CN105779520A - Method for promoting corynebacterium glutamicum to grow and to produce L-serine - Google Patents
Method for promoting corynebacterium glutamicum to grow and to produce L-serine Download PDFInfo
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- CN105779520A CN105779520A CN201510570110.6A CN201510570110A CN105779520A CN 105779520 A CN105779520 A CN 105779520A CN 201510570110 A CN201510570110 A CN 201510570110A CN 105779520 A CN105779520 A CN 105779520A
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- serine
- corynebacterium glutamicum
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Abstract
The invention provides a method for promoting corynebacterium glutamicum to grow and to produce L-serine with a high output the addition of protocatechuic acid (PCA). Based upon a result, by adding the protocatechuic acid to a culture medium, the growth of the corynebacterium glutamicum SYPS-062-33a [delta]SSA, corynebacterium glutamicum SYPS-062-33a [delta]SSA OD562 reaches 54.32, and the dry weight of the bacteria reaches 10.3g/L, which is 87% higher than that without the addition of the protocatechuic acid; and the yield of the L-serine reaches 19.75g/L, improved by 1.78 times in comparison with that of a control group, so that the efficiency of producing the L-serine by virtue of the corynebacterium glutamicum is improved.
Description
Technical field
Present invention relates particularly to a kind of protocatechuic acid that adds and promote Corynebacterium glutamicum growth and the side of high yield Serine
Method, belongs to biological technical field.
Background technology
Serine belongs to nonessential amino acid, has important physiological function and using value, be widely used in medicine,
Food and chemical field.Serine is applied to the composition of amino acid transfusion；Phonetic for purine, choline, thymus gland as intermediate
Pyridine and the synthesis of DOPA；Generating phosphatidyl serine after the hydroxyl of Serine is phosphorylated, this material has important life
Reason function；Serine has important medicinal function as the product of parent nucleus derivative reaction.Seromycin is a kind of wide spectrum
Antibiotic is used for treating tuberculosis, is also the intermediate of beta-Lactam antibiotic synthesis；The most important derivative is exactly
Azaserine, its except in addition to treating the medicine of tumour or the antimetabolite of glutamine and for acute leukemia with
The treatment of Ke Jie king's evil；Serine can stablize the pH of eye drops, and to eye nonirritant.The life of Serine at present
Product depends on albumen hydrolysis, enzymatic conversion method method and microorganism Precurosor fermentation method, however these methods to there is yield little, raw
Product cost is high, and the problems such as environmental pollution is big are not suitable for carrying out large-scale industrial production Serine.Microbe fermentation method utilizes
It is a kind of continuable mode of production of environmental protection that the direct metabolism of reproducible saccharine material produces Serine, has become as and grinds
Study carefully the focus of Serine industrialized production.But this production method is owing to being limited by bacterial strain, there is no all the time into
The breakthrough of one step, this also makes Serine become bottleneck amino acid.
Corynebacterium glutamicum (Corynebacterium glutamicum) is important amino acid preparation strain, extensively should
Glutamic acid, lysine and valine etc. are produced for fermentation method.At present in the bacterial strain research building high yield Serine, mainly
Concentrate on the releasing Serine feedback inhibition phenomenon to Serine route of synthesis key enzyme 3-phoshoglyceric acid (PGDH), fall
The degradation pathway etc. of low Serine.This laboratory is with Corynebacterium glutamicum SYPS-062-33a (the bacterial strain preservation of a strain mutagenesis
Number CGMCC No.8667) by releasing the Serine feedback inhibition to PGDH, the degraded etc. reducing Serine obtains energy
Sucrose direct fermentation is enough utilized to produce recombinant bacterial strain SYPS-062-33a Δ SSA (the bacterial strain preserving number of Serine
CGMCCNo.8668)。
The invention provides a kind of protocatechuic acid (PCA) that adds and promote Corynebacterium glutamicum growth and high yield Serine
Method, result shows to add protocatechuic acid to the life that can promote Corynebacterium glutamicum SYPS-062-33a Δ SSA in culture medium
Long, Corynebacterium glutamicum SYPS-062-33a Δ SSA OD562Reaching 54.32, dry cell weight reaches 10.3g/L, is relatively not added with former youngster
87% is improve during boheic acid；Serine yield reaches 19.75g/L, and relatively comparison improves 1.78 times, improves and utilizes glutamic acid
Bar bacterium produces the efficiency of Serine.
