CN114606276B - Method for improving fermentation yield of L-threonine - Google Patents
Method for improving fermentation yield of L-threonine Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 120
- 230000004151 fermentation Effects 0.000 title claims abstract description 119
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 title claims abstract description 97
- 239000004473 Threonine Substances 0.000 title claims abstract description 52
- 229960002898 threonine Drugs 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000002609 medium Substances 0.000 claims abstract description 43
- 241000588724 Escherichia coli Species 0.000 claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims description 49
- 239000000243 solution Substances 0.000 claims description 25
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 19
- 230000001965 increasing effect Effects 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 229910052760 oxygen Inorganic materials 0.000 claims description 11
- 239000001301 oxygen Substances 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- 229910052603 melanterite Inorganic materials 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 239000012266 salt solution Substances 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 241000209149 Zea Species 0.000 claims 2
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 60
- 235000015097 nutrients Nutrition 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 14
- 230000001276 controlling effect Effects 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 230000012010 growth Effects 0.000 description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 8
- 229940063746 oxygen 20 % Drugs 0.000 description 7
- 239000002253 acid Substances 0.000 description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000004806 packaging method and process Methods 0.000 description 5
- 238000010257 thawing Methods 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 229960003237 betaine Drugs 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- -1 iron ions Chemical class 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
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- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229960003403 betaine hydrochloride Drugs 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- HOPSCVCBEOCPJZ-UHFFFAOYSA-N carboxymethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC(O)=O HOPSCVCBEOCPJZ-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003068 pathway analysis Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000176 sodium gluconate Substances 0.000 description 1
- 235000012207 sodium gluconate Nutrition 0.000 description 1
- 229940005574 sodium gluconate Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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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
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Abstract
The invention provides a method for improving the fermentation yield of L-threonine, which takes escherichia coli (ESCHERICHIA COLI) as a fermentation strain, and adds PQQ into an initial fermentation culture medium or adds PQQ into the fermentation culture medium in the middle or later period of the fermentation process so as to improve the fermentation yield of L-threonine. According to the invention, the PQQ is added into the fermentation medium, so that the later metabolic activity of the fermentation strain is enhanced, the OD value of the thallus is improved, and the L-threonine yield is correspondingly improved.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for improving the fermentation yield of L-threonine.
Background
L-threonine is an important nutrition required by human beings and animals, has important significance for health, is one of 8 amino acids required by human bodies, increases global demand and yield year by year, achieves 70 ten thousand tons of world L-threonine yield in 2019, and is widely applied to the fields of foods, feeds, medicines and chemical industry.
The L-threonine is mainly produced by adopting escherichia coli fermentation, and CN106867952A replaces a promoter Pppc of a phosphoenolpyruvate carboxylase gene (ppc) of a starting strain with a promoter Pzwf of a glucose 6-phosphate dehydrogenase gene (zwf), so that the purpose of regulating the L-threonine production capacity by betaine is achieved. The betaine is added in the fermentation process, so that the yield of L-threonine produced by shake flask fermentation can reach 50-55g/L; the yield of the 5L fermentation tank reaches 120-150g/L, and the sugar acid conversion rate reaches 59-61%.
Su Yuewen et al (Effects of betaine supplementation on L-threonine fed-batch fermentation by Escherichia coli,BIOPROCESS AND BIOSYSTEMS ENGINEERING,2018) discloses a method for increasing the L-threonine concentration to 127.3g/L by a batch fermentation method, and the conversion rate reaches 58.12%.
The yield and the conversion rate of the L-threonine are improved by improving the escherichia coli genes and combining batch fermentation, and meanwhile, a large number of researches show that similar effects can be achieved by improving the fermentation conditions. Chen Ning et al (3 rd phase, 2008 of the university of Tianjin science and technology, based on optimization of the fermentation process of L-threonine by pathway analysis) increased the yield of L-threonine to 107g/L by metabolic flow analysis, adding sodium gluconate and controlling dissolved oxygen by 20%. Gold and the like (L-threonine fermentation temperature change control and yeast extract logistics process research, modern chemical engineering S2 phase, 2007) control the temperature to 37 ℃ for 12 hours before fermentation, and the temperature is raised to 39 ℃ after 12 hours, so that the maximum cell biomass and the L-threonine yield can be obtained simultaneously; on the basis, 2.0g/L yeast extract is continuously added in the later fermentation period, so that the generation of byproduct amino acid can be effectively reduced, the fermentation acid production level of L-threonine is stabilized, the maximum acid production mass concentration reaches 105.8g/L, and the sugar acid conversion rate reaches 47.5%. Xu Qingyang et al (effect of dissolved oxygen on L-threonine fermentation, microbiology report 2 nd, 2007) maintained 50% dissolved oxygen after the log phase by controlling 20% dissolved oxygen in the lag phase, 20% dissolved oxygen in the stationary phase, fed-batch fermentation by 10L tank for 36h, acid production up to 118.9g/L, sugar acid conversion of 47.6%. In CN107760734A, mixed fermentation promoter composed of betaine hydrochloride, VB4 and VB3 is added in the fermentation process of L-threonine, so that the yield of L-threonine can be improved by 10-12%, and the yield can reach 133.6g/L.
