CN116814504A - Screening method of high-yield succinic acid strain - Google Patents
Screening method of high-yield succinic acid strain Download PDFInfo
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- CN116814504A CN116814504A CN202311008581.9A CN202311008581A CN116814504A CN 116814504 A CN116814504 A CN 116814504A CN 202311008581 A CN202311008581 A CN 202311008581A CN 116814504 A CN116814504 A CN 116814504A
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 239000001384 succinic acid Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000012216 screening Methods 0.000 title claims abstract description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 56
- 238000002703 mutagenesis Methods 0.000 claims abstract description 54
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 54
- 238000000855 fermentation Methods 0.000 claims abstract description 38
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 239000002609 medium Substances 0.000 claims abstract description 30
- 239000011780 sodium chloride Substances 0.000 claims abstract description 28
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- 238000002156 mixing Methods 0.000 claims abstract description 3
- 238000012258 culturing Methods 0.000 claims description 4
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- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
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- 238000003786 synthesis reaction Methods 0.000 abstract description 8
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- 235000011044 succinic acid Nutrition 0.000 description 31
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 24
- 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 description 12
- 239000008103 glucose Substances 0.000 description 12
- 229910052742 iron Inorganic materials 0.000 description 12
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 7
- 238000012795 verification Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229940107700 pyruvic acid Drugs 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- HPQUMJNDQVOTAZ-UHFFFAOYSA-N 2,2-dihydroxypropanoic acid Chemical compound CC(O)(O)C(O)=O HPQUMJNDQVOTAZ-UHFFFAOYSA-N 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
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- OVYQSRKFHNKIBM-UHFFFAOYSA-N butanedioic acid Chemical compound OC(=O)CCC(O)=O.OC(=O)CCC(O)=O OVYQSRKFHNKIBM-UHFFFAOYSA-N 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
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- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a screening method of a high-yield succinic acid strain, which comprises the following steps: hybrid mutagenesis of strain BER308 using ARTP and microwaves; inoculating the strain BER308 after mutagenesis into succinic acid fermentation broth for anaerobic culture; inoculating the strain BER308 after anaerobic culture into LB culture medium with NaCl concentration above 0.6M for culture; selecting and mixing monoclone with good growth vigor; repeating steps (1) - (4), wherein after each mutagenesis, the concentration of NaCl in the NaCl-containing medium is higher than the concentration of NaCl in the NaCl-containing medium after the previous mutagenesis; until the NaCl concentration in the NaCl culture medium reaches 1-2M. The method can screen out the strain with the advantages of high succinic acid synthesis efficiency, high yield, good strain activity and the like, and the screened strain is suitable for industrial large-scale succinic acid fermentation synthesis.
Description
Technical Field
The invention relates to a screening method of a high-yield succinic acid strain.
Background
Succinic acid (Succinic acid) is a colorless crystal, an important organic acid, and has a chemical formula of C4H6O4, also called Succinic acid. Succinic acid is widely used in the fields of chemistry, medicine, food, cosmetics, etc., and the following are several main uses thereof: chemically, succinic acid can be used as a solvent, a catalyst, an intermediate, etc., and can be used for preparing various organic matters. The succinic acid can be used for preparing medicines for treating heart disease, liver disease, nephropathy, etc., and can also be used for treating anemia, fatigue syndrome, etc. On food, succinic acid can be used for preparing sour agent and flavoring agent, and can also be used as nutrition enhancer for baked food such as bread and cookies. The succinic acid can be used for preparing skin care products, cosmetics, etc., and has effects of keeping moisture and resisting oxidation. In the industrial field, succinic acid can be used for preparing resin, paint, plastic and the like, and can also be used in the industrial fields of water treatment, paper processing and the like.
The synthesis of succinic acid is largely divided into two modes, chemical synthesis and biological synthesis. Chemical synthesis mainly prepares acrylic acid through styrene oxidation reaction. Then, dihydroxypropionic acid is produced by reacting acrylic acid with ethylene glycol. Finally, dihydroxypropionic acid can be converted to succinic acid by heating and dehydration reactions. The method has the advantages of complicated operation, more byproducts and difficult separation and purification, so that succinic acid with high quality and high purity is difficult to obtain.
