CN115895976B - Escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan - Google Patents
Escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan Download PDFInfo
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 109
- 229960004799 tryptophan Drugs 0.000 title claims abstract description 68
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 35
- 238000000855 fermentation Methods 0.000 claims abstract description 76
- 230000004151 fermentation Effects 0.000 claims abstract description 76
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 22
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 36
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 235000013619 trace mineral Nutrition 0.000 claims description 10
- 239000011573 trace mineral Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
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- 238000002360 preparation method Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229930003451 Vitamin B1 Natural products 0.000 claims description 2
- 229930003756 Vitamin B7 Natural products 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 235000010374 vitamin B1 Nutrition 0.000 claims description 2
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- 239000011735 vitamin B7 Substances 0.000 claims description 2
- 239000002253 acid Substances 0.000 abstract description 19
- 238000009825 accumulation Methods 0.000 abstract description 8
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 abstract description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 28
- 238000004519 manufacturing process Methods 0.000 description 26
- 238000012216 screening Methods 0.000 description 16
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
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- 235000013922 glutamic acid Nutrition 0.000 description 8
- 239000004220 glutamic acid Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 239000001384 succinic acid Substances 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
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- 230000002068 genetic effect Effects 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- 238000005516 engineering process Methods 0.000 description 3
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- 239000011734 sodium Substances 0.000 description 3
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000080590 Niso Species 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 230000006861 primary carbon metabolism Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
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- 239000002002 slurry Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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Abstract
The invention discloses an escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan, and belongs to the technical field of fermentation. The invention aims to solve the problems of large accumulation of mixed acid and high extraction difficulty in the fermentation process of the L-tryptophan strain. The invention discloses a method for high-yield L-tryptophan, which comprises the specific steps of fermenting escherichia coli XYSH220517-04 at 36 ℃ for at least 32 hours. The escherichia coli is named as XYSH220517-04 and is preserved in China general microbiological culture collection center, the preservation date is 2022, 9 and 19 days, and the strain preservation number is CGMCC NO.25753. The strain has stable fermentation performance, the content of the mixed acid in the fermentation liquor is obviously reduced, the extraction yield of the L-tryptophan is improved from 68% to 80%, and the strain has the characteristics of short period, high acid yield and high extraction yield.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan.
Background
L-tryptophan is an essential amino acid in the vital activities of the human and animal bodies, plays an important role in the metabolism and growth and development processes of the human and animal bodies, and is called a second essential amino acid due to its irreplaceable physiological role. At present, L-tryptophan is widely applied to industries such as food, medicine and feed, and plays an important role in agricultural monitoring and environmental monitoring along with the technical development. L-tryptophan was first produced by proteolytic and chemical synthesis methods, but was phased out after 90 s of the last century due to defects such as complicated material supply, complicated production process, long cycle, unstable product quality, and the like. With the intensive research on the functions of microorganisms, the fermentation method for producing L-tryptophan is gradually and deeply popularized, and the fermentation method is practical and becomes the mainstream.
Compared with the traditional chemical synthesis method and protein hydrolysis method, the microbial fermentation method has the remarkable advantages of lower production cost, more environment protection, easier mass production and the like. In the process of producing L-tryptophan by a microbial fermentation method, the advantages and disadvantages of microbial strains determine the production process, the production cost and even the product quality in the whole fermentation production process.
At present, the difficult problem puzzling the industry is that the synthesis route of L-tryptophan is complex, a large amount of glutamic acid, organic acid and other fermentation byproducts can be accumulated in the fermentation process, the yield and conversion rate of the L-tryptophan are seriously influenced, and meanwhile, the L-tryptophan is extremely easy to decompose when coexisting with the hetero acid, so that the extraction difficulty of fermentation liquor is high, and the extraction yield is low. Therefore, the strain with low accumulation of glutamic acid and organic acid in the metabolic process is bred, and has important significance for improving the yield, acid production efficiency and extraction yield of L-tryptophan.