It can be seen that by enhancing or weakening gene expression, fermentation conditions such as temperature, pH, dissolved oxygen, etc., can be improved, and also L-threonine production and conversion can be improved by improving post-fermentation conditions such as fed-batch yeast extract.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation yield of L-threonine.
The invention is characterized in that: the microorganism has reduced activity in the late stage, and can improve the activity in the late stage by supplementing various nutritional factors such as amino acid, vitamin and the like. PQQ cannot be synthesized in Escherichia coli, but can be utilized by enzymes on the respiratory chain of Escherichia coli, and after PQQ is added, the respiratory efficiency of the latter bacteria can be improved, and the activity of the latter bacteria can be enhanced.
To achieve the object of the present invention, in a first aspect, the present invention provides a method for improving the fermentation yield of L-threonine, comprising: coli (ESCHERICHIA COLI) is used as a fermentation strain, and PQQ is added to an initial fermentation medium or added to the fermentation medium in the middle or later stage of the fermentation process to improve the fermentation yield of L-threonine.
The concentration of PQQ in the fermentation medium is 5 mug/L-5 mg/L.
Preferably, the fermentation strain is escherichia coli, and the composition of the fermentation medium is as follows: 30g/L glucose, 10g/L ammonium sulfate and corn steep liquor 6g/L,KH2PO4 0.5g/L,MgSO4·7H2O 0.5g/L,FeSO4·7H2O 0.01g/L,MnSO4·H2O 0.01g/L,pH7.0.
The method comprises the following steps:
1) Activating strains and preparing primary seed liquid;
2) Preparing secondary seed liquid;
3) And (3) inoculating the secondary seed liquid into a shake flask or a fermentation tank for fermentation culture.
The fermentation culture conditions of the step 3) are as follows: controlling pH7.0 of a fermentation system at 37 ℃ with dissolved oxygen of 20-50%. Taking a fermenter with a volume of 50L as an example, the liquid loading can be 20-30L.
Further, step 1) includes: the activated strain is inoculated into a 500ml triangular shake flask filled with 100ml LB culture medium, and shake culture is carried out for 5-6 h at 37 ℃ and 220rpm by a shaking table until the seed liquid OD 600 = 10, and the strain is used as first-stage seed liquid.
Further, step 2) includes: the first seed liquid was inoculated into a 10L fermenter containing 4L of seed medium, and cultured at 37℃under the conditions of 20% -50% dissolved oxygen and pH7.0 until the seed liquid OD 600 = 20, to obtain a second seed liquid.
Wherein the composition of the seed culture medium is as follows: 25g/L glucose, 10g/L ammonium sulfate and corn steep liquor 20g/L,KH2PO4 1.5g/L,MgSO4·7H2O 0.5g/L,FeSO4·7H2O 0.01g/L,MnSO4·H2O 0.01g/L,pH7.0.
Preferably, step 3) comprises: inoculating the secondary seed liquid into the fermentation medium according to the volume ratio of 1:10-1:7.
Further, after 13 to 17 hours (preferably 15 hours) of fermentation, PQQ may be added to the fermentation medium at once or fed at a constant rate. In order to prevent the PQQ from being disturbed in the growth stage and lost in the fermentation process, and simultaneously to ensure the maximum effect of the PQQ, the PQQ is added in the middle and later stages of bacterial growth, and the activity of the strain in the fermentation liquid is high at this time, so that the PQQ can be efficiently utilized.