Summary of the invention
The present invention provides a kind of protocatechuic acid that adds to promote that Corynebacterium glutamicum growth puies forward the method producing Serine yield.
For solving above-mentioned technical problem, concrete technical scheme provided by the present invention is as follows:
It is that every liter of culture medium adds 10mg, 30mg, 50mg protocatechuic acid by addition, adds Corynebacterium glutamicum fermentation to
Produce in the full-synthetic culture medium of Serine, Corynebacterium glutamicum SYPS-062-33a Δ SSA is carried out fermented and cultured, 30 DEG C,
220rpm, detection Corynebacterium glutamicum SYPS-062-33a Δ SSA growth and the situation of product Serine.
Beneficial effects of the present invention is as follows:
The protocatechuic acid that the present invention provides is added to Corynebacterium glutamicum full-synthetic culture medium carries out shaker fermentation training
Supporting, Corynebacterium glutamicum biomass in fully synthetic fermentation medium can be made to improve about 87%, Serine yield (g/L) carries
High nearly 1.78 times, improve the efficiency utilizing Corynebacterium glutamicum to produce Serine.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the present invention above-mentioned basic fundamental premise, it is also possible to make the amendment of other various ways, replace or change.Such as by former catechu
Acid is dissolved in different solvents the protocatechuic acid etc. adding, adding variable concentrations.
The foregoing of the present invention is described in further details by detailed description of the invention below by way of example forms again, but
This should not be interpreted as the scope of the above-mentioned theme of the present invention is only limitted to following instance, all realized based on foregoing of the present invention
Technical field belongs to the scope of the present invention.
Detailed description of the invention
Embodiment 1: be not added with Corynebacterium glutamicum growth and the ability of product Serine during protocatechuic acid
1. bacterial classification: with Corynebacterium glutamicum SYPS-062-33a Δ SSA (Corynebacterium glutamicum) bacterium
Strain (preserving number CGMCCNo.8668) carries out the production of Serine as the bacterium that sets out.
2. seed culture medium: sucrose 2%；BHI 3.7%；Ammonium sulfate 1%；MgSO4·7H2O 0.05%；
NaH2PO40.03%；K2HPO40.02%；
Fermentation medium: sucrose 10.0%；(NH4)2SO43.0%；CaCO33.0%；K2HPO40.3%；MgSO4·7H2O
0.05%；FeSO4·7H2O 0.002%；MnSO4·H2O 0.002%；Biotin 50μg/L；Thiamine·HCl 450μ
g/L；pH 7.0-7.2；3. Corynebacterium glutamicum fermented and cultured: utilize oese that bacterial strain lines seed culture medium solid and put down
In plate, being placed in by flat board in 30 DEG C of constant incubators and cultivate to growing single bacterium colony, oese is drawn respectively and is taken a ring thalline and be seeded to
In 20ml seed culture medium, 30 DEG C, 120rpm cultivates to mid log phase, is seeded to 25ml shaking flask according to the inoculation ratio of 5%
In fermentation medium, 30 DEG C, 120rpm fermented and cultured.Often group does 3 repetitions.
4. the detection of biomass: every 12h fermentation sampling, utilizes spectrophotometric determination biomass (OD562).Biomass (g/
L)=0.1925*OD562-0.0994；
5. the detection of amino acid concentration: it is as follows that HPLC method measures amino acid concentration condition in zymotic fluid；
By zymotic fluid ddH2O is diluted to suitable concn, using phenyl isothiocyanate (PITC) as derivating agent by amino acid
Derivatization, product phenylamino acid formyl sulfide derivative (PTC-AA) has stable strong UV absorption at 254nm, uses Venusil-
AA amino acid analysis dedicated columns (4.6 × 250mm, 5 μm), detects derivative under ultraviolet.
Mobile phase A phase: 92.6mM sodium acetate, is adjusted to 6.50 ± 0.05 with glacial acetic acid by pH；Mobile phase B phase: 80% second
Nitrile, flow velocity 1.0ml/min, column temperature: 40 DEG C, UV-detector wavelength 254nm.Gradient elution program is: 0～4min, 100%A；
4～16min, 97%A；16～17min, 89%；17～32min, 79%A；32～34min, 66%A；34～38min, 0%A；
38.01,100%A.
