. In some preferred
embodiments, the genetically modified nonpathogenic microorganism
is capable of producing (R)-3-hydroxybutyric acid in a one-step
fermentation process of this invention.
[0021] Specifically, the present invention provides a process for
producing (R)-3-hydroxybutyric acid by fermenting with a genetical
modified nonpathogenic microorganism in a fermentation broth. In
this process, the fermentation broth comprises carbon and nitrogen
sources and at least one 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, and the
(R)-3-hydroxybutyric acid was recovered from fermentation broth
after it excreted into the broth during fermentation process. The
nonpathogenic microorganism is selected from a group consisting of
Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium
lactofermentum, Brevibacterium difficile, Brevibacterium flavum and
Brevibacterium breve.
[0022] In some embodiments, the carbon source comprises a member
selected from the group consisting of glucose, sucrose, maltose,
molasses, starch and glycerol. In some other embodiments, the
nitrogen source comprises a member selected from the group
consisting of an organic nitrogen source and an inorganic nitrogen
source. Examples of suitable organic nitrogen source include, but
are not limited to, corn steep liquor, bran hydrolyzate, soybean
cake hydrolyzate, yeast extract, yeast powder, peptone, and urea;
wherein Examples of suitable inorganic nitrogen source include, but
are not limited to, ammonium sulfate, ammonium nitrate, and aqueous
ammonia.
[0023] In some embodiments, the (R)-3-hydroxybutyric acid produced
by the process of this invention is free of bacterial endotoxin and
has a purity of 95% or more.
[0024] In some embodiments, the (R)-3-hydroxybutyric acid prepared
by the process of this invention is 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.
[0025] In another aspect, this invention provides a food grade
(R)-3-hydroxybutyric acid and salts thereof.
[0026] In still another aspect, this invention also provides food
grade racemic 3-hydroxybutyric acid and its salts to meet the needs
of the market. The racemic 3-hydroxybutyric acid is prepared by
racemization treatment of (R)-3-hydroxybutyric acid or a
(R)-3-hydroxybutyrate salt produced in accordance of the process of
this invention.
[0027] In yet another aspect, the invention provides a recombinant
nucleic acid segment encoding succinyl-CoA transferase,
acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
[0028] The invention comprises the following technical scheme:
[0029] Fermentation of (R)-3-hydroxybutyric acid is carried out by
using a genetically modified non-pathogenic microorganism, which
directly converts the carbon and nitrogen sources into
(R)-3-hydroxybutyric acid which is excreted into the fermentation
broth and could be recovered directly. The microorganism can be one
or more members selected from the group consisting of
Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium
lactofermentum, Brevibacterium difficile, Brevibacterium flavum and
Brevibacterium breve. These microorganisms have the following
biotransformation functions: converting pyruvic acid and coenzyme A
to acetyl-CoA; converting acetyl-CoA into acetoacetyl-CoA;
converting acetoacetyl-CoA to acetoacetic acid; and then converting
acetoacetic acid to (R)-3-hydroxybutyric acid.
[0030] Preferably, the microorganism over-expresses one or more
enzymes selected from the group consisting of succinyl-CoA
transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase (3-HB
Dehydrogenase, 3-HBDH).
[0031] More preferably, the microorganism overexpresses
succinyl-CoA transferase, acetoacetyl-CoA synthase and/or 3-HB
dehydrogenase.
[0032] Different media may be used for different microorganisms in
the fermentation process. The carbon source may be selected from
the group consisting of glucose, sucrose, maltose, molasses, starch
and glycerol. One or more organic or inorganic nitrogen sources may
be used in a fermentation medium. The organic nitrogen source may
come from the group consisting of corn steep liquor, bran
hydrolyzate, soybean cake hydrolyzate, yeast extract, yeast meal,
peptone and urea; and the inorganic nitrogen source may include one
or more compounds selected from the groups consisting of ammonium
sulfate, ammonium nitrate, and aqueous ammonia.
[0033] In a preferred embodiment of the present invention, the
fermentation medium includes glucose 75 g/L, corn steep liquor
25-30 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, KH.sub.2PO.sub.4 1.5
g/L, MgSO.sub.4.7H.sub.2O 0.5 g/L, urea 1.0 g/L, histidine 30 mg/L,
molasses 25 g/L, biotin 100 .mu.g/L, and defoamer 0.2 g/L when
Corynebacterium glutamicum was applied.