The method further comprises the steps of: after 13-17h (preferably 15 h) of fermentation, an inorganic salt solution is fed into the fermentation medium until the fermentation is completed. At the moment, the addition of elements such as iron ions and the like in the electron transfer process promotes the action of PQQ and simultaneously ensures the activity of post fermentation of strains; in addition, iron ions play a role in promoting the growth of thalli and the production of L-threonine, and if the thalli is excessively high in growth vigor, excessive sugar and nutrition are consumed, the generation of amino acid is not facilitated, and the functions of promoting the growth of thalli and the production of L-threonine are achieved by selecting the middle and later stages of growth pairs.
Wherein the inorganic salt solution is prepared from phosphoric acid and ferrous sulfate heptahydrate.
The concentration of ferrous sulfate heptahydrate in the fermentation culture medium is controlled to be 0.01-0.1g/L, and the concentration of phosphoric acid is controlled to be 0.1-1g/L.
In the present invention, the E.coli means a bacterium having L-threonine-producing ability, and preferably the E.coli is E.coli (ESCHERICHIA COLI) MHZ-0215-2. The strain MHZ-0215-2 is preserved in China general microbiological culture Collection center, the address of North Star Xiyu No.1, 3 of the Korean area of Beijing, the national academy of sciences microbiological institute, mail code 100101, the preservation number CGMCC No.13403, and the preservation date 2016, 11, 30.
In a second aspect, the invention provides the use of PQQ in the fermentative production of L-threonine or for increasing the fermentative production of L-threonine.
In the application, the escherichia coli is used as a fermentation strain.
According to the invention, the PQQ is added into the fermentation medium, so that the later metabolic activity of the fermentation strain is enhanced, the OD value of the thallus is improved, and the L-threonine yield is correspondingly improved.
Drawings
FIGS. 1 to 4 show the growth of cells in the experimental group and the control group according to the preferred embodiment of the present invention. Wherein, 1: experimental group OD, 2: OD value of control group.
FIG. 5 shows the growth of cells in the experimental and control groups according to the preferred embodiment of the present invention. Wherein 1-4 respectively represent the addition of PQQ of 5 μg/L, 50 μg/L, 500 μg/L and 5mg/L, respectively, to the initial fermentation medium, and 5 is the OD of the control group.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The E.coli MHZ-0215-2 used in the following examples was purchased from Langfang plum blossom Biotechnology development Co.
The L-threonine content of the fermentation broth was measured by a High Performance Liquid Chromatography (HPLC) method, and the derivation method is PITC-trifluoroacetic acid (see the pottery champion, etc., HPLC quantitative analysis of glutamine in proteins and peptides, zhengzhou food college, vol. 20, 4, 1999), and a C18 column of Agilent was used. The OD was measured by a model 721 spectrophotometer.
Example 1 method for improving fermentation yield of L-threonine
1. Preparing culture medium
(1) Culture medium for preparing primary seed liquid
LB medium (g/L): yeast powder 5, peptone 10 and sodium chloride 5.
(2) Culture medium for preparing secondary seed liquid
Seed medium (g/L): glucose 25, ammonium sulfate 10, corn steep liquor 20,KH2PO4 1.5,MgSO4·7H2O0.5,FeSO4·7H2O 0.01,MnSO4·H2O 0.01;pH7.0,121℃, and sterilizing for 20min.
(3) Fermentation medium (g/L): glucose 30, ammonium sulfate 10, corn steep liquor 6,KH2PO4 0.5,MgSO4·7H2O,0.5,FeSO4·7H2O 0.01,MnSO4·H2O 0.01;pH7.0,121℃, and sterilizing for 20min.
2. Preparing fed-batch nutrient solution
1000. Mu.g/L of PQQ nutrient solution was prepared with ultrapure water.
3. Experimental method
Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121deg.C for 20min, sucking 500 μl, packaging into 2ml glycerol tube, and freezing seed solution with OD 600 of 10 at-70deg.C for use. Taking out the frozen glycerol tube for thawing, sucking 100 μl of seed liquid, coating LB inclined surface for activation, and culturing at 37deg.C for 12 hr; the inclined plane is transferred into a triangular shake flask (100 ml of LB culture medium is filled in the shake flask) with the volume of 500ml, and after inoculation, a shaking table at 37 ℃ is placed, and a 220rpm rotary shaking table is used for shaking culture for 5-6 h. Shake flask seeds OD 600 grow to 10, namely primary seed liquid; the first seed liquid was inoculated into a seed tank (4L seed medium in the seed tank) having a volume of 10L, and culturing conditions were: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and dissolving oxygen 20% -50%; when the seed liquid OD 600 grows to 20, the second-level seed liquid is obtained; 2.5L of the secondary seed liquid was inoculated into a 50L-volume fermenter (17.5L of fermentation medium was placed in the fermenter) for fermentation culture under the following conditions: controlling pH7.0 with 25-28% ammonia water at 37deg.C, dissolving oxygen 20% -50%, and adding 500mL of PQQ nutrient solution into the fermenter during 15h fermentation. The total fermentation time is 36h.