6. amino acid standard items and analyte derivative step:
(1) accurately measure amino acid hybrid standard liquid and sample 200 μ l, be respectively placed in 1.5ml centrifuge tube；
(2) pipe often accurately adds nor-leucine internal standard liquid 20 μ l；
(3) in each centrifuge tube, triethylamine acetonitrile solution 100 μ l, phenyl isothiocyanate acetonitrile solution 100 μ are added respectively
L, mixing, room temperature placing response 1h；
(4) add n-hexane 400 μ l, acutely place 10min after concussion；
(5) take off a layer PTC-AA solution, filter by 0.45 μm syringe filter；
(6) take filtration clear liquid 100 μ l, add 400 μ l ddH2O dilutes, and shakes up, sample introduction 20 μ l
Solution required in analyte derivativeization reaction is as follows:
Triethylamine acetonitrile: triethylamine 1.4ml+ acetonitrile 8.6ml, mixing；-20 DEG C of preservations.
Phenyl isothiocyanate acetonitrile solution: PITC 25 μ l, adds acetonitrile 2ml, mixing.(airtight, 4 DEG C of placement shelf-lifves are
7 days)
Nor-leucine internal standard liquid: weigh nor-leucine 10mg, adds 0.1M hydrochloric acid solution 10ml and makes it dissolve, mixing, 4 DEG C
Preserve.
Experimental result is shown in Table 1, when without protocatechuic acid, Corynebacterium glutamicum SYPS-062-33a Δ SSA poor growth
And it is inefficient to produce acid.
Table 1 is not added with Corynebacterium glutamicum SYPS-062-33a Δ SSA growth and the ability of product acid during protocatechuic acid
| Time (h) | Biomass (g/L) | Serine (g/L) |
| 0 | 0.0931 | ND |
| 60 | 2.0945 | 1.8259 |
| 120 | 5.5132 | 7.1257 |
Embodiment 2: add not same amount protocatechuic acid and Corynebacterium glutamicum is grown and the impact of Serine yield
1. bacterial classification: with Corynebacterium glutamicum SYPS-062-33a Δ SSA (Corynebacterium glutamicum) bacterium
Strain (preserving number CGMCCNo.8668) carries out the production of Serine as the bacterium that sets out.
2. seed culture medium: sucrose 2%；BHI 3.7%；Ammonium sulfate 1%；MgSO4·7H2O 0.05%；
NaH2PO40.03%；K2HPO40.02%；
Fermentation medium: sucrose 10.0%；(NH4)2SO43.0%；CaCO33.0%；K2HPO40.3%；MgSO4·7H2O
0.05%；FeSO4·7H2O 0.002%；MnSO4·H2O 0.002%；Biotin 50μg/L；Thiamine·HCl 450μ
g/L；pH 7.0-7.2；3. the interpolation of protocatechuic acid: be that every liter of culture medium adds the former catechu of 10mg, 30mg, 50mg by addition
Acid, adds in the full-synthetic culture medium that Serine is produced in Corynebacterium glutamicum fermentation；
4. Corynebacterium glutamicum fermented and cultured: with reference to embodiment example one；
5. the detection of biomass: with reference to embodiment example one；
6. the detection of amino acid concentration: with reference to embodiment example one；
7. amino acid standard items and analyte derivative step: with reference to embodiment example one；
Related experiment the results are shown in Table 2, table 3, table 4, and result shows that every liter of culture medium adds 30mg protocatechuic acid best results,
Corynebacterium glutamicum SYPS-062-33a Δ SSA growth OD562 reaches 54.32, and biomass reaches 10.3g/L, is relatively not added with former youngster
87% is improve during boheic acid；Serine yield reaches 19.75g/L, and relatively comparison improves 1.78 times.
