WO2024130983A1 - Method for producing ectoine using halomonas strain - Google Patents

Method for producing ectoine using halomonas strain Download PDF

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WO2024130983A1
WO2024130983A1 PCT/CN2023/100310 CN2023100310W WO2024130983A1 WO 2024130983 A1 WO2024130983 A1 WO 2024130983A1 CN 2023100310 W CN2023100310 W CN 2023100310W WO 2024130983 A1 WO2024130983 A1 WO 2024130983A1
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fermentation
ectoine
acetate
propionate
sodium
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Chinese (zh)
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周明新
周振涛
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深圳中科翎碳生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/10—Process efficiency
    • Y02P20/133—Renewable energy sources, e.g. sunlight

Definitions

  • the invention relates to the technical field of biological fermentation, and in particular to a new halomonas strain for producing ectoine and a method for producing ectoine by using the strain.
  • Ectoine (Ect) and its derivative hydroxyectoine (5-Hect) are an important class of compatible solutes originally discovered from halophilic and salt-tolerant microorganisms. Ectoine can help halophilic and salt-tolerant microorganisms adapt to environments such as high salt, high osmotic pressure and ultraviolet radiation, and maintain their normal growth in adversity. Ectoine can enhance the tolerance of cells in a variety of adversities (such as high salt, heat, dryness and freezing, etc.), so it has shown broad application prospects in the fields of biological protection, biomedicine and biotechnology. With the rapid development of commercial applications of ectoine, the study of microbial synthesis of ectoine has always been the focus and hotspot of ectoine research.
  • the first is to use wild strains to synthesize ectoine. Wild strains can quickly generate ectoine in a high-salt environment to maintain the balance of cell osmotic pressure. When the cells are in a low-salt environment, the cells can sense the change in osmotic pressure and release ectoine to the outside of the cell to maintain the balance of cell osmotic pressure. By repeatedly cycling the cells in high-salt and low-salt environments, the cells can continuously generate ectoine externally. This production method is vividly called "bacterial milking.”
  • the second method is to screen strains that can naturally synthesize ectoine in a low-salt environment, and use the natural strains' ability to produce ectoine in a low-salt environment and secrete it into the extracellular space to achieve continuous production of ectoine.
  • the third method is to use synthetic biology to perform heterologous synthesis of ectoine, transferring the ectABC gene cluster of ectoine synthesis into heterologous strains such as Escherichia coli and Corynebacterium glutamicum.
  • heterologous synthesis is that there is no degradation pathway of ectoine in heterologous strains, and high production of ectoine can be achieved without knocking out genes related to the degradation pathway.
  • the fourth method is to use synthetic biology to transform halophilic microorganisms to increase the production of ectoine.
  • the production of ectoine is mainly increased by increasing the expression of genes in the ectoine synthesis pathway and knocking out genes related to ectoine degradation.
  • the above four commonly used fermentation methods basically use glucose as the main carbon source for fermentation, and use glutamate, glycerol and L-aspartic acid as fermentation carbon sources in small amounts.
  • Glutamate, glycerol and L-aspartic acid are expensive and are often used as auxiliary materials in fermentation.
  • the production cost of using them as the main carbon source is high, and large-scale industrial application cannot be achieved.
  • the main source of glucose is starch, which is an important food resource. Therefore, finding a microbial fermentation method that uses non-glucose as the main carbon source to produce ectoine is an urgent problem that the industry needs to solve at this stage.
  • Short-chain fatty acids such as propionic acid and acetic acid are widely available and can be produced by microorganisms through fermentation of kitchen waste, high-concentration organic wastewater, and urban garbage.
  • electrocatalytic acetic acid production which can be used to convert carbon dioxide into propionic acid and acetic acid, which not only solves the problem of carbon dioxide emissions, but also generates usable biological carbon sources, helping carbon emission reduction and carbon neutrality. Therefore, the use of propionic acid, acetic acid, etc. as carbon sources to ferment and produce tetrahydropyrimidine can not only solve a series of problems caused by the use of glucose fermentation, but also contribute to carbon neutrality and carbon emission reduction.
  • the inventors of the present application obtained a high-yield ectoine-producing Halomonas sp. YL01 after sampling salt lake silt. Metagenomic sequencing and metabolic pathway analysis showed that it can metabolize using acetate as the only carbon source and has a high tolerance to acetate. Further research found that Halomonas sp. YL01 can produce ectoine using a carbon source consisting of acetate, propionate, or a mixture of the two.
  • the inventors have studied and optimized the fermentation medium, and finally achieved high-yield ectoine using a carbon source mainly composed of acetate, thus solving the problem that ectoine can only be produced using food crops such as glucose, glutamate, and glycerol.
  • the technical problem to be solved by the present invention is to provide a Halomonas sp. YL01 strain. By detecting and analyzing the genome of the strain, it is found that the strain can produce ectoine by fermentation with acetate as the main carbon source. Further research, through the screening of carbon sources and the optimization of fermentation processes, a culture medium formula and a fermentation method for efficiently producing ectoine are obtained.
  • the invention provides a fermentation method for producing ectoine by using a Halomonas sp. YL01 strain.
  • the carbon source used includes acetate or a mixed carbon source consisting of acetate and propionate.
  • the Halomonas sp. YL01 strain is used as a base bacteria for fermentation preparation.
  • the concentration of acetate in the fermentation broth is 10-40 g/L, and the concentration of propionate in the fermentation broth is not higher than 10 g/L.
  • Biomaterial deposit information YL01, taxonomic name: Halomonas sp. YL01 is deposited in the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Microbiological Analysis and Testing Center), the strain accession number is GDMCC.No62420, the deposit date is April 24, 2022, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Microbiological Analysis and Testing Center).
  • the acetate can be one of sodium acetate, potassium acetate, and ammonium acetate, or a mixture of several of them.
  • the propionate may be one of sodium propionate, potassium propionate, and ammonium propionate, or a mixture of the two.
  • the carbon source may also include glucose.
  • the fermentation temperature is 32°C-37°C.
  • the pH value of the culture medium used in the fermentation is 6-11.
  • Halomonas sp. YL01 is inoculated into the shake flask medium for fermentation culture.
  • the shake flask medium composition includes: sodium acetate 10-40 g/L, sodium propionate 0-10 g/L, glucose 0-40 g/L, urea 0.5-10 g/L, aspartic acid 0-10 g/L, yeast powder 1-15 g/L, anhydrous magnesium sulfate 0.05-0.6 g/L, potassium dihydrogen phosphate 1.5-5.5 g/L, sodium chloride 50-80 g/L, Fe(III)-NH 4 -Citrate 0.05-0.1 g/L, CaCl 2 ⁇ 2H 2 O 0.02-0.2 g/L, ZnSO 4 ⁇ 7H 2 O 0.01-0.1 g/L, MnCl 2 ⁇ 4H 2 O 0.002-0.02 g/L, H 3 BO 3 0.01-0.05 g/L, CoCl 2 ⁇ 6H 2 O 0.005-0.02 g/L, CuSO 4 ⁇ 5H 2 O 0.01-0.08 g/L, NiCl 2
  • the strain When fermentation is carried out in a fermenter, the strain is inoculated at a rate of 5% to 15% into the fermenter culture medium; during the fermentation process, the dissolved oxygen is controlled at 20% to 40%; during fermentation and culture, the acetic acid content is detected, and when the acetic acid content is lower than 10 g/L, feed culture medium is added, and feed is supplemented according to the growth situation.
  • the fermentation tank culture medium composition (g/L):
  • component I Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ⁇ 2H 2 O 2 g/L, HCl 5M, prepared with deionized water
  • component II ZnSO 4 ⁇ 7H 2 O 0.1 g/L; MnCl 2 ⁇ 4H 2 O 0.03g/L; H 3 BO 3 0.3g/L; CoCl 2 ⁇ 6H 2 O 0.2 g/L; CuSO 4 ⁇ 5H 2 O 0.01 g/L; NiCl 2 ⁇ 6H 2 O 0.02 g/L; NaMoO 4 ⁇ 2H 2 O 0.03 g/L, prepared with deionized water.
  • the Halomonas sp. YL01 of the present invention has a higher pH tolerance, which can tolerate a pH of 6-11, and can tolerate a high pH environment caused by high concentrations of acetate. Conventional strains cannot tolerate a culture environment with a pH greater than 9;
  • the present invention provides a carbon source mainly based on acetate as a fermentation medium, which can significantly reduce the fermentation cost of ectoine.
  • Figure 1 Metabolic pathway diagram of Halomonas sp.YL01 producing ectoine using acetate and propionate as mixed carbon sources;
  • FIG. 3a is a spectrum of a standard product of ectoine
  • FIG. 3b is a spectrum of ectoine produced by Halomonas sp. YL01 using acetate.
  • the strain used for fermentation in the present invention is Halomonas sp. YL01, which is deposited in the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbiological Analysis and Testing Center), the deposit date is April 24, 2022, and the deposit number is GDMCC.No62420.
  • the present invention firstly samples salt lake sludge in Qinghai province, screens halophilic bacteria in the salt lake using halophilic bacteria screening medium 60LB, obtains a strain of Halomonas sp.YL01 by screening, and then performs biochemical identification of carbon source utilization of Halomonas sp.YL01. It is verified that Halomonas sp.YL01 can grow by utilizing acetate, propionate, etc. as carbon sources.
  • the inventors used a conventional culture medium for producing ectoine to determine the ability of the strain to produce ectoine, and then used acetate as the main carbon source, designed a 60MM culture medium as the basic nutrient source, and performed shake flask fermentation to produce ectoine.
  • the fermentation medium was designed, fermented in a fermenter, and a feed medium was designed to obtain a batch feed fermentation method for producing ectoine by fermenting the strain with acetate as the main carbon source.
  • the present invention provides a fermentation method for producing ectoine using Halomonas sp. YL01, wherein the carbon source used includes acetate or a mixed carbon source composed of acetate and propionate, and the ectoine is prepared by fermentation using Halomonas sp. YL01, wherein the concentration of acetate in the fermentation broth is 10-40 g/L, and the concentration of propionate in the fermentation broth is not higher than 10 g/L.
  • the inventors have found through experiments that high concentrations of propionate will inhibit the growth of bacteria and reduce the yield of ectoine.
  • the acetate may be one or a mixture of sodium acetate, potassium acetate, and ammonium acetate.
  • the propionate may be one or a mixture of sodium propionate, potassium propionate, and ammonium propionate.
  • Acetate and propionate may be obtained in a commercially available manner, or may be obtained by converting carbon dioxide through thermal catalysis, electrocatalysis, photocatalysis, and the like, thereby realizing the reuse of carbon dioxide.
  • the carbon source may also include glucose.
  • the fermentation temperature is preferably 32° C.-37° C., and the pH value of the culture medium used for fermentation is 6-11.
  • the Halomonas sp. YL01 is inoculated into the shake flask medium, and the fermentation culture is carried out with a stirring speed of 150-300 rpm, and the fermentation lasts for 24 hours to 48 hours.
  • Shake flask medium 60MM composition sodium acetate 10-40 g/L, sodium propionate 0-10 g/L, glucose 0-40 g/L, urea 0.5-10 g/L, aspartic acid 0-10 g/L, yeast powder 1-15 g/L, anhydrous magnesium sulfate 0.05-0.6 g/L, potassium dihydrogen phosphate 1.5-5.5 g/L, sodium chloride 50-80 g/L, Fe(III)-NH 4 -Citrate 0.05-0.1 g/L, CaCl 2 ⁇ 2H 2 O 0.02-0.2 g/L, ZnSO 4 ⁇ 7H 2 O 0.01-0.1 g/L, MnCl 2 ⁇ 4H 2 O 0.002-0.02 g/L, H 3 BO 3 0.01-0.05 g/L, CoCl 2 ⁇ 6H 2 O 2 O 0.005-0.02 g/L, CuSO 4 ⁇ 5H 2 O 0.01-0.08 g/L, NiC
  • the strain When fermentation is carried out in a fermenter, the strain is inoculated at a rate of 5% to 15% into the fermenter culture medium; during the fermentation process, the dissolved oxygen is controlled at 20% to 40%; the stirring speed is 200 to 600 rpm; the fermentation lasts for 24 to 48 hours, and the acetic acid content is detected during the fermentation.