Patent CN 110643559B (application of transport carrier gene for improving L-tryptophan production efficiency in escherichia coli) mentions that L tryptophan accumulation amount of genetically modified tryptophan strain fermented for 24h in shake flask is 15.2g/L, and the shake flask adopts a mode of feeding glucose and ammonia water.
Zhao Chunguang et al in 2017 (rational construction of L-tryptophan-producing strains with deletion of acetate kinase-encoding genes) report that glutamic acid is accumulated due to imbalance of energy metabolism and reducing power metabolism during fermentation of tryptophan strains, the accumulation amount of glutamic acid is 7-9g/L, and the accumulation amount of byproduct glutamic acid is high, which results in decrease of tryptophan yield and affects tryptophan extraction yield and purity.
Succinic acid and citric acid are organic acids in TCA (ternary ammonium salt) circulation, and in the fermentation process of escherichia coli, the accumulation of succinic acid and citric acid is greatly caused by unsmooth TCA circulation, and the accumulation of succinic acid and citric acid in fermentation liquor of an original strain TRTH (bacterial strain TRTH) of the patent is more than 15g/L, so that the yield of tryptophan is seriously influenced, and the extraction of the fermentation liquor is greatly difficult.
The BoXiong et al document 2021 (Flux redistribution of central carbon metabolism for efficient production of L-tryptophan in Escherichia coli) reports that strain SX11 was grown in a 5 liter fermenter for 40 hours with a tryptophan yield of 41.7g/L, a conversion of 22.7% and an acid production rate of 1.04g/L/h.
Disclosure of Invention
The invention aims to solve the problems of large accumulation of mixed acid and high extraction difficulty in the fermentation process of the L-tryptophan strain, and the extraction yield of the L-tryptophan is obviously improved, so that the production cost is greatly reduced.
The invention provides Escherichia coli (Escherichia coli), which is named XYSH220517-04 and is preserved in China general microbiological culture collection center (CGMCC) with a preservation date of 2022, 9 months and 19 days and a strain preservation number of CGMCC NO.5753.
The present invention provides a microbial preparation containing the above Escherichia coli.
The invention provides the application of the escherichia coli or the microbial preparation in improving the yield, the conversion rate and the extraction yield of the L-tryptophan.
The invention provides a method for high-yield L-tryptophan, which comprises the specific steps of fermenting the escherichia coli at 36 ℃ for at least 32 hours.
Further defined, the fermentation conditions of the escherichia coli also comprise a pH value of 7.0, 25% -30% of dissolved oxygen and a tank pressure of 0.02MPa.
Further defined, the medium used for fermentation is glucose as a carbon source.
Further defined, the medium used for fermentation is yeast powder as the nitrogen source.
Further defined, the fermentation medium for fermentation contains glucose, yeast powder, citric acid, (NH 4) 2 SO 4 、KH 2 PO 4 、MgSO 4 ·7H 2 O、FeSO 4 ·7H 2 O, vitamin B1, vitamin H and trace elements.
Further defined, the fermentation medium for fermentation contains 10-20g/L glucose, 4-6g/L yeast powder, 1-4g/L citric acid, (NH) 4 ) 2 SO 4 2-8g/L，KH 2 PO 4 4-6g/L，MgSO 4 ·7H 2 O 1-2g/L，FeSO 4 ·7H 2 50-80mg/L of O, 1 5-7mg/L of VB, 0.2-0.6mg/L of VH and 1-2mL/L of trace element mixed solution.
The invention provides application of the method in improving the conversion rate and the extraction yield of L-tryptophan and reducing the fermentation period.