Every liter of culture medium of table 2 adds 10mg protocatechuic acid to Corynebacterium glutamicum SYPS-062-33a Δ SSA
Growth and the impact of product acid
| Time (h) | Biomass (g/L) | Serine (g/L) |
| 0 | 0.0931 | 0 |
| 12 | 0.1362 | 0.0972 |
| 24 | 0.2915 | 0.2617 |
| 36 | 1.3932 | 0.8251 |
| 48 | 2.8910 | 1.6239 |
| 60 | 5.5132 | 3.9812 |
| 72 | 6.1982 | 7.1284 |
| 84 | 6.9584 | 9.1920 |
| 96 | 7.2983 | 10.4107 |
| 108 | 7.5271 | 11.2063 |
| 120 | 7.6829 | 12.5785 |
Every liter of culture medium of table 3 adds 30mg protocatechuic acid to Corynebacterium glutamicum SYPS-062-33a Δ SSA
Growth and the impact of product acid
| Time (h) | Biomass (g/L) | Serine (g/L) |
| 0 | 0.0931 | 0 |
| 12 | 0.2413 | 0.1832 |
| 24 | 0.4820 | 0.5636 |
| 36 | 1.7024 | 1.2525 |
| 48 | 4.4494 | 2.5937 |
| 60 | 6.8248 | 5.6443 |
| 72 | 8.5881 | 10.1383 |
| 84 | 9.4505 | 16.2729 |
| 96 | 9.3832 | 17.4378 |
| 108 | 10.2244 | 18.5409 |
| 120 | 10.3572 | 19.7511 |
Every liter of culture medium of table 4 adds 50mg protocatechuic acid to Corynebacterium glutamicum SYPS-062-33a Δ SSA
Growth and the impact of product acid
| Time (h) | Biomass (g/L) | Serine (g/L) |
| 0 | 0.0931 | 0 |
| 12 | 0.1926 | 0.1535 |
| 24 | 0.3505 | 0.4926 |
| 36 | 1.5247 | 1.0129 |
| 48 | 3.9122 | 2.2078 |
| 60 | 6.1356 | 5.1204 |
| 72 | 7.8492 | 9.3728 |
| 84 | 8.5391 | 13.8617 |
| 96 | 9.1920 | 15.3168 |
| 108 | 9.6235 | 16.4521 |
| 120 | 9.8542 | 17.2190 |
Claims (3)
1. the method promoting Corynebacterium glutamicum to grow and improve Serine yield, is characterized by, adds in the medium
Enter protocatechuic acid.
2. the method described in claim 1, is characterized by, is that every liter of culture medium adds the former youngster of 10mg, 30mg, 50mg by addition
Boheic acid, adds in the full-synthetic culture medium that Serine is produced in Corynebacterium glutamicum fermentation, to Corynebacterium glutamicum SYPS-062-
33a Δ SSA carries out fermented and cultured, 30 DEG C, 220rpm.
3. the method described in claim 1, is characterized by, preferably adds the amount of protocatechuic acid, is added to for every liter of culture medium
The protocatechuic acid of 30mg.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486133A (en) * | 2018-06-29 | 2018-09-04 | 江南大学 | A kind of application process of Serine transport protein |
| CN110229853A (en) * | 2019-07-02 | 2019-09-13 | 武汉轻工大学 | A kind of preparation method of Serine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966704A (en) * | 2006-11-21 | 2007-05-23 | 江南大学 | Strain for production of L-serine and method for production of L-serine by using same |
| CN104561076A (en) * | 2014-11-28 | 2015-04-29 | 江南大学 | Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum |
-
2015
- 2015-09-09 CN CN201510570110.6A patent/CN105779520A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966704A (en) * | 2006-11-21 | 2007-05-23 | 江南大学 | Strain for production of L-serine and method for production of L-serine by using same |
| CN104561076A (en) * | 2014-11-28 | 2015-04-29 | 江南大学 | Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum |
Non-Patent Citations (3)
| Title |
|---|
| UNTHAN S.等: "Beyond Growth Rate 0.6: What Drives Corynebacterium glutamicum to Higher Growth Rates in Defined Medium", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 张晓娟等: "维生素对谷氨酸棒杆菌SYPS-062直接发酵合成L-丝氨酸的影响", 《中国生物工程杂志》 * |
| 罗玉常: "产L_丝氨酸谷氨酸棒杆菌SYPS_062的代谢工程", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486133A (en) * | 2018-06-29 | 2018-09-04 | 江南大学 | A kind of application process of Serine transport protein |
| CN108486133B (en) * | 2018-06-29 | 2021-06-01 | 江南大学 | Application method of L-serine transport protein |
| CN110229853A (en) * | 2019-07-02 | 2019-09-13 | 武汉轻工大学 | A kind of preparation method of Serine |
| CN110229853B (en) * | 2019-07-02 | 2021-06-01 | 武汉轻工大学 | Preparation method of L-serine |
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