During the biosynthesis of succinic acid, microorganisms produce succinic acid by tricarboxylic acid cycle. In this process, the microorganism first converts glucose or other carbon source into pyruvic acid and pyruvic acid. Then, pyruvic acid and pyruvic acid carboxylic acid react through carboxylation to generate succinic acid. Microorganisms for biosynthesis of succinic acid include many kinds of bacteria and fungi such as Escherichia coli, actinomycetes, yeast, and the like. The biosynthesis method has the advantages of wide raw materials, mild reaction conditions, high product purity and the like, and is widely applied to industrial production.
Disclosure of Invention
The invention aims to provide a screening method of a high-yield succinic acid strain.
The invention adopts the technical scheme that: the screening method of the high-yield succinic acid strain is characterized by comprising the following steps of:
(1) Hybrid mutagenesis of strain BER308 using ARTP and microwaves;
(2) Inoculating the strain BER308 after mutagenesis into succinic acid fermentation broth for anaerobic culture;
(3) Inoculating the strain BER308 after anaerobic culture into LB culture medium with NaCl concentration above 0.6M for culture;
(4) Selecting one or more of the monoclonal antibodies with good growth vigor, and mixing the monoclonal antibodies;
(5) Repeating the steps (1) - (4), wherein the NaCl concentration in the NaCl-containing medium after each mutagenesis is higher than that in the NaCl-containing medium after the previous mutagenesis until the NaCl concentration in the NaCl-containing medium reaches 1-2M; the succinic acid fermentation broth used in the inoculation after each mutagenesis in the step (2) is the succinic acid fermentation broth of the anaerobic culture of the previous mutagenesis.
Preferably, the strain BER308 is the strain BER308 described in China patent CN 201710443617.4.
Preferably, the ARTP and microwave mixed mutagenesis refers to the steps of firstly carrying out ARTP mutagenesis on a strain, culturing the strain in an LB culture medium for a period of time after mutagenesis is finished, and then culturing the strain in the LB culture medium for a period of time after mutagenesis is finished by using microwave mutagenesis.
Preferably, the strain is subjected to ARTP mutagenesis for 30-60s at a power of 120W, microwave mutagenesis for 30-60s at a power of 100W, and cultured in LB medium for 1-3 hours.
Preferably, the succinic acid fermentation broth is a product of a strain BER308 after succinic acid is fermented for 72 hours, and the strain is removed after centrifugation, so as to obtain a supernatant fermentation broth.
Preferably, anaerobic culture conditions are such that sufficient CO is introduced 2 Shake-culturing overnight at 37 ℃.
Preferably, the LB medium containing NaCl is an LB solid medium.
The method can screen out the strain with the advantages of high succinic acid synthesis efficiency, high yield, good strain activity and the like, and the screened strain is suitable for industrial large-scale succinic acid fermentation synthesis.
Drawings
FIG. 1 is a schematic diagram of screening principle of high-yield succinic acid strain.
FIG. 2 shows a graph comparing activities of strains before and after screening in a medium at a late stage of fermentation.
FIG. 3 is a graph showing comparison of the yields of strains on fermented succinic acid before and after screening.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
In order to improve the tolerance of BER308 to high concentration succinic acid and the utilization rate of glucose in the later fermentation period, the mutagenesis and domestication of the escherichia coli strain BER308 described in Chinese patent CN201710443617.4 are carried out, and the domestication process is shown in figure 1.BER308 is cultured in LB culture medium at 37 ℃ and 220rpm until OD600 value reaches 0.6, 10ul of the culture medium is coated on an iron sheet, and 120W of the culture medium is subjected to mutagenesis for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Putting the iron sheet into an EP pipe, adding 500ul BER308 succinic acid fermentation broth fermentation medium in the late fermentation stage, adopting the fermentation medium recorded in CN201710443617.4, wherein the carbon source is replaced by 10g/L glucose, and the culture is carried out at 37 ℃ and 220rpm until the OD600 value reaches 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 0.6M NaCl, and cultured at 37℃overnight. The better growth vigor monoclonal (the larger the growth vigor of the monoclonal is, the better, usually 1-10 monoclonal are selected) is selected for fermentation verification.