The beneficial effects are that: an Escherichia coli producing L-tryptophan and a method for producing L-tryptophan, wherein the Escherichia coli is Escherichia coli (Escherichia coli) XYSH220517-04, the Escherichia coli XYSH220517-04 is preserved in China general microbiological culture collection center (CGMCC), the preservation date is 2022, 9 months and 19 days, and the strain preservation number is CGMCC NO.25753. The invention combines the ARTP mutation breeding technology and the chemical mutation screening technology of escherichia coli E.coli TRTH, the strain is obtained by screening, and L-tryptophan can be accumulated to 18.26g/L within 24h by using the strain for shake flask fermentation, thus the strain is the highest shake flask tryptophan yield reported in China at present; the strain can accumulate L-tryptophan to 60.56g/L after fermentation for 32 hours in a 50L fermentation tank, and is improved by 32.20% compared with the original strain, and has the characteristics of short period and high acid production. In industrial production, the strain XYSH220517-04 has stable fermentation performance, the content of the hetero acid in the fermentation liquor is obviously reduced, the extraction yield of the L-tryptophan is greatly reduced from 68% to 80%, and the technology is greatly improved.
[ biological preservation ]: the biological material, (Escherichia coli) XYSH220517-04, the Escherichia coli XYSH220517-04 is preserved in China general microbiological culture collection center (CGMCC), the preservation date is 2022, 9 months and 19 days, and the preservation address is: the collection number of the strain is CGMCC No.25753 in the national institute of microbiology, national institute of sciences, no. 3, north Chen West Lu, no. 1, chaoyang, beijing city.
Drawings
FIG. 1 is a flow chart of a mutagenesis screening of E.coli XYSH 220517-04.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
A solid medium consisting of the following components: 10g/L tryptone, 10g/L beef extract, 5g/L yeast powder, 5g/L sodium chloride and 1-4g/L, KH glucose 2 PO 4 1-5g/L, and the balance of water, pH 6.8-7.2.
The primary screening medium (deep-hole plate fermentation medium) consists of the following components: glucose 20-40g/L, (NH) 4 ) 2 SO 4 1-5g/L、KH 2 PO 4 1-5g/L、MgSO 4 ·7H 2 O 0.5-2g/L、FeSO 4 ·7H 2 O 20-40mg/L、MnSO 4 ·H 2 O 1-3mg/L、Na 2 HPO 4 4-6g/L2-4g/L of yeast powder, 2-4g/L of sodium citrate, 20-80g/L of propanesulfonic acid and the balance of water, wherein the pH value is 6.8-7.2. The rotation speed of the shaking table is 600-800rpm/min, and the culture temperature is 36-37 ℃.
Re-screening culture medium (shake flask culture medium), seed shake flask culture medium comprises the following components: glucose 20-40g/L, (NH) 4 ) 2 SO 4 1 5g/L，KH 2 PO 4 1-5g/L，MgSO 4 ·7H 2 O0.5-2 g/L, yeast powder 2-5g/L, feSO 4 · 7 H 2 O20-40mg/L，MnSO 4 ·H 2 O 1-3mg/L，VH 0.1-0.5mg/L，VB 1 0.5-1.0mg/L, 1-3ml/L of trace element mixed solution and the balance of water, and sterilizing by a high-pressure steam kettle at 115 ℃ for 15min, wherein the pH value is 7.0-7.2.
The fermentation shake flask culture medium consists of the following components: glucose 20-40g/L, (NH) 4 ) 2 SO4 2-6g/L，KH 2 PO 4 1-5g/L，MgSO 4 · 7 H 2 O0.5-2 g/L, yeast powder 1-5g/L, feSO 4 · 7 H 2 O 30-60mg/L，MnSO 4 · 7 H 2 O 1-5mg/L，VH0.1-0.5mg/L，VB 1 0.5-1.0mg/L, 1-3ml/L of trace element mixed solution, 15-30g/L of phenol red, and the balance of water, wherein the pH value is 7.0-7.2, and the sterilizing is carried out in a 115 ℃ high-pressure steam pot for 15min.