[0034] Preferably, the feed medium includes ammonium sulfate 500
g/L and glucose 650 g/L when batch feed fermentation was
performed.
[0035] The purity of the (R)-3-hydroxybutyric acid prepared by a
fermentation method of the present invention could be greater than
95%, greater than 96%, greater than 97%, greater than 98%, or even
greater than 99%.
[0036] Pure (R)-3-hydroxybutyric acid thus prepared does not
contain bacterial endotoxin and chemical odor such as
bitterness.
[0037] (R)-3-hydroxybutyric acid is an acid which forms a salt with
a base. Alternatively, the (R)-3-hydroxybutyric acid prepared by
present invention may exist in the form of a salt, such as sodium
salt, potassium salt, magnesium salt, or calcium salt, preferably
in the form of a sodium salt. These salts may all be optically
active compounds.
[0038] Since racemic 3-hydroxybutyric acid and its sodium salt have
traditionally been accepted by food and pharmaceutical
manufacturers and consumers, (R)-3-hydroxybutyric acid and its
salts can be subjected to racemic treatment to prepare racemic
3-hydroxybutyric acid and its salts. For example, racemization can
be achieved by heating (R)-3-hydroxybutyric acid in an alkaline
solution such as sodium hydroxide solution for a certain time.
[0039] The racemic 3-hydroxybutyrate salt may be sodium
3-hydroxybutyrate, potassium 3-hydroxybutyrate, magnesium
3-hydroxybutyrate, or calcium 3-hydroxybutyrate.
[0040] (R)-3-hydroxybutyric acid and its salts prepared by the
present invention are free of bacterial endotoxin and toxic
chemicals, therefore ensuring food safety. In addition,
(R)-3-hydroxybutyric acid can be used directly to manufacture
pharmaceuticals and health products as it contains no chemical
residues or chemical reaction impurities, and it does not have
chemical odor such as bitterness as well.
[0041] The genetically engineered microorganisms constructed in the
present invention can effectively produce and accumulate
(R)-3-hydroxybutyric acid in the fermentation broth during the
fermentation process, and could give rise to food-grade (R) level
by downstream process, which has broad industrial prospects.
[0042] Corynebacterium glutamicum has been engineered to produce
(R)-3-hydroxybutyric acid at high yield. A strain of this
microorganism has been deposited under the accession number CGMCC
No. 13111 on Oct. 14, 2016, at China General Microbiological
Culture Collection Center, located in Institution of Microbiology,
Chinese Academy of Sciences, Building 3, No. 1 Beicheng West Road,
Chaoyang District, Beijing, China 100101 (www.cgmee.net).
[0043] As used herein, the term "one-step fermentation" refers to a
fermentation process that includes adding one or more fermenting
agents (e.g., a microorganism) to a fermentation medium which is
fermented to give the desired product, without having to add a
second round of fermenting agent and then going through a second
round of fermentation process.
[0044] As used herein, the term "nonpathogenic microorganism"
refers to a microorganism that generally does not cause disease,
harm or death to another microorganism, an organism, or human
being.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows an exemplary pathway for producing
(R)-3-hydroxybutyric acid. The first step is a native pathway of
the microorganism, and the other steps are genetically modified
pathways, in which enzymes are overexpressed. The overexpressed
enzymes catalyzing the pathway reactions are acetoacetyl-CoA
synthase, Succinyl-CoA transferase and 3-HB dehydrogenase.
[0046] FIG. 2 shows the plasmid map for the vector
pXMJ19-t3-OXCT1-BDH1.
[0047] FIG. 3 shows the plasmid map for the vector
pMA5-t3-OXCT1-BDH1.
[0048] FIG. 4 shows an HPLC spectrum showing the production of
(R)-3-hydroxybutyric acid from the genetically modified
nonpathogenic microorganism (Top: reference, Bottom: sample).
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.degree. 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..about.32.degree. C. during the initial stage of
fermentation and then increased to 34.sup..about.37.degree. 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..about.8.0,
preferably at pH 6.5.sup..about.7.0, during the initial stage of
fermentation. It can then be adjusted to 6.8.sup..about.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..about.3.0%
during fermentation process, more precisely at
1.5%.sup..about.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.degree. C. or lower, preferably at
25.degree. C. or lower, and more preferably at 20.degree. 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.
FIG. 1 depicts the exemplary pathway (with the native pathway of
the microorganism in the black frame; the engineered pathway, in
which enzyme are overexpressed, in the red frame; and the
overexpressed enzymes labeled near the red arrows.