Meanwhile, the monoclonals with better growth vigor are mixed together, shake-cultured in LB culture medium at 37 ℃ and 220rpm until the OD600 value reaches 0.6, 10ul of the mixture is coated on an iron sheet, and 120W of mutagenesis is performed for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Iron plates were placed in EP tubes and 500ul of fermentation broth was added for anaerobic culture after the last round of mutagenesis, followed by shaking culture at 37℃and 220rpm until OD600 reached 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 0.8M NaCl, and cultured at 37℃overnight. And selecting a monoclonal with better growth vigor, and carrying out fermentation verification.
Meanwhile, the monoclonals with better growth vigor are mixed together, shake-cultured in LB culture medium at 37 ℃ and 220rpm until the OD600 value reaches 0.6, 10ul of the mixture is coated on an iron sheet, and 120W of mutagenesis is performed for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Iron plates were placed in EP tubes and 500ul of fermentation broth was added for anaerobic culture after the last round of mutagenesis, followed by shaking culture at 37℃and 220rpm until OD600 reached 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 1M NaCl, and cultured at 37℃overnight. And selecting a monoclonal with better growth vigor, and carrying out fermentation verification.
Meanwhile, the monoclonals with better growth vigor are mixed together, shake-cultured in LB culture medium at 37 ℃ and 220rpm until the OD600 value reaches 0.6, 10ul of the mixture is coated on an iron sheet, and 120W of mutagenesis is performed for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Iron plates were placed in EP tubes and 500ul of fermentation broth was added for anaerobic culture after the last round of mutagenesis, followed by shaking culture at 37℃and 220rpm until OD600 reached 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 1.2M NaCl, and cultured at 37℃overnight. And selecting a monoclonal with better growth vigor, and carrying out fermentation verification.
Meanwhile, the monoclonals with better growth vigor are mixed together, shake-cultured in LB culture medium at 37 ℃ and 220rpm until the OD600 value reaches 0.6, 10ul of the mixture is coated on an iron sheet, and 120W of mutagenesis is performed for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Iron plates were placed in EP tubes and 500ul of fermentation broth was added for anaerobic culture after the last round of mutagenesis, followed by shaking culture at 37℃and 220rpm until OD600 reached 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 1.4M NaCl, and cultured at 37℃overnight. And selecting a monoclonal with better growth vigor, and carrying out fermentation verification.
Meanwhile, the monoclonals with better growth vigor are mixed together, shake-cultured in LB culture medium at 37 ℃ and 220rpm until the OD600 value reaches 0.6, 10ul of the mixture is coated on an iron sheet, and 120W of mutagenesis is performed for 40s (about 80% -90% of mortality) on an ARTP mutagenesis instrument. Iron plates were placed in EP tubes and 500ul of fermentation broth was added for anaerobic culture after the last round of mutagenesis, followed by shaking culture at 37℃and 220rpm until OD600 reached 0.3. In a microwave mutagenesis instrument, ice bath microwaves for 60s (about 80% -90% mortality) were continued to culture until the OD600 reached 0.8. Spread on LB medium containing 1.6M NaCl, and cultured at 37℃overnight. And selecting a monoclonal with better growth vigor, and carrying out fermentation verification.