After the fermentation, the L-threonine treatment in the fermentation broth of the experimental group (PQQ added) was increased by 1.5g/L and the glucose conversion rate was increased by 1% as compared with the control group (PQQ not added) (Table 1). The growth of the cells in the experimental group and the control group is shown in FIG. 1.
TABLE 1L-threonine production enhancement after PQQ addition
Example 2 method for improving fermentation yield of L-threonine
1. Preparing inorganic salt nutrient solution
A phosphoric acid solution having a concentration of 10g/L was prepared, and ferrous sulfate heptahydrate was dissolved so that the final concentration of ferrous sulfate heptahydrate was 0.1g/L, to prepare an inorganic salt solution (inorganic salt nutrient solution). 1L of an inorganic salt solution was prepared. Sterilizing at 121deg.C for 20 min.
2. LB medium, seed medium, fermentation medium and fed-batch nutrient solution were prepared separately, see example 1.
3. Experimental method
Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121deg.C for 20min, sucking 500 μl, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70deg.C for use. Taking out the frozen glycerol tube for thawing, sucking 100 μl of seed liquid, coating LB inclined surface for activation, and culturing at 37deg.C for 12 hr; the inclined plane is transferred into a triangular shake flask (100 ml of LB culture medium is filled in the shake flask) with the volume of 500ml, and after inoculation, a shaking table at 37 ℃ is placed, and a 220rpm rotary shaking table is used for shaking culture for 5-6 h. Shake flask seeds OD 600 grow to 10, namely primary seed liquid; the first seed liquid was inoculated into a seed tank (4L seed medium in the seed tank) having a volume of 10L, and culturing conditions were: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and dissolving oxygen 20% -50%; when the seed liquid OD 600 grows to 20, the second-level seed liquid is obtained; 2.5L of the secondary seed liquid was inoculated into a 50L-volume fermenter (17.5L of fermentation medium was placed in the fermenter) for fermentation culture under the following conditions: the pH value is controlled to be 7.0 by 25-28% ammonia water at 37 ℃, dissolved oxygen is 20% -50%, 500mL of PQQ nutrient solution and 500mL of inorganic salt nutrient solution are added into a fermentation tank during 15h of fermentation. The total fermentation time is 36h.
After the fermentation, the L-threonine content in the fermentation broth was increased by 3g/L and the glucose conversion rate was increased by 2% as compared with the control group (without PQQ and inorganic salt nutrient solution added) (Table 2). The growth of the cells in the experimental group and the control group is shown in FIG. 2.
TABLE 2L-threonine production enhancement by PQQ and inorganic salt addition
Example 3 method for improving fermentation yield of L-threonine
1. Inorganic salt nutrient solutions were formulated, see in particular example 2.
2. LB medium, seed medium, fermentation medium and PQQ nutrient solution were prepared separately, see example 1.
3. Experimental method
Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121deg.C for 20min, sucking 500 μl, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70deg.C for use. Taking out the frozen glycerol tube for thawing, sucking 100 μl of seed liquid, coating LB inclined surface for activation, and culturing at 37deg.C for 12 hr; the inclined plane is transferred into a triangular shake flask (100 ml of LB culture medium is filled in the shake flask) with the volume of 500ml, and after inoculation, a shaking table at 37 ℃ is placed, and a 220rpm rotary shaking table is used for shaking culture for 5-6 h. Shake flask seeds OD 600 grow to 10, namely primary seed liquid; the first seed liquid was inoculated into a seed tank (4L seed medium in the seed tank) having a volume of 10L, and culturing conditions were: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and dissolving oxygen 20% -50%; when the seed liquid OD 600 grows to 20, the second-level seed liquid is obtained; 2.5L of the secondary seed liquid was inoculated into a 50L-volume fermenter (17.5L of fermentation medium was placed in the fermenter) for fermentation culture under the following conditions: controlling pH to 7.0 with 25-28% ammonia water at 37deg.C, adding 500mL of inorganic salt nutrient solution into the fermenter at 15h of fermentation, and fermenting for 36h.