  • the acetic acid content is lower than 10 g/L, feed culture medium is added. Depending on the growth conditions, feed I is used for the first 0 to 24 hours, and feed II is used for the last 24 to 48 hours.
  • the fermentation tank culture medium composition (g/L):
  • the first trace element component specifically includes Fe(III)-NH 4 -Citrate 3-7 g/L; CaCl 2 ⁇ 2H 2 O 1-3 g/L; HCl 3-7 M, prepared by deionized water;
  • the second trace element component specifically includes ZnSO 4 ⁇ 7H 2 O 0.1-0.2 g/L; MnCl 2 ⁇ 4H 2 O 0.02-0.04 g/L; H 3 BO 3 0.2-0.4 g/L; CoCl 2 ⁇ 6H 2 O 0.1-0.3 g/L; CuSO 4 ⁇ 5H 2 O 0.01-0.03 g/L; NiCl 2 ⁇ 6H 2 O 0.01-0.03 g/L; NaMoO 4 ⁇ 2H 2 O 0.02-0.05 g/L, and is prepared with deionized water.
  • the first trace element component Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ⁇ 2H 2 O 2 g/L, HCl 5M, prepared with deionized water;
  • the second trace element component ZnSO 4 ⁇ 7H 2 O 0.1g/L; MnCl 2 ⁇ 4H 2 O 0.03g/L; H 3 BO 3 0.3g/L; CoCl 2 ⁇ 6H 2 O 0.2 g/L; CuSO 4 ⁇ 5H 2 O 0.01g/L; NiCl 2 ⁇ 6H 2 O 0.02 g/L; NaMoO 4 ⁇ 2H 2 O 0.03g/L, prepared with deionized water.
  • feed medium I acetate 300-1000 g/L, urea 15-100 g/L, aspartic acid 0-15 g/L.
  • feed medium II sodium acetate 500-1000 g/L, urea 2-20 g/L, wherein the type of acetate can be one of sodium acetate, potassium acetate, ammonium acetate or a mixture of several of them.
  • 60LB medium peptone 5-20, yeast powder 3-10, sodium chloride 50-80, pH adjusted to 6-10, used for screening and culture of Halomonas sp. YL01; 1.5-2% agarose should be added when preparing the plate.
  • 60MM culture medium (g/L): sodium acetate 10-40, sodium propionate 0-10, glucose 0-40, urea 0.5-10, aspartic acid 0-10, yeast powder 1-15, anhydrous magnesium sulfate 0.05-0.6, potassium dihydrogen phosphate 1.5-5.5, sodium chloride 50-80, Fe(III)-NH 4 -Citrate 0.05-0.1, CaCl 2 ⁇ 2H 2 O 0.02-0.2, ZnSO 4 ⁇ 7H 2 O 0.01-0.1, MnCl 2 ⁇ 4H 2 O 0.002-0.02, H 3 BO 3 0.01-0.05, CoCl 2 ⁇ 6H 2 O 0.005-0.02, CuSO 4 ⁇ 5H 2 O 0.01-0.08, NiCl 2 ⁇ 6H 2 O 0.005-0.01, NaMoO 4 ⁇ 2H 2 O 0.02-0.1, pH 6-11.
  • peptone provides the nitrogen source required for microbial growth
  • yeast powder provides the nitrogen source, vitamins, growth factors and other nutrients required for microbial growth
  • Mg 2+ is an important activator of multiple enzymes such as the TCA pathway and the EMP pathway
  • phosphorus is an important component of protein and DNA.
  • the medium first creates a high-salt environment (5%-8%) to stimulate the bacteria to synthesize ectoine and maintain osmotic pressure balance; urea is converted into ammonium, and ammonium is converted into L-glutamate to enter the ectoine synthesis pathway to increase the yield of ectoine.
  • Aspartic acid is a precursor of ectoine and can be quickly converted into ectoine to increase the yield of ectoine.
  • high concentrations of urea will inhibit the production of PHB by the bacteria, further increasing the yield of ectoine.
  • Feed I g/L: acetate 300-1000, urea 15-100, aspartic acid 0-15;
  • Feed II acetate 500-1000, urea 2-20.
  • the first trace element component Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ⁇ 2H 2 O 2 g/L, HCl 5M, prepared with deionized water
  • the second trace element component ZnSO 4 ⁇ 7H 2 O 0.1 g/L; MnCl 2 ⁇ 4H 2 O 0.03 g/L; H 3 BO 3 0.3 g/L; CoCl 2 ⁇ 6H 2 O 0.2 g/L; CuSO 4 ⁇ 5H 2 O 0.01 g/L; NiCl 2 ⁇ 6H 2 O 0.02 g/L; NaMoO 4 ⁇ 2H 2 O 0.03 g/L, prepared with deionized water.
  • 60LB medium The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
  • Biolog's PM high-throughput microbial metabolic phenotype chip system was used to detect the carbon source utilization of Halomonas sp. YL01. The test results of the carbon source utilization of the strain were obtained, see Table 1.
  • Halomonas sp.YL01 can utilize carbon sources such as propionic acid, acetic acid, and pyruvic acid to carry out physiological metabolic activities, and has the basis for developing fermentation using short-chain fatty acids as carbon sources.
  • the components (g/L) of 60MM culture medium are as follows: urea 1, aspartic acid 0, yeast powder 10, anhydrous magnesium sulfate 2, potassium dihydrogen phosphate 4, sodium chloride 60, Fe(III)-NH 4 -Citrate 0.05, CaCl 2 ⁇ 2H 2 O 0.02, ZnSO 4 ⁇ 7H 2 O 0.05, MnCl 2 ⁇ 4H 2 O 0.005, H 3 BO 3 0.01, CoCl 2 ⁇ 6H 2 O 0.005, CuSO 4 ⁇ 5H 2 O 0.01, NiCl 2 ⁇ 6H 2 O 0.007, NaMoO 4 ⁇ 2H 2 O 0.02.
  • the components (g/L) of 60MM culture medium are as follows: urea 1, aspartic acid 0, yeast powder 10, anhydrous magnesium sulfate 2, potassium dihydrogen phosphate 4, sodium chloride 60, Fe(III)-NH 4 -Citrate 0.05, CaCl 2 ⁇ 2H 2 O 0.02, ZnSO 4 ⁇ 7H 2 O 0.05, MnCl 2 ⁇ 4H 2 O 0.005, H 3 BO 3 0.01, CoCl 2 ⁇ 6H 2 O 0.005, CuSO 4 ⁇ 5H 2 O 0.01, NiCl 2 ⁇ 6H 2 O 0.007, NaMoO 4 ⁇ 2H 2 O 0.02.
  • 60LB medium The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
  • ectoine content Take the fermented bacterial liquid, use an ultrasonic cell disruptor to disrupt the cells, centrifuge the disrupted liquid at 12200g for 10 minutes, take the supernatant, filter it with a 0.22 ⁇ m filter membrane, and complete the sample processing.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 20 g/L sodium acetate + 10 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 20 g/L sodium acetate + 5 g/L potassium acetate + 5 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 10 g/L sodium acetate + 20 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 25 g/L sodium acetate + 5 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 5 g/L sodium acetate + 25 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 15 g/L glucose + 10 g/L sodium acetate + 5 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 10 g/L glucose + 10 g/L sodium acetate + 10 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 30 g/L sodium acetate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 30 g/L sodium propionate.
  • Example 2 The same method as in Example 2 was used, except that the carbon source used was 30 g/L glucose.
  • the yield of the strain was significantly lower than that of propionate and acetate. This may be because when the bacteria use two salts at the same time, different carbon sources can enter different metabolic pathways, improve the efficiency of carbon source utilization, and enhance the synthesis of ectoine.
  • Example 2 The same method as in Example 2 was used, except that the pH value was adjusted to 5.5.
  • Example 2 The same method as in Example 2 was used, except that the temperature was adjusted to 32°C.
  • Example 2 The same method as in Example 2 was used, except that the temperature was adjusted to 35°C.
  • Example 2 The same method as in Example 2 was used, except that the temperature was adjusted to 20°C.
  • Example 2 The same method as in Example 2 was used, except that the temperature was adjusted to 25°C.
  • Example 2 The same method as in Example 2 was used, except that the fermentation strain was Halomonas elongate (strain collection number: ATCC33173).
  • Example 2 The same method as in Example 2 was used, except that the fermentation strain was Halomonas salina (strain collection number: ATCC49509).
  • the other bacteria cannot ferment acetate and propionate to produce ectoine well. This is related to the different metabolic pathways of different bacteria.
  • the bacteria lack enzymes related to acetate or propionate metabolism, high concentrations of acetate or propionate will enter the bacteria, destroy the stability of the internal environment of the bacteria, have a toxic effect on the bacteria, inhibit the growth of the bacteria, or even kill the bacteria.
  • Example 15 Fermentation in a fermenter to produce tetrahydropyrimidine
  • the first trace element component Fe(III)-NH 4 -Citrate 5 g/L, CaCl 2 ⁇ 2H 2 O 2 g/L, HCl 5M, prepared with deionized water.
  • the second trace element component ZnSO 4 ⁇ 7H 2 O 0.1 g/L; MnCl 2 ⁇ 4H 2 O 0.03 g/L; H 3 BO 3 0.3 g/L; CoCl 2 ⁇ 6H 2 O 0.2 g/L; CuSO 4 ⁇ 5H 2 O 0.01 g/L; NiCl 2 ⁇ 6H 2 O 0.02 g/L; NaMoO 4 ⁇ 2H 2 O 0.03 g/L, prepared with deionized water.
  • Feed I (g/L): acetate 500, urea 50, aspartic acid 2;
  • Feed II (g/L): acetate 800, urea 15.
  • 60LB medium The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
  • the fermentation medium was prepared according to the above fermentation medium formula.
  • the secondary seed liquid was inoculated at 8% into the culture medium in a 15L fermenter (total culture medium volume was 10L), the fermentation temperature was adjusted to 37°C, the pH was 9, the ventilation was 1vvm, the fermentation dissolved oxygen was controlled to 30%, the stirring speed was linked to the dissolved oxygen, the fermentation time was 48h, and the acetic acid content was detected during the fermentation.
  • the acetic acid content was lower than 10g/L, feed culture medium was added.
  • feed I was used before 24h and feed II was used after 24h.
  • the acetate type of the feed was potassium acetate.
  • Example 16 The same method as Example 15 is adopted, except that the fermentation dissolved oxygen is 20%.
  • Example 17 The same method as Example 15 is adopted, except that the fermentation dissolved oxygen is 40%.
  • the dissolved oxygen content during fermentation has little effect on the yield of ectoine, and the appropriate range of dissolved oxygen is 20-40%, with the optimal being 30%.
  • Halomonas sp.YL01 By analyzing the metabolic pathway of Halomonas sp.YL01, it was found that the bacterium has a pathway for metabolizing acetic acid. Further experiments verified that Halomonas sp.YL01 can ferment acetate and propionate to produce ectoine, and the fermentation of a mixed carbon source consisting of acetate and propionate has no significant difference in the yield of ectoine compared with glucose, which is a good means to replace glucose to produce ectoine.
  • the Halomonas sp. YL01 of the present invention has a higher pH tolerance, can tolerate a pH of 6-11, and can tolerate a high pH environment brought about by high concentrations of acetate. Conventional strains cannot tolerate a culture environment with a pH greater than 9.
  • the present invention provides a carbon source mainly based on acetate as a fermentation medium, which can greatly reduce the fermentation cost of ectoine.

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Abstract

The present invention provides a method for producing ectoine by fermentation of a Halomonas strain Halomonas sp. YL01. The strain is used as a zymogenic strain, acetate is used as a main carbon source, and a culture medium and fermentation conditions are optimized, thus achieving the industrial fermentation production of ectoine. By means of replacing glucose that is traditionally used as the carbon source, the fermentation method greatly reduces the cost for producing ectoine.