The trace element mixed solution comprises the following components: coSO 4 ·7H 2 O 0.4-0.8g/L，ZnSO 4 ·7H 2 O 6-8g/L，CuSO 4 ·5H 2 O 4-6g/L，Al 2 (SO 4 ) 3 ·18H 2 O 2-4g/L，MnSO 4 ·H 2 O 4-6g/L，Na 2 MoO 4 ·2H 2 O 2-4g/L，NiSO 4 ·6H 2 O 2-4g/L，H 3 BO 3 1-2g/L。
Example 1: method for screening escherichia coli XYSH220517-04 by mutagenesis
As shown in FIG. 1, the specific procedure for obtaining Escherichia coli XYSH220517-04 is as follows:
1. preparing a bacterial suspension: taking fresh inclined plane of Escherichia coli TRTH cultured at 37 ℃ for 1d at constant temperature, adding 15mL of sterile water, scraping off the strain, and transferring into a 250mL triangle with glass beadsIn the bottle, 15ml of glycerin containing 1% is added into a triangular bottle to prepare bacterial suspension, and the bacterial concentration is regulated to 10 8 individual/mL; irradiating the bacterial suspension by using an ARTP mutagenesis instrument for 60s, diluting the bacterial suspension after mutagenesis, coating the bacterial suspension in a solid plate culture medium, and culturing the bacterial suspension in an inversion way for 1d at 37 ℃;
3. and (3) primary screening: inoculating the single colonies obtained by mutagenesis in the step 1 into a 96-deep hole plate, wherein the deep hole plate contains 600 microlitres of screening culture medium, sealing the 96-deep hole plate by using a sealing plate film, then placing the deep hole plate on a high-speed shaking table for culturing, detecting the content of L-tryptophan in fermentation liquor, and primarily screening the single colonies with the content of L-tryptophan in the fermentation liquor being more than 2g/L, wherein the rotation speed of the shaking table is 600-800rpm, the culturing temperature is 36-37 ℃, the humidity is 80%, and the culturing is 24 hours.
4. And (3) re-screening: the strain preserved by the primary screening glycerol tube is respectively scratched on an inclined plane and cultured for 20 hours at 36 ℃. Selecting one-loop fungus from the inclined plane to a seed shake flask, culturing at 180r/min and 36.5 ℃ for 5h, transferring to fermentation shake flasks, wherein each fermentation shake flask contains 30 milliliters of fermentation culture medium, the rotation speed of a shaking table is 200rpm, and the pH value is maintained at 7.0-7.2 by adding ammonia water in the fermentation process; taking phenol red as an indicator, regarding the fermentation broth as sugar deficiency when the color of the fermentation broth is no longer changed, and supplementing 1-2mL of 60% (m/v) glucose solution when the color of the fermentation broth is sugar deficiency; and (3) detecting the content of L-tryptophan in the fermentation broth after the fermentation period is 20-24 hours, and screening out single colonies with the content of L-tryptophan higher than 12g/L in the fermentation broth and preserving the single colonies.
5. Activating the single colony obtained by re-screening in the step 4 with an inclined plane, culturing for 1d, scraping bacteria into physiological saline by using an inoculating loop to prepare bacterial suspension with the concentration of 10 9 And (3) adding 2ml of the bacterial suspension into a 50ml sterile centrifuge tube, adding 4 ml of 0.5mg/L nitrosoguanidine solution, repeating the steps (3) to (4), and primarily screening to select single colonies with acid production of more than 2.5g/L for preservation, and secondarily screening to select single colonies with acid production of more than 15g/L for preservation, wherein the single colonies comprise a strain XYSH220517-04 and a shake flask with acid production of the strain XYSH220517-04 reaching 18g/L.
The following experiment was used to verify the experimental effect:
genetic stability test:
and (3) separating single bacterial colony from the tryptophan high-yield strain obtained by screening, continuously shaking and passaging for 10 generations, and firstly carrying out seed culture on each generation of strain, and selecting the strain with stable heredity and high acid production for further research. Shake flask passaging method: the tryptophan high-yield strain is transferred into a shake flask from an inclined plane, cultured to a logarithmic growth phase and then transferred into a next generation shake flask.
The final tryptophan high-yield strain XYSH220517-04 was continuously propagated ten times, and the L tryptophan yield was examined by using 500mL shake flask fermentation culture. The results were as follows:
TABLE 1 genetic stability of Strain XYSH220517-04
As can be seen from Table 1, the mutant strain XYSH220517-04 has good genetic stability, and the tryptophan yield after 10 continuous passages in a 500mL fermenter is substantially stabilized at about 18g/L, and the strain XYSH220517-04 has good genetic stability.