After the fermentation, the L-threonine treatment in the fermentation broth of the experimental group (added with inorganic salt nutrient solution) was increased by 0.5g/L, and the glucose conversion rate was increased by 0.5% as compared with the control group (not added with inorganic salt nutrient solution) (Table 3). The growth of the cells in the experimental group and the control group is shown in FIG. 3.
TABLE 3L-threonine production enhancement by inorganic salt addition
Example 4 method for improving fermentation yield of L-threonine
1. Inorganic salt nutrient solutions were formulated, see in particular example 2.
2. LB medium, seed medium, fermentation medium and PQQ nutrient solution were prepared separately, see example 1.
3. Experimental method
Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121deg.C for 20min, sucking 500 μl, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70deg.C for use. Taking out the frozen glycerol tube for thawing, sucking 100 μl of seed liquid, coating LB inclined surface for activation, and culturing at 37deg.C for 12 hr; the inclined plane is transferred into a triangular shake flask (100 ml of LB culture medium is filled in the shake flask) with the volume of 500ml, and after inoculation, a shaking table at 37 ℃ is placed, and a 220rpm rotary shaking table is used for shaking culture for 5-6 h. Shake flask seeds OD 600 grow to 10, namely primary seed liquid; the first seed liquid was inoculated into a seed tank (4L seed medium in the seed tank) having a volume of 10L, and culturing conditions were: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and dissolving oxygen 20% -50%; when the seed liquid OD 600 grows to 20, the second-level seed liquid is obtained; 2.5L of the secondary seed liquid was inoculated into a 50L-volume fermenter (17.5L of fermentation medium was placed in the fermenter) for fermentation culture under the following conditions: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and adding PQQ nutrient solution into the fermenter at constant speed of 50mL/h at 15 hr, and adding inorganic salt nutrient solution into the fermenter at constant speed of 50mL/h until fermentation is completed. The total fermentation time is 36h.
After the fermentation, the L-threonine content in the fermentation broth was increased by 2g/L and the glucose conversion rate was increased by 1.5% as compared with the control (PQQ and inorganic salt nutrient solution not fed-batch) (Table 4). The growth of the cells in the experimental group and the control group is shown in FIG. 4.
TABLE 4 enhancement of L-threonine production by PQQ addition and mineral salt addition
Example 5 method for improving fermentation yield of L-threonine
1. LB medium, seed medium, fermentation medium were prepared separately, and 5. Mu.g/L, 50. Mu.g/L, 500. Mu.g/L and 5mg/L of PQQ were added to the initial fermentation medium, respectively, as described in example 1.
2. Experimental method
Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121deg.C for 20min, sucking 500 μl, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70deg.C for use. Taking out the frozen glycerol tube for thawing, sucking 100 μl of seed liquid, coating LB inclined surface for activation, and culturing at 37deg.C for 12 hr; the inclined plane is transferred into a triangular shake flask (100 ml of LB culture medium is filled in the shake flask) with the volume of 500ml, and after inoculation, a shaking table at 37 ℃ is placed, and a 220rpm rotary shaking table is used for shaking culture for 5-6 h. Shake flask seeds OD 600 grow to 10, namely primary seed liquid; the first seed liquid was inoculated into a seed tank (6L seed culture medium in the seed tank) having a volume of 10L, and culture conditions were: controlling pH7.0 with 25-28% ammonia water at 37deg.C, and dissolving oxygen 20% -50%; when the OD600 of the seed liquid grows to 20, the second-level seed liquid is obtained; two-stage seed solutions (2.5L) were inoculated into two 50L-volume fermenters (17.5L of PQQ fermentation medium containing 5. Mu.g/L and 5mg/L, respectively) and cultured under the following conditions: controlling pH7.0 with 25-28% ammonia water at 37deg.C, dissolving oxygen 20% -50%, and fermenting for 36 hr.
After fermentation, compared with a control group (without PQQ), the addition of 5 mu g/L of L-threonine in the fermentation broth is improved by 0.5g/L, and the glucose conversion rate is improved by 0.2%; the addition of 50. Mu.g/L of L-threonine in the broth increased by 1.5g/L, the conversion to glucose increased by 1%, the addition of 500. Mu.g/L of L-threonine in the broth increased by 1.6g/L, the conversion to glucose increased by 1.1%, the addition of 5mg/L of L-threonine in the broth increased by 0.4g/L, and the conversion to glucose increased by 0.15% (Table 5). The growth of the cells in the experimental group and the control group is shown in FIG. 5.