Description

采用盐单胞菌株生产四氢嘧啶的方法Method for producing ectoine using Halomonas strain 技术领域Technical Field
本发明涉及生物发酵技术领域，具体涉及一种全新的生产四氢嘧啶用盐单胞菌株及采用该菌生产四氢嘧啶的方法。The invention relates to the technical field of biological fermentation, and in particular to a new halomonas strain for producing ectoine and a method for producing ectoine by using the strain.
背景技术Background technique
四氢嘧啶(ectoine，Ect)及其衍生物羟基四氢嘧啶(hydroxyectoine，5-Hect)最初是从嗜盐和耐盐微生物中发现的一类重要的相容性溶质 (compatible solutes)。四氢嘧啶可以帮助嗜盐和耐盐微生物适应高盐、高渗透压和紫外辐射等环境，维持其在逆境中的正常生长。四氢嘧啶可以增强细胞在多种逆境(如高盐、热、干燥和冷冻等)中的耐受性，因此在生物保护、生物医药以及生物科技等领域都展现出广阔的应用前景。伴随着四氢嘧啶商业化应用的快速发展，四氢嘧啶的微生物合成研究一直是四氢嘧啶研究的重点和热点。Ectoine (Ect) and its derivative hydroxyectoine (5-Hect) are an important class of compatible solutes originally discovered from halophilic and salt-tolerant microorganisms. Ectoine can help halophilic and salt-tolerant microorganisms adapt to environments such as high salt, high osmotic pressure and ultraviolet radiation, and maintain their normal growth in adversity. Ectoine can enhance the tolerance of cells in a variety of adversities (such as high salt, heat, dryness and freezing, etc.), so it has shown broad application prospects in the fields of biological protection, biomedicine and biotechnology. With the rapid development of commercial applications of ectoine, the study of microbial synthesis of ectoine has always been the focus and hotspot of ectoine research.
现有四氢嘧啶生物合成主要有以下四种方法，第一种是利用野生菌株合成四氢嘧啶。野生菌株在高盐环境中可以快速生成四氢嘧啶以维持细胞渗透压的平衡，当细胞处于低盐环境中，细胞可感知渗透压的变化并将四氢嘧啶释放至胞外，保持细胞渗透压平衡。将细胞在高盐及低盐环境中反复循环，细胞可持续对外生成四氢嘧啶。这种生产方式被形象的称为“细菌挤奶”。There are four main methods for the biosynthesis of ectoine. The first is to use wild strains to synthesize ectoine. Wild strains can quickly generate ectoine in a high-salt environment to maintain the balance of cell osmotic pressure. When the cells are in a low-salt environment, the cells can sense the change in osmotic pressure and release ectoine to the outside of the cell to maintain the balance of cell osmotic pressure. By repeatedly cycling the cells in high-salt and low-salt environments, the cells can continuously generate ectoine externally. This production method is vividly called "bacterial milking."
第二种是筛选天然可在低盐环境中合成四氢嘧啶的菌株，利用天然菌株在低盐环境中可生产四氢嘧啶并分泌到胞外特性，实现四氢嘧啶的连续生产。The second method is to screen strains that can naturally synthesize ectoine in a low-salt environment, and use the natural strains' ability to produce ectoine in a low-salt environment and secrete it into the extracellular space to achieve continuous production of ectoine.
第三种是利用合成生物学手段进行四氢嘧啶的异源合成，将四氢嘧啶合成基因簇ectABC转入大肠杆菌、谷氨酸棒杆菌等异源菌种。异源合成优势在于异源菌种中不存在四氢嘧啶的降解通路，不需对降解通路相关基因进行敲除即可实现四氢嘧啶的高产。The third method is to use synthetic biology to perform heterologous synthesis of ectoine, transferring the ectABC gene cluster of ectoine synthesis into heterologous strains such as Escherichia coli and Corynebacterium glutamicum. The advantage of heterologous synthesis is that there is no degradation pathway of ectoine in heterologous strains, and high production of ectoine can be achieved without knocking out genes related to the degradation pathway.
第四种是利用合成生物学手段改造嗜盐微生物提升四氢嘧啶产量。主要是通过提升四氢嘧啶合成通路基因的表达及敲除四氢嘧啶降解相关基因来提升四氢嘧啶的产量。The fourth method is to use synthetic biology to transform halophilic microorganisms to increase the production of ectoine. The production of ectoine is mainly increased by increasing the expression of genes in the ectoine synthesis pathway and knocking out genes related to ectoine degradation.
以上四种常用的发酵方式基本上采用葡萄糖为主要碳源进行发酵，少量利用谷氨酸盐、甘油和L-天门冬氨酸等作为发酵碳源。谷氨酸盐、甘油及L-天门冬氨酸价格高昂，在发酵中往往作为辅助材料，以这些为主要碳源生产成本较高，无法实现大规模的产业化应用。The above four commonly used fermentation methods basically use glucose as the main carbon source for fermentation, and use glutamate, glycerol and L-aspartic acid as fermentation carbon sources in small amounts. Glutamate, glycerol and L-aspartic acid are expensive and are often used as auxiliary materials in fermentation. The production cost of using them as the main carbon source is high, and large-scale industrial application cannot be achieved.
葡萄糖主要来源是淀粉，而淀粉是重要的粮食资源。因此寻找一种以非葡萄糖为主要碳源的微生物发酵产四氢嘧啶是现阶段行业亟需解决的问题。The main source of glucose is starch, which is an important food resource. Therefore, finding a microbial fermentation method that uses non-glucose as the main carbon source to produce ectoine is an urgent problem that the industry needs to solve at this stage.
北京化工大学生命学院谭天伟院士研究团队和软物质高精尖中心兼职教授延斯尼尔森院士研究团队在 Nature Catalysis 刊文，提出了第三代生物炼制的概念，旨在利用微生物细胞工厂将可再生能源和二氧化碳转化为燃料和化学品。与传统生物炼制路线相比，第三代生物炼制更加环保，环境友好性高，且可以极大的降低原料加工成本。The research team of Academician Tianwei Tan from the School of Life Sciences of Beijing University of Chemical Technology and the research team of Academician Jens Nielsen, an adjunct professor at the Advanced Center for Soft Matter, published an article in Nature Catalysis, proposing the concept of the third-generation biorefinery, which aims to use microbial cell factories to convert renewable energy and carbon dioxide into fuels and chemicals. Compared with the traditional biorefinery route, the third-generation biorefinery is more environmentally friendly and can greatly reduce the cost of raw material processing.
短链脂肪酸如丙酸、乙酸等来源广泛，可被微生物利用厨余垃圾、高浓度有机废水、城市垃圾等发酵生产。同时也存在电催化产乙酸，可利用电催化技术将二氧化碳转化为丙酸、乙酸，不仅解决了二氧化碳排放问题，同时生成可利用的生物碳源，助力碳减排碳中和。因此利用丙酸、乙酸等为碳源发酵生产四氢嘧啶不仅能解决利用葡萄糖发酵带来的一系列问题，同时能为碳中和碳减排贡献部分力量。Short-chain fatty acids such as propionic acid and acetic acid are widely available and can be produced by microorganisms through fermentation of kitchen waste, high-concentration organic wastewater, and urban garbage. There is also electrocatalytic acetic acid production, which can be used to convert carbon dioxide into propionic acid and acetic acid, which not only solves the problem of carbon dioxide emissions, but also generates usable biological carbon sources, helping carbon emission reduction and carbon neutrality. Therefore, the use of propionic acid, acetic acid, etc. as carbon sources to ferment and produce tetrahydropyrimidine can not only solve a series of problems caused by the use of glucose fermentation, but also contribute to carbon neutrality and carbon emission reduction.
本申请发明人在进行盐湖淤泥采样后获得一株高产四氢嘧啶的盐单胞菌 Halomonassp. YL01，对其进行宏基因组测序及代谢通路分析表明，其可利用乙酸为唯一碳源进行代谢，且具有较高的乙酸盐耐受度。进一步通过研究发现，盐单胞菌 Halomonassp. YL01可利用以乙酸盐、丙酸盐或者两者构成的混合碳源生产四氢嘧啶。 The inventors of the present application obtained a high-yield ectoine-producing Halomonas sp. YL01 after sampling salt lake silt. Metagenomic sequencing and metabolic pathway analysis showed that it can metabolize using acetate as the only carbon source and has a high tolerance to acetate. Further research found that Halomonas sp. YL01 can produce ectoine using a carbon source consisting of acetate, propionate, or a mixture of the two.
本发明人通过研究对发酵培养基的调整优化，最终实现了利用以乙酸盐为主的碳源高产四氢嘧啶，解决了现有只能利用葡萄糖、谷氨酸盐、甘油等粮食作物生产四氢嘧啶的困境。The inventors have studied and optimized the fermentation medium, and finally achieved high-yield ectoine using a carbon source mainly composed of acetate, thus solving the problem that ectoine can only be produced using food crops such as glucose, glutamate, and glycerol.
技术问题technical problem
本发明所要解决的技术问题是提供一种盐单胞菌株 Halomonassp. YL01，通过对该菌株基因组进行检测分析，发现其可利用以乙酸盐为主要碳源发酵生产四氢嘧啶，并进一步研究，经过对碳源的筛选和发酵工艺的优化，获得高效生产四氢嘧啶的培养基配方及发酵方法。 The technical problem to be solved by the present invention is to provide a Halomonas sp. YL01 strain. By detecting and analyzing the genome of the strain, it is found that the strain can produce ectoine by fermentation with acetate as the main carbon source. Further research, through the screening of carbon sources and the optimization of fermentation processes, a culture medium formula and a fermentation method for efficiently producing ectoine are obtained.
技术解决方案Technical Solutions
本发明提供一种采用盐单胞菌株 Halomonassp. YL01生产四氢嘧啶的发酵方法，所采用的碳源包括乙酸盐或乙酸盐与丙酸盐中构成的混合碳源，以盐单胞菌株 Halomonassp. YL01为底盘菌进行发酵制备，所述乙酸盐在发酵液中的浓度10-40g/L，丙酸盐在发酵液中的浓度不高于10g/L。 The invention provides a fermentation method for producing ectoine by using a Halomonas sp. YL01 strain. The carbon source used includes acetate or a mixed carbon source consisting of acetate and propionate. The Halomonas sp. YL01 strain is used as a base bacteria for fermentation preparation. The concentration of acetate in the fermentation broth is 10-40 g/L, and the concentration of propionate in the fermentation broth is not higher than 10 g/L.
生物材料保藏信息：YL01，分类命名为 Halomonas sp.  YL01，保藏于广东省科学院微生物研究所(广东省微生物分析检测中心)，菌种保藏号为GDMCC.No62420，保藏日期为2022年04月24日，保藏地址为广东省科学院微生物研究所(广东省微生物分析检测中心)。 Biomaterial deposit information: YL01, taxonomic name: Halomonas sp. YL01 is deposited in the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Microbiological Analysis and Testing Center), the strain accession number is GDMCC.No62420, the deposit date is April 24, 2022, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Microbiological Analysis and Testing Center).
其中，所述乙酸盐可以为乙酸钠、乙酸钾、乙酸铵中的一种或几种混合。Wherein, the acetate can be one of sodium acetate, potassium acetate, and ammonium acetate, or a mixture of several of them.
其中，所述丙酸盐可以为丙酸钠、丙酸钾、丙酸铵中的一种或几种混合。The propionate may be one of sodium propionate, potassium propionate, and ammonium propionate, or a mixture of the two.
其中，所述碳源还可以包括葡萄糖。Wherein, the carbon source may also include glucose.
其中，所述发酵温度为32℃-37℃。Wherein, the fermentation temperature is 32°C-37°C.
其中，所述发酵所采用的培养基的pH值为6-11。Wherein, the pH value of the culture medium used in the fermentation is 6-11.
其中，当采用摇瓶发酵时，接种盐单胞菌株 Halomonassp. YL01至摇瓶培养基，发酵培养。 When shake flask fermentation is used, the Halomonas sp. YL01 is inoculated into the shake flask medium for fermentation culture.