[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. FIG. 2 is the plasmid map for
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.degree. 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 .mu.g/L of biotin, pH 7.0). The cells were
cultured at 30.degree. 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..about.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.degree. 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.degree. C.
The dissolved oxygen concentration (dO.sub.2) was controlled at
15.sup..about.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..about.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
FIG. 1.
[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. FIG. 3 is
the plasmid map for 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.degree. 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.degree. 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..about.7.5 after autoclaving. The tank
pressure was maintained at 0.05 Mpa, and the cultivation
temperature was controlled at 37.degree. 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..about.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..about.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.times.4.6). The mobile phase
consisted of acetonitrile: water (v/v)=15:85, UV detection
wavelength was 210 nm, injection volume was 20 .mu.L, flow rate was
1 mL/min, column temperature was 10.degree. C. The purity of
(R)-3-hydroxybutyric acid was 98% and the specific optical value
was [.alpha.] D20=-25.degree. (C=6%, H.sub.2O).
[0095] FIG. 4 is HPLC spectrum showing the production of
(R)-3-hydroxybutyric acid from the genetically modified
microorganism (Top: reference, Bottom: sample).
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.degree. 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.degree. C. and a specific
optical value [.alpha.].sub.D.sup.20 of -14.1.degree. (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.
Sequence CWU 1
1
311560DNAArtificial SequenceCodon Optimized Nucleic Acid Sequence
1atggcagctc tgaaactgct gtcttctggt ctgcgtctgt gtgcttctgc tcgcggttct
60ggtgccactt ggtacaaagg ctgcgtctgt agcttcagca cctctgctca tcgccacacc
120aaattctaca ctgaccctgt tgaagctgtg aaagacatcc cggatggtgc
taccgtactg 180gtaggtggtt tcggcctgtg cggtatcccg gagaatctga
tcgacgctct gctgaaaacc 240ggcgtaaaag gtctgaccgc agtaagcaac
aacgcaggcg tggacaattt cggtctgggc 300ctgctgctgc gttctaaaca
gatcaaacgc atggtatcct cctacgttgg tgagaacgca 360gaattcgaac
gtcagtacct gagcggtgag ctggaagtgg aactgacccc gcagggtacc
420ctggcagaac gtatccgtgc tggtggtgcg ggtgtgcctg ctttctatac
cccgactggc 480tatggcactc tggtacagga aggtggctcc ccgatcaaat
acaacaaaga cggctctgtg 540gcaatcgcat ccaaacctcg tgaagtgcgt
gaattcaacg gccagcactt catcctggaa 600gaagcaatta ccggcgactt
cgcactggta aaagcatgga aagcggatcg tgcgggtaac 660gttatctttc
gcaaatctgc acgcaacttc aatctgccga tgtgcaaagc ggcagaaacc
720accgttgttg aggtagagga aatcgtggac attggcgctt tcgcgccgga
agacatccac 780attccgcaga tctacgttca ccgtctgatc aaaggcgaaa
aatatgaaaa acgtattgaa 840cgcctgtcta tccgtaagga aggtgatggc
gaggccaaaa gcgcgaaacc gggcgatgac 900gtacgtgaac gcatcatcaa
acgtgcagca ctggaattcg aagacggcat gtacgccaac 960ctgggcattg
gcattccgct gctggcctcc aacttcatct ccccgaatat cactgtgcac
1020ctgcagtctg aaaacggtgt gctgggtctg ggtccgtatc cgcgtcagca