EXAMPLE 2 Process for producing L-tryptophan
Using escherichia coli XYSH220517-04 and the original strain TRTH of the present invention, 50L tank fermentation culture was performed respectively in combination with the related process of the present invention, five batches were cultured respectively, and the average value of the five batches was calculated as follows:
(1) Seed pot medium composition: 40-60g/L glucose, 2-6g/L yeast powder, 0.5-2g/L citric acid,
(NH 4 ) 2 SO 4 2-8g/L，KH 2 PO 4 5-7g/L，VB 1 1-2mg/L，VH 0.3-0.5mg/L，MgSO 4 ·7H 2 O 1.5-2g/L，FeSO 4 · 7 H 2 o20-40 mg/L and trace elements are mixed and dissolved in 1-2mL/L.
(2) Fermentation medium composition: 10-20g/L glucose, 4-6g/L yeast powder, 1-4g/L citric acid, (NH) 4 ) 2 SO 4 2-8 g/L，KH 2 PO 4 4-6g/L，MgSO 4 ·7H 2 O 1-2g/L，FeSO 4 ·7H 2 O 50-80mg/L，VB 1 5-7mg
and/L, VH is 0.2-0.6mg/L, and the trace element mixed solution is 1-2mL/L.
(3) The trace element mixed solution comprises the following components: coSO 4 · 7 H 2 O 0.4-0.8g/L，ZnSO 4 · 7 H 2 O 6-8g/L，CuSO 4 · 5 H 2 O 4-6g/L，Al 2 (SO 4 ) 3 · 18 H 2 O 2-4g/L，MnSO 4 ·H 2 O 4-6g/L，Na 2 MoO 4 · 2 H 2 O 2-4g/L，NiSO 4 · 6 H 2 O 2-4g/L，H 3 BO 3 1-2g/L。
(4) Culturing strain in seed tank, sterilizing seed culture medium, cooling to 36 deg.C, regulating pH to 7.0, inoculating Escherichia coli XYSH220517-04 strain into seed culture medium, and culturing to logarithmic phase.
Strain culture conditions: the temperature is 36 ℃, the pH value is about 7.0, the dissolved oxygen is 25-30%, and the tank pressure is 0.02MPa.
(5) Sterilizing the fermentation medium, cooling to about 36 ℃, adjusting the pH to about 7.0, and inoculating the logarithmic growth phase strain prepared in the step (1) into the fermentation medium for fermentation culture to prepare the L tryptophan fermentation broth. Fermentation culture conditions: the temperature is 36 ℃, the pH value is 7.0, the dissolved oxygen is 25% -30%, the tank pressure is 0.02MPa, and the content of residual sugar in the fermentation process is controlled to be 0.04%. And (5) culturing until the culture time is 32-40 h, and obtaining the L tryptophan fermentation broth.
(6) Tryptophan content, glutamic acid content, acetic acid content, citric acid content and succinic acid content are detected by using a liquid chromatography, and ammonia nitrogen is detected by using a Kjeldahl nitrogen determination instrument.
(7) Conversion rate calculation formula = (fermentation liquor volume l×fermentation acid production content g/L)/fermentation glucose dosage g×100%.
Table 2 comparison of Tryptophan production Performance in 50 liter fermentors of mutant strain and starting strain
As can be seen from Table 2, the strain XYSH220517-04 of the invention has greatly improved L-tryptophan yield, conversion rate and fermentation period compared with the original strain TRTH, the content of hetero acid at the fermentation end point is obviously reduced, the content of glutamic acid is reduced to 1.17g/L, the content of citric acid is reduced to 2.12g/L, and the content of succinic acid is reduced to 2.34g/L.