其中，摇瓶培养基组成：乙酸钠10-40 g/L、丙酸钠0-10 g/L、葡萄糖0-40 g/L、尿素0.5-10g/L，天冬氨酸 0-10 g/L，酵母粉1-15 g/L，无水硫酸镁0.05-0.6 g/L，磷酸二氢钾1.5-5.5 g/L，氯化钠50-80 g/L，Fe(III)-NH 4-Citrate 0.05-0.1 g/L，CaCl 2·2H 2O 0.02-0.2 g/L，ZnSO 4·7H 2O 0.01-0.1 g/L，MnCl 2·4H 2O 0.002-0.02 g/L，H 3BO 30.01-0.05 g/L，CoCl 2·6H 2O 0.005-0.02 g/L，CuSO 4·5H 2O 0.01-0.08 g/L，NiCl 2·6H 2O 0.005-0.01 g/L，NaMoO 4·2H 2O 0.02-0.1 g/L。 The shake flask medium composition includes: sodium acetate 10-40 g/L, sodium propionate 0-10 g/L, glucose 0-40 g/L, urea 0.5-10 g/L, aspartic acid 0-10 g/L, yeast powder 1-15 g/L, anhydrous magnesium sulfate 0.05-0.6 g/L, potassium dihydrogen phosphate 1.5-5.5 g/L, sodium chloride 50-80 g/L, Fe(III)-NH 4 -Citrate 0.05-0.1 g/L, CaCl 2 ·2H 2 O 0.02-0.2 g/L, ZnSO 4 ·7H 2 O 0.01-0.1 g/L, MnCl 2 ·4H 2 O 0.002-0.02 g/L, H 3 BO 3 0.01-0.05 g/L, CoCl 2 ·6H 2 O 0.005-0.02 g/L, CuSO 4 ·5H 2 O 0.01-0.08 g/L, NiCl 2 ·6H 2 O 0.005-0.01 g/L, NaMoO 4 ·2H 2 O 0.02-0.1 g/L.
其中，当采用发酵罐发酵时，将菌种按接种量为5％-15％接入发酵罐培养基；发酵过程中控制溶氧为20％～40％；发酵培养，发酵期间检测乙酸含量，当乙酸含量低于10g/L添加补料培养基，依据生长情况补料。When fermentation is carried out in a fermenter, the strain is inoculated at a rate of 5% to 15% into the fermenter culture medium; during the fermentation process, the dissolved oxygen is controlled at 20% to 40%; during fermentation and culture, the acetic acid content is detected, and when the acetic acid content is lower than 10 g/L, feed culture medium is added, and feed is supplemented according to the growth situation.
其中，发酵罐培养基组成(g/L)：Among them, the fermentation tank culture medium composition (g/L):
乙酸钠10-40、丙酸钠0-10、葡萄糖0-40、氯化钠50-80 ；酵母膏 1-10 、MgSO 4  1-10；尿素1-4 ；磷酸二氢钾3-10；组分I 5-15、组分Ⅱ1-5。 Sodium acetate 10-40, sodium propionate 0-10, glucose 0-40, sodium chloride 50-80; yeast extract 1-10, MgSO4 1-10; urea 1-4; potassium dihydrogen phosphate 3-10; component I 5-15, component II 1-5.
其中，组分I：Fe(III)-NH 4-Citrate 5g/L、 CaCl 2·2H 2O 2 g/L、HCl 5M ，去离子水配制；组分Ⅱ：ZnSO 4·7H 2O 0.1 g/L;MnCl 2·4H 2O   0.03g/L;H 3BO 30.3g/L;CoCl 2·6H 2O 0.2 g/L;CuSO 4·5H 2O  0.01 g/L;NiCl 2·6H 2O 0.02 g/L;NaMoO 4·2H 2O 0.03 g/L ，去离子水配制。 Among them, component I: Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ·2H 2 O 2 g/L, HCl 5M, prepared with deionized water; component II: ZnSO 4 ·7H 2 O 0.1 g/L; MnCl 2 ·4H 2 O 0.03g/L; H 3 BO 3 0.3g/L; CoCl 2 ·6H 2 O 0.2 g/L; CuSO 4 ·5H 2 O 0.01 g/L; NiCl 2 ·6H 2 O 0.02 g/L; NaMoO 4 ·2H 2 O 0.03 g/L, prepared with deionized water.
有益效果Beneficial Effects
与现有技术相比，本发明技术方案具有以下优点：Compared with the prior art, the technical solution of the present invention has the following advantages:
1、与现有菌种相比，本发明 Halomonas sp.YL01菌具有较高的pH耐受度，可耐受pH为6-11，可耐受高浓度乙酸盐带来的高pH环境，常规的菌种无法耐受pH大于9的培养环境； 1. Compared with existing strains, the Halomonas sp. YL01 of the present invention has a higher pH tolerance, which can tolerate a pH of 6-11, and can tolerate a high pH environment caused by high concentrations of acetate. Conventional strains cannot tolerate a culture environment with a pH greater than 9;
2、与现有技术相比，本发明提供了一种以乙酸盐为主的碳源作为发酵培养基，可大幅度降低四氢嘧啶的发酵成本。2. Compared with the prior art, the present invention provides a carbon source mainly based on acetate as a fermentation medium, which can significantly reduce the fermentation cost of ectoine.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1： Halomonas sp.YL01利用乙酸盐和丙酸盐作为混合碳源生产四氢嘧啶代谢通路图； Figure 1: Metabolic pathway diagram of Halomonas sp.YL01 producing ectoine using acetate and propionate as mixed carbon sources;
图2： Halomonas sp.YL01碳源利用生化鉴定图； Figure 2: Biochemical identification of carbon source utilization of Halomonas sp.YL01;
图3a为四氢嘧啶标准品谱图、图3b为 Halomonas sp.YL01利用乙酸盐产四氢嘧啶谱图。 FIG. 3a is a spectrum of a standard product of ectoine, and FIG. 3b is a spectrum of ectoine produced by Halomonas sp. YL01 using acetate.
本发明的最佳实施方式Best Mode for Carrying Out the Invention
本发明所采用的用于发酵的菌株为盐单胞菌株 Halomonas sp. YL01，其保藏于广东省科学院微生物研究所(广东省微生物分析检测中心)，保藏日期为2022年4月24日，保藏编号为GDMCC.No62420。 The strain used for fermentation in the present invention is Halomonas sp. YL01, which is deposited in the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbiological Analysis and Testing Center), the deposit date is April 24, 2022, and the deposit number is GDMCC.No62420.
本发明首先在青海省盐湖淤泥取样，利用嗜盐菌筛选培养基60LB对盐湖中的嗜盐菌进行筛选，筛选获得一株盐单胞菌 Halomonas sp.YL01，然后对 Halomonas sp.YL01菌种进行碳源利用的生化鉴定，经验证 Halomonas sp.YL01可以利用乙酸盐、丙酸盐等作为碳源生长。 The present invention firstly samples salt lake sludge in Qinghai Province, screens halophilic bacteria in the salt lake using halophilic bacteria screening medium 60LB, obtains a strain of Halomonas sp.YL01 by screening, and then performs biochemical identification of carbon source utilization of Halomonas sp.YL01. It is verified that Halomonas sp.YL01 can grow by utilizing acetate, propionate, etc. as carbon sources.
随后，发明人利用常规的生产四氢嘧啶的培养基确定该菌种产四氢嘧啶的能力，再以乙酸盐为主作为碳源，设计60MM培养基为基础营养源，进行摇瓶发酵产四氢嘧啶。最后设计发酵培养基，利用发酵罐发酵，并设计补料培养基，获得菌种发酵以乙酸盐为主碳源生产四氢嘧啶的分批补料发酵方法。Subsequently, the inventors used a conventional culture medium for producing ectoine to determine the ability of the strain to produce ectoine, and then used acetate as the main carbon source, designed a 60MM culture medium as the basic nutrient source, and performed shake flask fermentation to produce ectoine. Finally, the fermentation medium was designed, fermented in a fermenter, and a feed medium was designed to obtain a batch feed fermentation method for producing ectoine by fermenting the strain with acetate as the main carbon source.
具体的，本发明提供一种采用盐单胞菌株 Halomonassp. YL01生产四氢嘧啶的发酵方法，所采用的碳源包括乙酸盐或乙酸盐与丙酸盐中构成的混合碳源，通过盐单胞菌株 Halomonassp. YL01进行发酵制备，所述乙酸盐在发酵液中的浓度10-40g/L，丙酸盐在发酵液中的浓度不高于10g/L。发明人经过实验发现，高浓度丙酸盐将抑制菌体的生长，降低四氢嘧啶产量。 Specifically, the present invention provides a fermentation method for producing ectoine using Halomonas sp. YL01, wherein the carbon source used includes acetate or a mixed carbon source composed of acetate and propionate, and the ectoine is prepared by fermentation using Halomonas sp. YL01, wherein the concentration of acetate in the fermentation broth is 10-40 g/L, and the concentration of propionate in the fermentation broth is not higher than 10 g/L. The inventors have found through experiments that high concentrations of propionate will inhibit the growth of bacteria and reduce the yield of ectoine.
所述乙酸盐可以为乙酸钠、乙酸钾、乙酸铵中的一种或几种混合。所述丙酸盐可以为丙酸钠、丙酸钾、丙酸铵中的一种或几种混合。乙酸盐和丙酸盐可以通过市售的方式获得，也可以通过将二氧化碳通过热催化、电催化、光催化等手段转化而成，实现二氧化碳的再利用。The acetate may be one or a mixture of sodium acetate, potassium acetate, and ammonium acetate. The propionate may be one or a mixture of sodium propionate, potassium propionate, and ammonium propionate. Acetate and propionate may be obtained in a commercially available manner, or may be obtained by converting carbon dioxide through thermal catalysis, electrocatalysis, photocatalysis, and the like, thereby realizing the reuse of carbon dioxide.
所述碳源还可以包括葡萄糖。The carbon source may also include glucose.
发酵温度优选为32℃-37℃，发酵所采用的培养基的pH值为6-11。The fermentation temperature is preferably 32° C.-37° C., and the pH value of the culture medium used for fermentation is 6-11.
其中，当采用摇瓶发酵时，接种盐单胞菌株 Halomonassp. YL01至摇瓶培养基，发酵培养，搅拌转速150-300rpm，发酵持续24h～48h。 When shake flask fermentation is adopted, the Halomonas sp. YL01 is inoculated into the shake flask medium, and the fermentation culture is carried out with a stirring speed of 150-300 rpm, and the fermentation lasts for 24 hours to 48 hours.
摇瓶培养基60MM组成：乙酸钠10-40 g/L、丙酸钠0-10 g/L、葡萄糖0-40 g/L、尿素0.5-10g/L，天冬氨酸 0-10 g/L，酵母粉1-15 g/L，无水硫酸镁0.05-0.6 g/L，磷酸二氢钾1.5-5.5 g/L，氯化钠50-80 g/L，Fe(III)-NH 4-Citrate 0.05-0.1 g/L，CaCl 2·2H 2O 0.02-0.2 g/L，ZnSO 4·7H 2O 0.01-0.1 g/L，MnCl 2·4H 2O 0.002-0.02 g/L，H 3BO 30.01-0.05 g/L，CoCl 2·6H 2O 0.005-0.02 g/L，CuSO 4·5H 2O 0.01-0.08 g/L，NiCl 2·6H 2O 0.005-0.01 g/L，NaMoO 4·2H 2O 0.02-0.1 g/L。 Shake flask medium 60MM composition: sodium acetate 10-40 g/L, sodium propionate 0-10 g/L, glucose 0-40 g/L, urea 0.5-10 g/L, aspartic acid 0-10 g/L, yeast powder 1-15 g/L, anhydrous magnesium sulfate 0.05-0.6 g/L, potassium dihydrogen phosphate 1.5-5.5 g/L, sodium chloride 50-80 g/L, Fe(III)-NH 4 -Citrate 0.05-0.1 g/L, CaCl 2 ·2H 2 O 0.02-0.2 g/L, ZnSO 4 ·7H 2 O 0.01-0.1 g/L, MnCl 2 ·4H 2 O 0.002-0.02 g/L, H 3 BO 3 0.01-0.05 g/L, CoCl 2 ·6H 2 O 2 O 0.005-0.02 g/L, CuSO 4 ·5H 2 O 0.01-0.08 g/L, NiCl 2 ·6H 2 O 0.005-0.01 g/L, NaMoO 4 ·2H 2 O 0.02-0.1 g/L.