cgaggcagac 1080gcagatctga ttaatgccgg taaggaaact gtgacgattc
tgccgggcgc gtcctttttc 1140agctccgacg aatcttttgc tatgatccgt
ggcggtcacg tggatctgac catgctgggt 1200gcgatgcagg tatctaagta
cggcgacctg gcgaactgga tgatccctgg taaaatggta 1260aaaggtatgg
gcggcgcaat ggacctggtt agctctgcaa aaaccaaagt ggtggtcact
1320atggaacact ctgccaaagg taacgctcac aaaatcatgg aaaagtgcac
cctgccgctg 1380actggtaaac agtgtgtaaa tcgtatcatc actgagaaag
cagttttcga cgtagataag 1440aagaagggtc tgaccctgat cgaactgtgg
gaaggcctga ccgtcgatga tgttcagaaa 1500agcaccggtt gcgatttcgc
cgtttcccca aagctgatgc cgatgcagca gatcgcgaac 156021176DNAArtificial
SequenceCodon Optimized Nucleic Acid Sequence 2atgcaggaag
ttgtaatcgt tgcagctcgt cgtactgcag taggcgcgtt cggtggttct 60ctggcaggta
tcccggcgag cgatctgggt gcactggtta tcaaagacat tctggcgagc
120actggtgtag ctccggatca ggttgacgaa gttctgctgg gtcaggtact
gaccgcaggt 180gtgggtcaga atccggctcg tcaggcggct atcaaaggtg
gtctgtccga ttccgtgccg 240gctatgacta tcaacaaagt gtgcggctct
ggtctgaaag ctctgcatct ggcgacccaa 300gctatccgtt gcggtgacgc
ggaactgatc ctggctggtg gccaagaaaa catgagcgcg 360tctccgcacg
tgctgccgaa ctctcgtaac ggccagcgta tgggcgattg gaaagctatc
420gactccatgg tgcacgatgg tctgtgggac gctttcaaca actaccacat
gggcattacg 480gctgaaaacc tggcggaaaa gtactccatt acccgtgaag
ctatggacga gttcgcggct 540gcatctcagc agaaagctgc tgcggctatc
cgcgaaggca aatttaaaag ccagatcgta 600cctgttgaaa ttccacaacg
caaaggtgat ccagttgtat ttgacaccga cgaaaacccg 660cgtgaggtta
ccgcggagaa actgggtggc atgcgtccgg cttttaaaaa ggacggtact
720gtaactgcag gcaacgcttc cgcgctgaac gacggtgctg ctatcgtaat
gctgtgctct 780gcggagaaag cgaaacaact gggtctggaa ccgctggcac
gtatcgcggc gtacgcgaac 840gctggtgttg acccagctat tatgggtatc
ggtccagctc cggctacgcg tcgttgtctg 900gagaaagcgg gttggtccct
ggacgacctg gatctggttg aggcgaacga agcgtttgct 960gctcaggccc
tgtctgttaa taaagaactg ggctgggatg tatccaaagt taacgttaac
1020ggtggtgcga tcgctctggg tcacccaatt ggcgcttccg gttgccgtgt
tctgattacc 1080ctgctgcacg agatgattgc ccgtgatgcg aaaaaaggtc
tggcaactct gtgcatcggc 1140ggtggtcaag gcgttgctct ggcgattgaa cgtccg
117631029DNAArtificial SequenceCodon Optimized Nucleic Acid
Sequence 3atgctggcta ctcgtctgtc tcgtcctctg tctcgtctgc ctggtaaaac
tctgtccgcg 60tgcgatcgtg aaaatggtgc acgtcgtcca ctgctgctgg gttccacctc
tttcatccca 120atcggtcgtc gtacctacgc ttccgcagca gaacctgttg
gttctaaggc tgtgctggtg 180accggttgtg attctggttt tggtttctcc
ctggcaaaac atctgcacag caaaggtttt 240ctggttttcg cgggctgcct
gatgaaggat aagggccatg atggcgtgaa agaactggac 300tctctgaaca
gcgatcgtct gcgtaccgtg cagctgaacg tgtgttcttc tgaagaagtg
360gagaaagttg tggagattgt tcgttcctct ctgaaagacc cggagaaagg
tatgtggggt 420ctggtcaaca acgcgggtat ctctactttc ggtgaagttg
aattcaccag cctggaaacc 480tacaaacagg tggcggaagt gaacctgtgg
ggtaccgtgc gtatgactaa atccttcctg 540ccgctgattc gtcgcgctaa
aggtcgtgtg gtcaacattt cctctatgct gggtcgtatg 600gcaaacccag
cgcgttcccc gtactgtatt accaaattcg gcgttgaagc gttctccgac
660tgcctgcgct atgaaatgta tccgctgggt gtaaaagtat ctgttgttga
accgggtaac 720tttatcgctg cgactagcct gtatagcccg gaaagcatcc
aggctatcgc taaaaaaatg 780tgggaggaac tgccggaagt agttcgtaaa
gactatggta agaagtattt cgacgaaaaa 840attgcgaaga tggaaaccta
ctgctcctct ggttccaccg acacctcccc ggttatcgac 900gcggttaccc
acgctctgac cgccactacc ccgtacaccc gttaccaccc gatggattac
960tactggtggc tgcgtatgca gattatgact cacctgccgg gtgcgatttc
tgacatgatc 1020tacatccgt 1029
* * * * *