The bacterial liquid is streaked on LB solid medium plate for monoclone, and cultured for 15h-18h at 37 ℃ until larger monoclone colony grows. The colonies were selected and inoculated into a test tube containing 5mL of LB medium, and cultured at 37℃and 200rpm/min for about 8-12 hours as the primary seed liquid (OD 550 2-4) (the test tube was required to be placed obliquely in a shaker). The primary seed solution was transferred to a 1000mL flask containing 200mL of LB medium (a three-pin flask was recommended), and cultured at 37℃and 200rpm for 15-18 hours (OD 550-8) to obtain a secondary seed solution. The secondary seed liquid was inoculated to the fermentation medium ((NH) at an inoculum size of 10% 4 H 2 PO 4 3.87g/L，KCl 0.3g/L，MgSO 4 ·7H 2 0.7g/L of trace element (FeCl) 1/1000 (v/v) 3 ·6H 2 O 3.4g/L，CoCl 2 ·2H 2 O 1.3g/L，CuCl 2 ·2H 2 O 0.15g/L，ZnCl 2 ·4H 2 O 1g/L，MnCl 2 ·4H 2 O1 g/L), and the glucose concentration is controlled to be 10 g/L). The fermentation temperature is 37 ℃, the pH is 6.8, the DO is 20 percent, and the bacterial OD600 is as long as 5, and the anaerobic fermentation is carried out. CO2 is continuously introduced, the temperature is 37 ℃, and the anaerobic fermentation is carried out at 250rpm until the anaerobic fermentation is finished. Glucose is fed in batches after being sterilized independently, the initial concentration of the glucose in the aerobic stage is 30g/L, the glucose concentration and the thallus OD500 are detected every 4-6 hours, when the first concentration of the glucose is 10g/L, 600g/L glucose is continuously fed in to control the glucose concentration to be about 10g/L, the glucose concentration and the thallus OD600 are continuously detected every 4-6 hours, and the fermentation can be ended (about 100 hours) when the glucose is basically not consumed. By high performance liquid chromatograph HPLC (High Performance Liquid Chromatography)
(UITIMate 3000HPLC system,Dionex,USA) detecting concentration of succinic acid in fermentation broth, and detecting the type of the used organic acid column is Bio Rad Aminex HPX-87H. The mobile phase A used for detection is 0.25g/L dilute sulfuric acid, and is a single-pipeline single-mobile phase, and the needle washing liquid is 10% methanol. The results are shown in fig. 2 and 3, and the results shown in fig. 2 and 3 are the 9 best growing monoclonal cells finally selected from the 1.6M NaCl medium, and it can be seen that the strain after multiple induction and acclimatization has better activity and higher succinic acid yield.
Claims (7)
1. The screening method of the high-yield succinic acid strain is characterized by comprising the following steps of:
(1) Hybrid mutagenesis of strain BER308 using ARTP and microwaves;
(2) Inoculating the strain BER308 after mutagenesis into succinic acid fermentation broth for anaerobic culture;
(3) Inoculating the strain BER308 after anaerobic culture into LB culture medium with NaCl concentration above 0.6M for culture;
(4) Selecting one or more of the monoclonal antibodies with good growth vigor, and mixing the monoclonal antibodies;
(5) Repeating the steps (1) - (4), wherein the NaCl concentration in the NaCl-containing medium after each mutagenesis is higher than that in the NaCl-containing medium after the previous mutagenesis until the NaCl concentration in the NaCl-containing medium reaches 1-2M; the succinic acid fermentation broth used in the inoculation after each mutagenesis in the step (2) is the succinic acid fermentation broth of the anaerobic culture of the previous mutagenesis.
2. The method for screening a high-yield succinic acid strain according to claim 1, wherein the strain BER308 is strain BER308 described in chinese patent CN 201710443617.4.
3. The method for screening a high-yield succinic acid strain according to claim 2, wherein the mixed mutagenesis of ARTP and microwave is to subject the strain to ARTP mutagenesis, culture it in LB medium for a while after the mutagenesis is completed, and culture it in LB medium for a while after the mutagenesis is completed by using microwave mutagenesis.
4. The method for screening a high-yield succinic acid strain according to claim 3, wherein the strain is subjected to ARTP mutagenesis for 30-60s at a power of 60-120W, microwave mutagenesis for 30-60s at a power of 50-100W, and cultured in LB medium for 1-3 hours.
5. The method for screening a high-yield succinic acid strain according to claim 2, wherein the succinic acid fermentation broth is a product of the strain BER308 after succinic acid fermentation for 72 hours, and the strain is removed after centrifugation, and the supernatant fermentation broth is obtained.
6. The method for screening a high-yield succinic acid strain according to claim 5, wherein the anaerobic culture is conducted under conditions in which CO is introduced in an amount sufficient for the culture 2 Shake-culturing overnight at 37 ℃.
7. The method for screening a high-yield succinic acid strain according to claim 2, wherein the LB medium containing NaCl is an LB solid medium.
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