其中，当采用发酵罐发酵时，将菌种按接种量为5％-15％接入发酵罐培养基；发酵过程中控制溶氧为20％～40％；搅拌转速200-600rpm；发酵持续24h～48h，发酵期间检测乙酸含量，当乙酸含量低于10g/L添加补料培养基，依据生长情况，前0-24h补料使用补料Ⅰ，后24-48h补料使用补料Ⅱ。When fermentation is carried out in a fermenter, the strain is inoculated at a rate of 5% to 15% into the fermenter culture medium; during the fermentation process, the dissolved oxygen is controlled at 20% to 40%; the stirring speed is 200 to 600 rpm; the fermentation lasts for 24 to 48 hours, and the acetic acid content is detected during the fermentation. When the acetic acid content is lower than 10 g/L, feed culture medium is added. Depending on the growth conditions, feed I is used for the first 0 to 24 hours, and feed II is used for the last 24 to 48 hours.
其中，发酵罐培养基组成(g/L)：Among them, the fermentation tank culture medium composition (g/L):
乙酸钠10-40、丙酸钠0-10、葡萄糖0-40、氯化钠50-80 ；酵母膏 1-10 、MgSO 4  1-10；尿素1-4 ；磷酸二氢钾3-10；第一微量元素组分 5-15、第二微量元素组分1-5。 Sodium acetate 10-40, sodium propionate 0-10, glucose 0-40, sodium chloride 50-80; yeast extract 1-10, MgSO4 1-10; urea 1-4; potassium dihydrogen phosphate 3-10; the first trace element component 5-15, the second trace element component 1-5.
第一微量元素组分具体包括 Fe(III)-NH 4-Citrate 3-7 g/L； CaCl 2·2H 2O 1-3 g/L；HCl 3-7 M    ，通过去离子水配制； The first trace element component specifically includes Fe(III)-NH 4 -Citrate 3-7 g/L; CaCl 2 ·2H 2 O 1-3 g/L; HCl 3-7 M, prepared by deionized water;
第二微量元素组分具体包括ZnSO 4·7H 2O 0.1-0.2 g/L; MnCl 2·4H 2O       0.02-0.04g/L; H 3BO 3 0.2-0.4g/L;CoCl 2·6H 2O 0.1-0.3 g/L;CuSO 4·5H 2O   0.01-0.03 g/L;NiCl 2·6H 2O 0.01-0.03 g/L;NaMoO 4·2H 2O 0.02-0.05 g/L ，通过去离子水配制。 The second trace element component specifically includes ZnSO 4 ·7H 2 O 0.1-0.2 g/L; MnCl 2 ·4H 2 O 0.02-0.04 g/L; H 3 BO 3 0.2-0.4 g/L; CoCl 2 ·6H 2 O 0.1-0.3 g/L; CuSO 4 ·5H 2 O 0.01-0.03 g/L; NiCl 2 ·6H 2 O 0.01-0.03 g/L; NaMoO 4 ·2H 2 O 0.02-0.05 g/L, and is prepared with deionized water.
进一步优选，第一微量元素组分： Fe(III)-NH 4-Citrate 5g/L、 CaCl 2·2H 2O 2 g/L、HCl 5M ，去离子水配制； Further preferably, the first trace element component: Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ·2H 2 O 2 g/L, HCl 5M, prepared with deionized water;
第二微量元素组分：ZnSO 4·7H 2O 0.1g/L; MnCl 2·4H 2O 0.03g/L; H 3BO 3 0.3g/L; CoCl 2·6H 2O 0.2 g/L; CuSO 4·5H 2O  0.01g/L; NiCl 2·6H 2O 0.02 g/L; NaMoO 4·2H 2O  0.03g/L ，去离子水配制。 The second trace element component: ZnSO 4 ·7H 2 O 0.1g/L; MnCl 2 ·4H 2 O 0.03g/L; H 3 BO 3 0.3g/L; CoCl 2 ·6H 2 O 0.2 g/L; CuSO 4 ·5H 2 O 0.01g/L; NiCl 2 ·6H 2 O 0.02 g/L; NaMoO 4 ·2H 2 O 0.03g/L, prepared with deionized water.
其中，补料培养基Ⅰ：乙酸盐300-1000g/L，尿素 15-100 g/L，天冬氨酸0-15 g/L。Among them, feed medium I: acetate 300-1000 g/L, urea 15-100 g/L, aspartic acid 0-15 g/L.
其中，补料培养基Ⅱ：乙酸钠500-1000 g/L，尿素 2-20 g/L，其中乙酸盐的种类可以是乙酸钠、乙酸钾、乙酸铵中的一种或几种的混合物。Among them, feed medium II: sodium acetate 500-1000 g/L, urea 2-20 g/L, wherein the type of acetate can be one of sodium acetate, potassium acetate, ammonium acetate or a mixture of several of them.
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.
下文的公开提供了许多不同的实施例或例子用来实现本发明的不同实施方式。为了简化本发明的公开，下文中对特定实施例或示例进行描述。当然，他们仅仅为示例，并且目的不在于限制本发明。此外，本发明提供的各种特定工艺和材料的例子，本领域普通技术人员可以意识到其他工艺的可应用性和/或其他材料的使用。除非另有说明，本发明的实施将采用本领域技术人员的能力范围之内的化学、分子生物学等领域的传统技术。The disclosure below provides many different embodiments or examples to realize different implementation methods of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, those of ordinary skill in the art can be aware of the applicability of other processes and/or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the conventional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art.
下面通过说明性的具体实施例对本发明进行描述，这些实施例并不以任何方式限制本发明的范围。特别说明的是：本发明所用到的试剂除特别说明外均有市售。The present invention is described below by means of illustrative specific examples, which are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents used in the present invention are commercially available unless otherwise specified.
其他需要说明的是，除非另外定义，本发明实施例使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。It should also be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present invention should have the common meanings understood by persons having ordinary skills in the field to which the present disclosure belongs.
实验材料和试剂Experimental Materials and Reagents
1、菌种筛选所用培养基、乙酸盐发酵培养基及批次补料发酵营养物组分1. Culture medium for strain screening, acetate fermentation medium and nutrient components for batch fed-batch fermentation
1、菌种筛选培养基1. Culture medium for strain screening
（1）60LB培养基（g/L）：蛋白胨5-20，酵母粉3-10，氯化钠50-80，pH调至6-10，用于 Halomonas sp.YL01的筛分和菌种培养；配置平板即需加入1.5-2%的琼脂糖。 (1) 60LB medium (g/L): peptone 5-20, yeast powder 3-10, sodium chloride 50-80, pH adjusted to 6-10, used for screening and culture of Halomonas sp. YL01; 1.5-2% agarose should be added when preparing the plate.
（2）60MM培养基（g/L）：乙酸钠10-40、丙酸钠0-10、葡萄糖0-40、尿素0.5-10，天冬氨酸 0-10，酵母粉1-15，无水硫酸镁0.05-0.6，磷酸二氢钾1.5-5.5，氯化钠50-80，Fe(III)-NH 4-Citrate 0.05-0.1，CaCl 2·2H 2O 0.02-0.2，ZnSO 4·7H 2O 0.01-0.1，MnCl 2·4H 2O 0.002-0.02，H 3BO 30.01-0.05，CoCl 2·6H 2O 0.005-0.02，CuSO 4·5H 2O 0.01-0.08，NiCl 2·6H 2O 0.005-0.01，NaMoO 4·2H 2O 0.02-0.1，pH 6-11。 (2) 60MM culture medium (g/L): sodium acetate 10-40, sodium propionate 0-10, glucose 0-40, urea 0.5-10, aspartic acid 0-10, yeast powder 1-15, anhydrous magnesium sulfate 0.05-0.6, potassium dihydrogen phosphate 1.5-5.5, sodium chloride 50-80, Fe(III)-NH 4 -Citrate 0.05-0.1, CaCl 2 ·2H 2 O 0.02-0.2, ZnSO 4 ·7H 2 O 0.01-0.1, MnCl 2 ·4H 2 O 0.002-0.02, H 3 BO 3 0.01-0.05, CoCl 2 ·6H 2 O 0.005-0.02, CuSO 4 ·5H 2 O 0.01-0.08, NiCl 2 ·6H 2 O 0.005-0.01, NaMoO 4 ·2H 2 O 0.02-0.1, pH 6-11.
该用于 Halomonassp.YL01菌生产四氢嘧啶的基础培养基，在此基础上增加碳源用于生产四氢嘧啶。在培养基中蛋白胨提供微生物生长所需的氮源、酵母粉提供微生物生长所需的氮源、维生素、生长因子等营养；Mg 2+是TCA途径、EMP途径等多种酶的重要激活剂；磷元素是蛋白质、DNA的重要组成成分。该培养基首先创造高盐环境（5%-8%），刺激菌体合成四氢嘧啶维持渗透压平衡；尿素转化为铵，铵转化为L-谷氨酸进入四氢嘧啶合成途径提升四氢嘧啶产量，天冬氨酸是四氢嘧啶的前体物质，可快速转化为四氢嘧啶，提升四氢嘧啶产量，同时高浓度的尿素会抑制菌体产PHB，进一步提升四氢嘧啶产量。 This is a basic medium for Halomonas sp.YL01 to produce ectoine, and a carbon source is added to this medium to produce ectoine. In the medium, peptone provides the nitrogen source required for microbial growth, yeast powder provides the nitrogen source, vitamins, growth factors and other nutrients required for microbial growth; Mg 2+ is an important activator of multiple enzymes such as the TCA pathway and the EMP pathway; phosphorus is an important component of protein and DNA. The medium first creates a high-salt environment (5%-8%) to stimulate the bacteria to synthesize ectoine and maintain osmotic pressure balance; urea is converted into ammonium, and ammonium is converted into L-glutamate to enter the ectoine synthesis pathway to increase the yield of ectoine. Aspartic acid is a precursor of ectoine and can be quickly converted into ectoine to increase the yield of ectoine. At the same time, high concentrations of urea will inhibit the production of PHB by the bacteria, further increasing the yield of ectoine.
（3）补料Ⅰ（g/L）：乙酸盐300-1000，尿素 15-100，天冬氨酸 0-15；(3) Feed I (g/L): acetate 300-1000, urea 15-100, aspartic acid 0-15;
（4）补料Ⅱ（g/L）：乙酸盐500-1000，尿素 2-20。(4) Feed II (g/L): acetate 500-1000, urea 2-20.
（5）发酵培养基(5) Fermentation medium
第一微量元素组分： Fe(III)-NH 4-Citrate 5g/L、 CaCl 2·2H 2O 2g/L、HCl 5M，去离子水配制 The first trace element component: Fe(III)-NH 4 -Citrate 5g/L, CaCl 2 ·2H 2 O 2 g/L, HCl 5M, prepared with deionized water
第二微量元素组分：ZnSO 4·7H 2O 0.1 g/L；MnCl 2·4H 2O 0.03 g/L；H 3BO 3 0.3 g/L；CoCl 2·6H 2O 0.2 g/L；CuSO 4·5H 2O  0.01 g/L；NiCl 2·6H 2O 0.02 g/L；NaMoO 4·2H 2O 0.03 g/L 去离子水配制。 The second trace element component: ZnSO 4 ·7H 2 O 0.1 g/L; MnCl 2 ·4H 2 O 0.03 g/L; H 3 BO 3 0.3 g/L; CoCl 2 ·6H 2 O 0.2 g/L; CuSO 4 ·5H 2 O 0.01 g/L; NiCl 2 ·6H 2 O 0.02 g/L; NaMoO 4 ·2H 2 O 0.03 g/L, prepared with deionized water.