TABLE 5L-threonine production increase after PQQ addition to initial fermentation Medium
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for increasing the fermentation yield of L-threonine comprising: coli (ESCHERICHIA COLI) is used as a fermentation strain, and PQQ is added into an initial fermentation medium or is added into the fermentation medium in the middle or later period of the fermentation process so as to improve the fermentation yield of L-threonine;
the concentration of PQQ in the fermentation medium is 50-500 mug/L;
the Escherichia coli is Escherichia coli (ESCHERICHIA COLI) MHZ-0215-2, and the preservation number is CGMCC No.13403.
2. The method according to claim 1, wherein the fermentation strain is escherichia coli and the composition of the fermentation medium is as follows: glucose 30 g/L, ammonium sulfate 10 g/L, corn steep liquor 6 g/L,KH2PO4 0.5 g/L,MgSO4·7H2O 0.5 g/L,FeSO4·7H2O 0.01 g/L,MnSO4·H2O 0.01 g/L,pH7.0.
3. The method according to claim 2, characterized in that it comprises the steps of:
1) Activating strains and preparing primary seed liquid;
2) Preparing secondary seed liquid;
3) Inoculating the secondary seed liquid into a fermentation tank for fermentation culture;
The fermentation culture conditions of the step 3) are as follows: controlling pH7.0 of a fermentation system at 37 ℃ with dissolved oxygen of 20-50%.
4. A method according to claim 3, wherein step 1) comprises: and inoculating the activated strain into a 500ml triangular shake flask filled with 100ml LB culture medium, and shake culturing at 37 ℃ for 5-6 hours by a shaking table at 220rpm until the seed liquid OD 600 = 10, wherein the seed liquid serves as primary seed liquid.
5. The method of claim 4, wherein step 2) comprises: inoculating the first-stage seed solution into a 10L fermentation tank filled with 4L seed culture medium, and culturing at 37deg.C under the conditions of dissolved oxygen of 20% -50% and pH7.0 until the seed solution OD 600 = 20 to obtain a second-stage seed solution;
Wherein the composition of the seed culture medium is as follows: glucose 25 g/L, ammonium sulfate 10 g/L and corn steep liquor 20 g/L,KH2PO4 1.5 g/L,MgSO4·7H2O 0.5 g/L,FeSO4·7H2O 0.01 g/L,MnSO4 ·H2O 0.01 g/L,pH7.0.
6. The method of claim 5, wherein step 3) comprises: inoculating the secondary seed liquid into the fermentation medium according to the volume ratio of 1:10-1:7.
7. The method of claim 6, wherein the PQQ is added to the fermentation medium after 13-17 hours of fermentation.
8. The method according to any one of claims 2-6, further comprising: after fermentation for 13-17h, inorganic salt solution is added into the fermentation culture medium until the fermentation is finished;
Wherein the inorganic salt solution is prepared from phosphoric acid and ferrous sulfate heptahydrate; the concentration of ferrous sulfate heptahydrate in the fermentation culture medium is controlled to be 0.01-0.1g/L, and the concentration of phosphoric acid is controlled to be 0.1-1g/L.
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| CN102177246A (en) * | 2008-09-08 | 2011-09-07 | 味之素株式会社 | Microorganism capable of producing L-amino acid, and method for producing L-amino acid |
| CN106635945A (en) * | 2016-12-29 | 2017-05-10 | 廊坊梅花生物技术开发有限公司 | Recombinant strain and preparation method thereof and method for producing L-threonine |
| CN107760734A (en) * | 2017-11-07 | 2018-03-06 | 吉林大学 | A kind of method and its application for improving production amount of threonine |
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| CN102177246A (en) * | 2008-09-08 | 2011-09-07 | 味之素株式会社 | Microorganism capable of producing L-amino acid, and method for producing L-amino acid |
| CN106635945A (en) * | 2016-12-29 | 2017-05-10 | 廊坊梅花生物技术开发有限公司 | Recombinant strain and preparation method thereof and method for producing L-threonine |
| CN107760734A (en) * | 2017-11-07 | 2018-03-06 | 吉林大学 | A kind of method and its application for improving production amount of threonine |
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