The extraction process flow of the L-tryptophan comprises the following steps: removing thalli from the L tryptophan fermentation liquor through membrane filtration to obtain a filtered clear liquid, evaporating, concentrating and crystallizing the filtered clear liquid, cooling and crystallizing, further centrifuging to remove mother liquor to obtain wet crystals, and drying the wet crystals to obtain L tryptophan crystals. The membrane filtration equipment is a ceramic membrane, an organic membrane or a metal membrane, the aperture is 8nm, the evaporation concentration crystallization equipment is a multi-effect evaporation crystallizer, the cooling crystallization equipment is a cold crystallizer, the L tryptophan crystal drying equipment is a fluidized bed dryer or a vacuum bipyramid dryer, and the L tryptophan particle product drying equipment is a gunite granulation dryer.
In this way, a 350 ton fermenter is used for fermentation culture until the fermentation culture time reaches 32 hours, the tank volume is 230 tons, the yield of L tryptophan is 62.56g/L, the conversion rate is 30.12%, and the acid production rate is 1.96g/L/h. The strain XYSH220517-04 realizes the production characteristics of short period, high acid production and high acid production rate. The content of the mixed acid in the fermentation liquor is obviously reduced, the content of the glutamic acid is reduced to 1.15g/L, the content of the citric acid is reduced to 2.18g/L, and the content of the succinic acid is reduced to 2.52g/L. Concentrating the L tryptophan fermentation liquor, spraying slurry, and granulating to obtain 11.51 tons of L tryptophan granule products, wherein the extraction yield reaches 80%, and the extraction yield of the original strain TRTH is 68%. On the premise of unchanged extraction process, the L-tryptophan extraction yield of the strain XYSH220517-04 fermentation broth is improved by 12 percent compared with the fermentation broth of the strain TRTH, and the tryptophan purity and various indexes meet the national standard requirements of feed-grade tryptophan, so that the production performance of the mutant strain XYSH220517-04 is greatly broken through, and the industrial production performance is good.
Table 3 comparison of Tryptophan production Performance of mutant strain and starting strain in 350 ton fermenter
The extraction yield formula: the extraction yield calculation formula: tryptophan finished product mass/(fermentation liquor final volume×tryptophan concentration) ×100%.
Claims (10)
1. The Escherichia coli is characterized in that the Escherichia coli is named as XYSH220517-04 and is preserved in China general microbiological culture collection center (CGMCC) with a preservation date of 2022, 9 months and 19 days and a strain preservation number of CGMCC No.25753.
2. A microbial preparation comprising the Escherichia coli according to claim 1.
3. Use of the escherichia coli of claim 1 or the microbial preparation of claim 2 for increasing the yield, conversion rate and extraction yield of L-tryptophan.
4. A process for producing L-tryptophan at a high yield, which comprises the step of fermenting the Escherichia coli of claim 1 at 36℃for at least 32 hours.
5. The method according to claim 4, wherein the fermentation conditions of Escherichia coli further comprise pH 7.0, dissolved oxygen of 25% -30% and a tank pressure of 0.02MPa.
6. The method according to claim 4, wherein the medium for fermentation uses glucose as a carbon source.
7. The method according to claim 4, wherein the medium for fermentation uses yeast powder as nitrogen source.
8. The method according to claim 4, wherein the fermentation medium for fermentation contains glucose, yeast powder, citric acid, (NH) 4 ) 2 SO 4 、KH 2 PO 4 、MgSO 4 ·7H 2 O、FeSO 4 ·7H 2 O, vitamin B1, vitamin H and trace elements.
9. The method according to claim 4, wherein the fermentation medium for fermentation contains glucose 10-20g/L, yeast powder 4-6g/L, citric acid 1-4g/L, (NH) 4 ) 2 SO 4 2-8g/L，KH 2 PO 4 4-6g/L，MgSO 4 ·7H 2 O1-2g/L，FeSO 4 ·7H 2 50-80mg/L of O, 1 5-7mg/L of VB, 0.2-0.6mg/L of VH and 1-2mL/L of trace element mixed solution.
10. Use of the process of any one of claims 4-9 to increase L-tryptophan conversion, extraction yield and decrease fermentation cycle.
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