配料（g/L）：乙酸钠10-40、丙酸钠0-10、葡萄糖0-40、氯化钠 50-80 ；酵母膏 1-10 、MgSO 4  1-10；尿素1-4 ；磷酸二氢钾3-10；第一微量元素组分5-15、第二微量元素组分1-5。 Ingredients (g/L): sodium acetate 10-40, sodium propionate 0-10, glucose 0-40, sodium chloride 50-80; yeast extract 1-10, MgSO4 1-10; urea 1-4; potassium dihydrogen phosphate 3-10; the first trace element component 5-15, the second trace element component 1-5.
本发明的实施方式Embodiments of the present invention
实施例1Example 1 Halomonas Halomonas sp.YL01菌种碳源利用的生化鉴定Biochemical identification of carbon source utilization by sp.YL01
1、盐单胞菌 Halomonassp.YL01的获取 1. Acquisition of Halomonas sp.YL01
取青海省盐湖淤泥样1 g后用无菌水连续稀释，涂布于60LB 平板上，37 ℃培养48 h，待菌落长出后挑取单菌落，继续划线传代培养30 d，驯化筛选直至获得纯菌。将得到的纯菌16S rDNA 扩增、测序以及序列比对后，即获得本发明的盐单胞菌 Halomonas sp.YL01，生物保藏号为：GDMCC No. 62420。 Take 1 g of Qinghai Salt Lake sludge sample and dilute it with sterile water, spread it on a 60LB plate, and culture it at 37 °C for 48 h. After the colony grows, pick a single colony, continue to streak and subculture for 30 days, and acclimate and screen until pure bacteria are obtained. After amplifying, sequencing and aligning the 16S rDNA of the obtained pure bacteria, the Halomonas sp.YL01 of the present invention is obtained, and the biological preservation number is: GDMCC No. 62420.
60LB培养基组分如下（g/L）：蛋白胨10，酵母粉8，氯化钠60，pH调至8。The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
2、生理生化验证2. Physiological and biochemical verification
采用Biolog的PM高通量微生物代谢表型芯片系统对 Halomonassp.YL01的碳源利用进行检测，经过检测获得菌种碳源利用的检测结果，见表1。 Biolog's PM high-throughput microbial metabolic phenotype chip system was used to detect the carbon source utilization of Halomonas sp. YL01. The test results of the carbon source utilization of the strain were obtained, see Table 1.
表1 菌株 Halomonassp.YL01生理生化特性-碳源利用 Table 1 Physiological and biochemical characteristics of strain Halomonas sp.YL01 - carbon source utilization
通过对表1数据分析显示， Halomonas sp.YL01可以利用丙酸、乙酸、丙酮酸等碳源进行生理代谢活动，具备开发以短链脂肪酸为碳源发酵的基础。 Analysis of the data in Table 1 shows that Halomonas sp.YL01 can utilize carbon sources such as propionic acid, acetic acid, and pyruvic acid to carry out physiological metabolic activities, and has the basis for developing fermentation using short-chain fatty acids as carbon sources.
3、通过测定生长量测定菌株对丙酸、乙酸盐的利用3. Determine the utilization of propionate and acetate by measuring growth
①取接种环在超净工作台上将菌株盐单胞菌 Halomonas sp.YL01划线至60LB平板上，37 ℃活化24 h，待其长出单克隆； ① Take an inoculation loop and streak the strain Halomonas sp.YL01 onto a 60LB plate on a clean bench, activate it at 37 °C for 24 h, and wait for it to grow a single clone;
②在超净工作台中用无菌接种环挑取上述①的单克隆抗体，接种至装有5 mL 60LB培养基中的50 mL摇菌管中，37℃200 rpm培养12h，获得种子液。② In an ultra-clean workbench, pick up the monoclonal antibody in ① above with a sterile inoculation loop, inoculate it into a 50 mL shaking tube containing 5 mL 60LB culture medium, and culture at 37℃ and 200 rpm for 12 h to obtain seed solution.
③用移液器将种子液以5%添加量分别添加至含100 ml 60MM（对照组）、100 mL 60MM+30g/L乙酸钠、100 mL 60MM+15g/L乙酸钠+15g/L丙酸钠、100 mL 60MM+30g/L丙酸钠、100 mL 60MM+30g/L葡萄糖培养基的500 mL三角瓶中，其中培养基pH为8.0，37℃200 rpm培养12h，测定其生物量（OD 600），实验重复三次，结果见图1。 ③ Use a pipette to add 5% seed solution into 500 mL Erlenmeyer flasks containing 100 ml 60MM (control group), 100 mL 60MM+30g/L sodium acetate, 100 mL 60MM+15g/L sodium acetate +15g/L sodium propionate, 100 mL 60MM+30g/L sodium propionate, and 100 mL 60MM+30g/L glucose culture medium, respectively. The pH of the culture medium was 8.0. The culture was cultured at 37℃ and 200 rpm for 12 hours. The biomass ( OD600 ) was determined. The experiment was repeated three times. The results are shown in Figure 1.
60MM培养基个组分（g/L）如下：尿素1，天冬氨酸 0，酵母粉10，无水硫酸镁2，磷酸二氢钾4，氯化钠60，Fe(III)-NH 4-Citrate 0.05，CaCl 2·2H 2O 0.02，ZnSO 4·7H 2O 0.05，MnCl 2·4H 2O 0.005，H 3BO 3 0.01，CoCl 2·6H 2O 0.005，CuSO 4·5H 2O 0.01，NiCl 2·6H 2O 0.007，NaMoO 4·2H 2O 0.02。 The components (g/L) of 60MM culture medium are as follows: urea 1, aspartic acid 0, yeast powder 10, anhydrous magnesium sulfate 2, potassium dihydrogen phosphate 4, sodium chloride 60, Fe(III)-NH 4 -Citrate 0.05, CaCl 2 ·2H 2 O 0.02, ZnSO 4 ·7H 2 O 0.05, MnCl 2 ·4H 2 O 0.005, H 3 BO 3 0.01, CoCl 2 ·6H 2 O 0.005, CuSO 4 ·5H 2 O 0.01, NiCl 2 ·6H 2 O 0.007, NaMoO 4 ·2H 2 O 0.02.
图2结果表明，盐单胞菌 Halomonas sp.YL01可以在60MM培养基中维持基本生理活动，但因缺少碳源，生物量没有明显增长。与60MM组对比，以丙酸盐或乙酸盐为唯一碳源均出现了生物量的明显增长，表明盐单胞菌 Halomonas sp.YL01可以利用单独利用丙酸盐或乙酸盐进行发酵，具有对应的代谢途径。可进行相关的发酵研究。 The results in Figure 2 show that Halomonas sp.YL01 can maintain basic physiological activities in 60MM medium, but due to the lack of carbon source, the biomass did not increase significantly. Compared with the 60MM group, the biomass increased significantly when propionate or acetate was the only carbon source, indicating that Halomonas sp.YL01 can ferment using propionate or acetate alone and has the corresponding metabolic pathway. Related fermentation research can be carried out.
进一步研究发现，以丙酸盐或乙酸盐为唯一碳源条件下，其生物增长量显著低于乙酸盐+丙酸盐的培养基，且乙酸盐+丙酸盐的培养基生物生长量与利用葡萄糖培养基无显著差距。因此选用丙酸盐和乙酸盐构成的混合碳源进一步进行四氢嘧啶发酵研究，其代谢路径如图1所示。Further research found that when propionate or acetate was used as the sole carbon source, the biological growth was significantly lower than that of the medium containing acetate + propionate, and the biological growth of the medium containing acetate + propionate was not significantly different from that of the medium containing glucose. Therefore, a mixed carbon source consisting of propionate and acetate was selected to further study the fermentation of tetrahydropyrimidine, and its metabolic pathway is shown in Figure 1.
实施例2  摇瓶发酵产四氢嘧啶Example 2 Shake flask fermentation to produce ectoine
①用接种环在超净工作台上将菌株盐单胞菌 Halomonas sp.YL01划线至60LB平板上，37 ℃活化24 h，待其长出单克隆； ① Use an inoculation loop to streak the strain Halomonas sp.YL01 onto a 60LB plate on a clean bench, activate it at 37 °C for 24 h, and wait for it to grow into a single clone;
②在超净工作台中用无菌接种环挑取上述①的单克隆抗体，接种至装有5mL 60LB培养基中的50mL摇菌管中，37℃200 rpm培养12H，获得种子液。② In the clean bench, pick the monoclonal antibody in ① above with a sterile inoculation loop, inoculate it into a 50mL shaking tube containing 5mL 60LB culture medium, and culture at 37℃ and 200 rpm for 12 hours to obtain seed solution.
③在超净工作台上用移液器将种子液以5%添加量添加至含200 mL 60MM+20g/L乙酸钠+10g/L丙酸钠培养基（pH为9）的1000 mL三角瓶中，37℃，200 rpm培养24 h。③ On the clean bench, use a pipette to add 5% of the seed solution into a 1000 mL Erlenmeyer flask containing 200 mL of 60MM + 20 g/L sodium acetate + 10 g/L sodium propionate medium (pH 9), and culture at 37°C, 200 rpm for 24 h.
60MM培养基个组分（g/L）如下：尿素1，天冬氨酸 0，酵母粉10，无水硫酸镁2，磷酸二氢钾4，氯化钠60，Fe(III)-NH 4-Citrate 0.05，CaCl 2·2H 2O 0.02，ZnSO 4·7H 2O 0.05，MnCl 2·4H 2O 0.005，H 3BO 3 0.01，CoCl 2·6H 2O 0.005，CuSO 4·5H 2O 0.01，NiCl 2·6H 2O 0.007，NaMoO 4·2H 2O 0.02。 The components (g/L) of 60MM culture medium are as follows: urea 1, aspartic acid 0, yeast powder 10, anhydrous magnesium sulfate 2, potassium dihydrogen phosphate 4, sodium chloride 60, Fe(III)-NH 4 -Citrate 0.05, CaCl 2 ·2H 2 O 0.02, ZnSO 4 ·7H 2 O 0.05, MnCl 2 ·4H 2 O 0.005, H 3 BO 3 0.01, CoCl 2 ·6H 2 O 0.005, CuSO 4 ·5H 2 O 0.01, NiCl 2 ·6H 2 O 0.007, NaMoO 4 ·2H 2 O 0.02.
60LB培养基组分如下（g/L）：蛋白胨10，酵母粉8，氯化钠60，pH调至8。The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
四氢嘧啶含量测定：取发酵后菌液，用超声波细胞破碎仪破碎细胞，将破碎后液体12200g离心10min，取上清， 0.22μm滤膜过滤，完成样品处理。高效液相色谱法（HPLC）采用C18色谱柱；流动相为乙腈（A 液）和纯水（B 液）且A:B=70:30；进样量 10 μL；流速 1 mL/min；检测波长 210 nm。Determination of ectoine content: Take the fermented bacterial liquid, use an ultrasonic cell disruptor to disrupt the cells, centrifuge the disrupted liquid at 12200g for 10 minutes, take the supernatant, filter it with a 0.22μm filter membrane, and complete the sample processing. High performance liquid chromatography (HPLC) uses a C18 column; the mobile phase is acetonitrile (liquid A) and pure water (liquid B) and A:B=70:30; the injection volume is 10 μL; the flow rate is 1 mL/min; the detection wavelength is 210 nm.
从图3a的四氢嘧啶标准谱图和采用本实施例方法制备的四氢嘧啶谱图对比可以看出，两者相同，证明采用乙酸和丙酸作为碳源，采用盐单胞菌 Halomonas sp.YL01可以很好的生产四氢嘧啶。 From the comparison between the standard spectrum of ectoine in FIG. 3a and the spectrum of ectoine prepared by the method of this embodiment, it can be seen that the two are the same, which proves that ectoine can be well produced by using acetic acid and propionic acid as carbon sources and Halomonas sp. YL01.
实施例3Example 3
采用与实施例2相同的方法，区别在于，所采用的碳源为20g/L乙酸钠+10g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 20 g/L sodium acetate + 10 g/L sodium propionate.
实施例4Example 4
采用与实施例2相同的方法，区别在于，所采用的碳源为20g/L乙酸钠+5g/L乙酸钾+5g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 20 g/L sodium acetate + 5 g/L potassium acetate + 5 g/L sodium propionate.
实施例5Example 5
采用与实施例2相同的方法，区别在于，所采用的碳源为10g/L乙酸钠+20g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 10 g/L sodium acetate + 20 g/L sodium propionate.
实施例6Example 6
采用与实施例2相同的方法，区别在于，所采用的碳源为25g/L乙酸钠+5g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 25 g/L sodium acetate + 5 g/L sodium propionate.
实施例7Example 7
采用与实施例2相同的方法，区别在于，所采用的碳源为5g/L乙酸钠+25g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 5 g/L sodium acetate + 25 g/L sodium propionate.
实施例8Example 8
采用与实施例2相同的方法，区别在于，所采用的碳源为15g/L 葡萄糖+10g/L乙酸钠+5g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 15 g/L glucose + 10 g/L sodium acetate + 5 g/L sodium propionate.
实施例9Example 9
采用与实施例2相同的方法，区别在于，所采用的碳源为10g/L 葡萄糖+10g/L乙酸钠+10g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 10 g/L glucose + 10 g/L sodium acetate + 10 g/L sodium propionate.
比较例1Comparative Example 1
采用与实施例2相同的方法，区别在于，所采用的碳源为30g/L乙酸钠。The same method as in Example 2 was used, except that the carbon source used was 30 g/L sodium acetate.
比较例2Comparative Example 2
采用与实施例2相同的方法，区别在于，所采用的碳源为30g/L丙酸钠。The same method as in Example 2 was used, except that the carbon source used was 30 g/L sodium propionate.
比较例3Comparative Example 3
采用与实施例2相同的方法，区别在于，所采用的碳源为30g/L葡萄糖。The same method as in Example 2 was used, except that the carbon source used was 30 g/L glucose.
表2 不同乙酸盐和丙酸盐作为碳源条件下摇瓶发酵产四氢嘧啶的含量Table 2 The content of ectoine produced by shake flask fermentation under different conditions of acetate and propionate as carbon sources
由表2可知，采用葡萄糖作为碳源的比较例3的四氢嘧啶产量最高，而采用乙酸盐和丙酸盐作为混合碳源的实施例2的四氢嘧啶产量与比较例3较为接近，为8.1g/L，其次为实施例8。通过该表可以看出，当加丙酸盐浓度高于10g/L时，四氢嘧啶含量逐渐降低，表明在高浓度丙酸盐的刺激下，盐单胞菌的生长明显被抑制，四氢嘧啶产量随之下降。这是因为高浓度丙酸进入胞内时，可降低胞内pH值，同时破坏微生物细胞内环境稳态，抑制DNA复制、表达、抑制微生物增殖。As shown in Table 2, the ectoine yield of Comparative Example 3 using glucose as a carbon source is the highest, while the ectoine yield of Example 2 using acetate and propionate as a mixed carbon source is close to that of Comparative Example 3, which is 8.1 g/L, followed by Example 8. It can be seen from the table that when the concentration of propionate added is higher than 10 g/L, the ectoine content gradually decreases, indicating that under the stimulation of high concentrations of propionate, the growth of Halomonas is significantly inhibited, and the ectoine yield decreases accordingly. This is because when high concentrations of propionic acid enter the cell, it can reduce the intracellular pH value, while destroying the homeostasis of the microbial cell environment, inhibiting DNA replication and expression, and inhibiting microbial proliferation.
菌株在以乙酸盐或丙酸盐为唯一碳源下，产量明显低于丙酸盐和乙酸盐。可能是因为当菌体同时利用两种盐时，不同的碳源可以进入不同的代谢途径，提升碳源利用效率，增强四氢嘧啶的合成。When acetate or propionate was used as the sole carbon source, the yield of the strain was significantly lower than that of propionate and acetate. This may be because when the bacteria use two salts at the same time, different carbon sources can enter different metabolic pathways, improve the efficiency of carbon source utilization, and enhance the synthesis of ectoine.
不同pH条件下摇瓶发酵产四氢嘧啶的含量Content of ectoine produced by shake flask fermentation under different pH conditions
实施例10Example 10
采用与实施例2相同的方法，区别在于，pH值调整为6。The same method as in Example 2 was used, except that the pH value was adjusted to 6.
实施例11Embodiment 11
采用与实施例2相同的方法，区别在于，pH值调整为10。The same method as in Example 2 was used, except that the pH value was adjusted to 10.
比较例4Comparative Example 4
采用与实施例2相同的方法，区别在于，pH值调整为4。The same method as in Example 2 was used, except that the pH value was adjusted to 4.
比较例5Comparative Example 5
采用与实施例2相同的方法，区别在于，pH值调整为5.5。The same method as in Example 2 was used, except that the pH value was adjusted to 5.5.
表3 不同pH条件下摇瓶发酵产四氢嘧啶的含量Table 3 The content of ectoine produced by shake flask fermentation under different pH conditions
由表3可知，pH偏向酸性时，四氢嘧啶含量明显下降，且随着pH的逐渐下降，四氢嘧啶含量下降明显，当pH等于4时，菌株几乎不产生四氢嘧啶，而当pH偏碱性时，四氢嘧啶含量明显增加，当pH等于10时，四氢嘧啶含量最高。这是因为 Halomonas sp.YL01菌生长的原生环境为高盐碱环境，其最适生长的pH值为碱性，细胞壁结构更适应碱性环境，因此处于酸性环境时，细胞生长不良，对应四氢嘧啶产量较低。 As shown in Table 3, when the pH is acidic, the ectoine content decreases significantly, and as the pH gradually decreases, the ectoine content decreases significantly. When the pH is equal to 4, the strain produces almost no ectoine, and when the pH is alkaline, the ectoine content increases significantly, and when the pH is equal to 10, the ectoine content is the highest. This is because the native environment of Halomonas sp.YL01 is a high-salt-alkali environment, and its optimal growth pH is alkaline. The cell wall structure is more adapted to the alkaline environment. Therefore, in an acidic environment, the cells grow poorly, and the corresponding ectoine production is low.
不同发酵温度下摇瓶发酵产四氢嘧啶的含量Content of ectoine produced by shake flask fermentation at different fermentation temperatures
实施例13Example 13
采用与实施例2相同的方法，区别在于，温度调整为32℃。The same method as in Example 2 was used, except that the temperature was adjusted to 32°C.
实施例14Embodiment 14
采用与实施例2相同的方法，区别在于，温度调整为35℃。The same method as in Example 2 was used, except that the temperature was adjusted to 35°C.
比较例6Comparative Example 6
采用与实施例2相同的方法，区别在于，温度调整为20℃。The same method as in Example 2 was used, except that the temperature was adjusted to 20°C.
比较例7Comparative Example 7
采用与实施例2相同的方法，区别在于，温度调整为25℃。The same method as in Example 2 was used, except that the temperature was adjusted to 25°C.
表4 不同温度条件下摇瓶发酵产四氢嘧啶的含量Table 4 The content of tetrahydropyrimidine produced by shake flask fermentation under different temperature conditions
由表4可知，随着温度的逐渐下降，四氢嘧啶含量逐渐降低，随着温度的下降，菌株体内酶的活性逐渐降低，菌体生长缓慢，对应四氢嘧啶合成酶的活性降低，导致四氢嘧啶生成量较低。As shown in Table 4, as the temperature gradually decreases, the content of ectoine gradually decreases. As the temperature decreases, the activity of the enzyme in the strain gradually decreases, the bacterial growth slows down, and the activity of the corresponding ectoine synthase decreases, resulting in a lower amount of ectoine produced.
不同底盘菌对发酵生产四氢嘧啶的影响Effects of different substrate bacteria on fermentation production of ectoine
比较例8Comparative Example 8
采用与实施例2相同的方法，区别在于发酵菌种选用 Halomonas elongate（菌种保藏编号：ATCC33173） The same method as in Example 2 was used, except that the fermentation strain was Halomonas elongate (strain collection number: ATCC33173).
比较例9Comparative Example 9
采用与实施例2相同的方法，区别在于发酵菌种 Halomonas salina （菌种保藏编号：ATCC49509） The same method as in Example 2 was used, except that the fermentation strain was Halomonas salina (strain collection number: ATCC49509).
表5不同底盘菌对发酵生产四氢嘧啶的影响Table 5 Effects of different base bacteria on fermentation production of tetrahydropyrimidine
由表5可知，选用同样的发酵方法，除 Halomonas sp. YL01菌，其余菌不能很好的发酵乙酸盐和丙酸盐产四氢嘧啶。这与不同菌体代谢途径不同有关，当菌体缺少乙酸或丙酸代谢相关的酶时，高浓度的乙酸盐或丙酸盐会进入菌体内部，破坏菌体内环境的稳定，对菌体具有毒害作用，抑制菌体生长甚至杀死菌体。 As shown in Table 5, with the same fermentation method, except for Halomonas sp. YL01, the other bacteria cannot ferment acetate and propionate to produce ectoine well. This is related to the different metabolic pathways of different bacteria. When the bacteria lack enzymes related to acetate or propionate metabolism, high concentrations of acetate or propionate will enter the bacteria, destroy the stability of the internal environment of the bacteria, have a toxic effect on the bacteria, inhibit the growth of the bacteria, or even kill the bacteria.
实施例15  发酵罐发酵产四氢嘧啶Example 15 Fermentation in a fermenter to produce tetrahydropyrimidine
①用接种环在超净工作台上将菌株盐单胞菌 Halomonas sp.YL01划线至60LB平板上，37 ℃活化24 h，待其长出单克隆； ① Use an inoculation loop to streak the strain Halomonas sp.YL01 onto a 60LB plate on a clean bench, activate it at 37 °C for 24 h, and wait for it to grow into a single clone;
②在超净工作台中用无菌接种环挑取上述①的单克隆抗体，接种至装有5mL60LB培养基中的50mL摇菌管中，37℃200rpm培养12H，获得一级种子液。② In the clean bench, pick the monoclonal antibody of ① above with a sterile inoculation loop, inoculate it into a 50mL shaking tube containing 5mL 60LB culture medium, and culture at 37℃ 200rpm for 12 hours to obtain the primary seed solution.
③在超净工作台上用移液器将一级种子液以10%添加量接种至含有100ml 60LB培养基的中500mL三角瓶中，37℃200rpm培养12H，获得二级种子液。③ On the clean bench, use a pipette to inoculate the first-level seed solution at a 10% addition rate into a 500mL Erlenmeyer flask containing 100mL 60LB culture medium, and culture at 37℃ and 200rpm for 12 hours to obtain the second-level seed solution.
④发酵罐培养基配制④ Preparation of fermentation tank culture medium
配料（g/L）：乙酸钠20、丙酸钠10、葡萄糖0、氯化钠 50 ；酵母膏 1.5、MgSO 4  5；尿素3 ；磷酸二氢钾5；第一微量元素组分 10、第二微量元素组分2。 Ingredients (g/L): 20% sodium acetate, 10% sodium propionate, 0% glucose, 50% sodium chloride; 1.5% yeast extract, 5% MgSO 4 ; 3% urea; 5% potassium dihydrogen phosphate; 10% first trace element component, 2% second trace element component.
第一微量元素组分： Fe(III)-NH 4-Citrate 5g/L、 CaCl 2·2H 2O 2 g/L、HCl 5M，去离子水配制。 The first trace element component: Fe(III)-NH 4 -Citrate 5 g/L, CaCl 2 ·2H 2 O 2 g/L, HCl 5M, prepared with deionized water.
第二微量元素组分：ZnSO 4·7H 2O 0.1 g/L；MnCl 2·4H 2O 0.03 g/L；H 3BO 3 0.3 g/L；CoCl 2·6H 2O 0.2 g/L；CuSO 4·5H 2O   0.01 g/L；NiCl 2·6H 2O 0.02 g/L；NaMoO 4·2H 2O 0.03 g/L 去离子水配制。 The second trace element component: ZnSO 4 ·7H 2 O 0.1 g/L; MnCl 2 ·4H 2 O 0.03 g/L; H 3 BO 3 0.3 g/L; CoCl 2 ·6H 2 O 0.2 g/L; CuSO 4 ·5H 2 O 0.01 g/L; NiCl 2 ·6H 2 O 0.02 g/L; NaMoO 4 ·2H 2 O 0.03 g/L, prepared with deionized water.
⑤补料培养基配制⑤ Preparation of feed medium
补料Ⅰ（g/L）：乙酸盐500，尿素 50，天冬氨酸 2；Feed I (g/L): acetate 500, urea 50, aspartic acid 2;
补料Ⅱ（g/L）：乙酸盐800，尿素15。Feed II (g/L): acetate 800, urea 15.
60LB培养基组分如下（g/L）：蛋白胨10，酵母粉8，氯化钠60，pH调至8。The components of 60LB medium are as follows (g/L): peptone 10, yeast powder 8, sodium chloride 60, pH adjusted to 8.
按上述发酵培养基配方配制发酵培养基。The fermentation medium was prepared according to the above fermentation medium formula.
将二级种子液按8%接种于15L发酵罐（培养基总体积为10L）中的培养基中，调整发酵温度为37℃，pH为9，通气量为1vvm，控制发酵溶解氧为30%，搅拌转速联动溶解氧，发酵时间为48h，发酵期间检测乙酸含量，当乙酸含量低于10g/L添加补料培养基，依据生长情况， 24h前使用补料Ⅰ补料，24h后使用补料Ⅱ补料，其中补料的乙酸盐类型为乙酸钾。The secondary seed liquid was inoculated at 8% into the culture medium in a 15L fermenter (total culture medium volume was 10L), the fermentation temperature was adjusted to 37°C, the pH was 9, the ventilation was 1vvm, the fermentation dissolved oxygen was controlled to 30%, the stirring speed was linked to the dissolved oxygen, the fermentation time was 48h, and the acetic acid content was detected during the fermentation. When the acetic acid content was lower than 10g/L, feed culture medium was added. Depending on the growth conditions, feed I was used before 24h and feed II was used after 24h. The acetate type of the feed was potassium acetate.
实施例16  采用与实施例15相同的方法，区别在于发酵溶解氧为20%。Example 16: The same method as Example 15 is adopted, except that the fermentation dissolved oxygen is 20%.
实施例17  采用与实施例15相同的方法，区别在于发酵溶解氧为40%。Example 17: The same method as Example 15 is adopted, except that the fermentation dissolved oxygen is 40%.
表6 不同溶解氧下发酵罐发酵产四氢嘧啶的含量Table 6 The content of tetrahydropyrimidine produced by fermentation in fermentation tanks under different dissolved oxygen
由表6可知，发酵期间的溶解氧含量对四氢嘧啶产量影响较小，其溶解氧合适范围为20-40%，其中最优为30%。As shown in Table 6, the dissolved oxygen content during fermentation has little effect on the yield of ectoine, and the appropriate range of dissolved oxygen is 20-40%, with the optimal being 30%.
通过对 Halomonassp.YL01菌的代谢通路进行分析，可知该菌具有代谢乙酸的通路。进一步通过实验验证 Halomonas sp.YL01可发酵乙酸盐、丙酸盐产四氢嘧啶，且采用乙酸盐和丙酸盐构成的混合碳源进行发酵，使四氢嘧啶产量对比葡萄糖无显著差异，是一种很好的替代葡萄糖生产四氢嘧啶的手段。 By analyzing the metabolic pathway of Halomonas sp.YL01, it was found that the bacterium has a pathway for metabolizing acetic acid. Further experiments verified that Halomonas sp.YL01 can ferment acetate and propionate to produce ectoine, and the fermentation of a mixed carbon source consisting of acetate and propionate has no significant difference in the yield of ectoine compared with glucose, which is a good means to replace glucose to produce ectoine.
工业实用性Industrial Applicability
与现有菌种相比，本发明 Halomonas sp. YL01菌具有较高的pH耐受度，可耐受pH为6-11，可耐受高浓度乙酸盐带来的高pH环境，常规的菌种无法耐受pH大于9的培养环境；与现有技术相比，本发明提供了一种以乙酸盐为主的碳源作为发酵培养基，可大幅度降低四氢嘧啶的发酵成本。 Compared with existing strains, the Halomonas sp. YL01 of the present invention has a higher pH tolerance, can tolerate a pH of 6-11, and can tolerate a high pH environment brought about by high concentrations of acetate. Conventional strains cannot tolerate a culture environment with a pH greater than 9. Compared with the prior art, the present invention provides a carbon source mainly based on acetate as a fermentation medium, which can greatly reduce the fermentation cost of ectoine.

Claims (8)

  1. 一种采用盐单胞菌株生产四氢嘧啶的方法，其特征在于：所采用的碳源由乙酸盐与 丙酸盐构成混合碳源，通过盐单胞菌株Halomonas sp. YL01进行发酵制备，所述盐单胞菌株Halomonas sp. YL01保藏于广东省微生物菌种保藏中心，菌种保藏号为GDMCC No. 62420，保藏日期为2022年04月24日；所述乙酸盐在发酵液中的浓度20-25 g/L，丙酸盐在发酵液中的浓度5-10 g/L；所述发酵温度为35-37℃，所述发酵所采用的培养基的pH值为 9-11。 A method for producing ectoine using Halomonas sp., characterized in that: the carbon source used is a mixed carbon source composed of acetate and propionate, and is prepared by fermentation by Halomonas sp. YL01, the Halomonas sp. YL01 is deposited in Guangdong Microbiological Culture Collection Center, the culture collection number is GDMCC No. 62420, and the preservation date is April 24, 2022; the concentration of acetate in the fermentation broth is 20-25 g/L, and the concentration of propionate in the fermentation broth is 5-10 g/L; the fermentation temperature is 35-37°C, and the pH value of the culture medium used in the fermentation is 9-11.
  2. 如权利要求1所述生产四氢嘧啶的方法，其特征在于：所述乙酸盐为乙酸钠、乙酸钾、 乙酸铵中的一种或几种混合。 The method for producing tetrahydropyrimidine as described in claim 1 is characterized in that the acetate is one or a mixture of sodium acetate, potassium acetate, and ammonium acetate.
  3. 如权利要求1或2所述生产四氢嘧啶的方法，其特征在于：所述丙酸盐为丙酸钠、丙酸钾、丙酸铵中的一种或几种混合。 The method for producing tetrahydropyrimidine as described in claim 1 or 2 is characterized in that the propionate is one or a mixture of sodium propionate, potassium propionate, and ammonium propionate.
  4. 如权利要求1或2所述生产四氢嘧啶的方法，其特征在于：当采用摇瓶发酵时，接种盐单胞菌株Halomonas sp. YL01至摇瓶培养基，发酵培养。 The method for producing ectoine as described in claim 1 or 2 is characterized in that: when shake flask fermentation is adopted, the Halomonas sp. YL01 is inoculated into the shake flask culture medium for fermentation culture.
  5. 如权利要求4所述生产四氢嘧啶的方法，其特征在于：摇瓶培养基由乙酸钠20-25 g/ L、丙酸钠5-10 g/L、尿素0.5-10 g/L、天冬氨酸 0-10 g/L、酵母粉1-15 g/L、无水硫酸镁 0.05-0.6 g/L、磷酸二氢钾1.5-5.5 g/L、氯化钠50-80 g/L、三价铁-铵根-柠檬酸盐 0.05-0.1 g/L、二水合氯化钙 0.02-0.2 g/L、七水合硫酸锌 0.01-0.1 g/L、四水合二氯化锰 0.002-0.02 g/L、硼酸 0.01-0.05 g/L、六水合氯化钴 0.005-0.02 g/L、五水合硫酸铜 0.01-0.08 g/L、六水合氯化镍 0.005-0.01 g/L和二水合钼酸钠 0.02-0.1 g/L组成。 The method for producing ectoine according to claim 4, characterized in that the shake flask culture medium is composed of sodium acetate 20-25 g/L, sodium propionate 5-10 g/L, urea 0.5-10 g/L, aspartic acid 0-10 g/L, yeast powder 1-15 g/L, anhydrous magnesium sulfate 0.05-0.6 g/L, potassium dihydrogen phosphate 1.5-5.5 g/L, sodium chloride 50-80 g/L, trivalent iron-ammonium root-citrate 0.05-0.1 g/L, calcium chloride dihydrate 0.02-0.2 g/L, zinc sulfate heptahydrate 0.01-0.1 g/L, manganese dichloride tetrahydrate 0.002-0.02 g/L, boric acid 0.01-0.05 g/L, cobalt chloride hexahydrate 0.005-0.02 g/L, copper sulfate pentahydrate 0.01-0.08 g/L, nickel chloride hexahydrate 0.005-0.01 g/L and sodium molybdate dihydrate 0.02-0.1 g/L.
  6. 如权利要求1或2所述生产四氢嘧啶的方法，其特征在于：当采用发酵罐发酵时，将菌种按接种量为5％-15％接入发酵罐培养基；发酵过程中控制溶氧为20％-40％；发酵培养， 发酵期间检测乙酸含量，当乙酸含量低于10 g/L添加补料培养基，依据生长情况补料。 The method for producing ectoine as described in claim 1 or 2 is characterized in that: when fermentation is carried out in a fermenter, the bacteria are inoculated into the fermenter culture medium at an inoculation rate of 5%-15%; the dissolved oxygen is controlled at 20%-40% during the fermentation process; during the fermentation culture, the acetic acid content is detected, and when the acetic acid content is lower than 10 g/L, feed culture medium is added, and feed is supplemented according to the growth conditions.
  7. 如权利要求6所述生产四氢嘧啶的方法，其特征在于：发酵罐培养基由乙酸钠20-25 g/L、丙酸钠5-10 g/L、氯化钠50-80 g/L、酵母膏 1-10 g/L、硫酸镁1-10 g/L、尿素1-4 g/L、 磷酸二氢钾3-10 g/L、第一微量元素组分 5-15 g/L和第二微量元素组分1-5 g/L组成。 The method for producing ectoine as described in claim 6 is characterized in that the fermentation tank culture medium consists of 20-25 g/L sodium acetate, 5-10 g/L sodium propionate, 50-80 g/L sodium chloride, 1-10 g/L yeast extract, 1-10 g/L magnesium sulfate, 1-4 g/L urea, 3-10 g/L potassium dihydrogen phosphate, 5-15 g/L first trace element component and 1-5 g/L second trace element component.
  8. 如权利要求7所述生产四氢嘧啶的方法，其特征在于：第一微量元素组分具体包括三价铁-铵根-柠檬酸盐 3-7 g/L、二水合氯化钙 1-3 g/L、盐酸 3-7 M，通过去离子水配制；第二微量元素组分具体包括七水合硫酸锌 0.1-0.2 g/L、四水合二氯化锰 0.02-0.04 g/L、 硼酸 0.2-0.4 g/L、六水合氯化钴 0.1-0.3 g/L、五水合硫酸铜 0.01-0.03 g/L、六水合氯化镍 0.01-0.03 g/L、二水合钼酸钠 0.02-0.05 g/L，通过去离子水配制。The method for producing ectoine according to claim 7, characterized in that: the first trace element component specifically includes 3-7 g/L of trivalent iron-ammonium-citrate, 1-3 g/L of calcium chloride dihydrate, and 3-7 M of hydrochloric acid, which are prepared by deionized water; the second trace element component specifically includes 0.1-0.2 g/L of zinc sulfate heptahydrate, 0.02-0.04 g/L of manganese dichloride tetrahydrate, 0.2-0.4 g/L of boric acid, 0.1-0.3 g/L of cobalt chloride hexahydrate, 0.01-0.03 g/L of copper sulfate pentahydrate, 0.01-0.03 g/L of nickel chloride hexahydrate, and 0.02-0.05 g/L of sodium molybdate dihydrate, which are prepared by deionized water.
PCT/CN2023/100310 2022-12-24 2023-06-15 Method for producing ectoine using halomonas strain WO2024130983A1 (en)

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