CN113493761A - Fermentation process for increasing yield of 5-hydroxytryptophan - Google Patents
Fermentation process for increasing yield of 5-hydroxytryptophan Download PDFInfo
- Publication number
- CN113493761A CN113493761A CN202110714160.2A CN202110714160A CN113493761A CN 113493761 A CN113493761 A CN 113493761A CN 202110714160 A CN202110714160 A CN 202110714160A CN 113493761 A CN113493761 A CN 113493761A
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- China
- Prior art keywords
- hydroxytryptophan
- fermentation
- chelated
- yield
- fermentation process
- Prior art date
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- Granted
Links
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- 230000004151 fermentation Effects 0.000 title claims abstract description 70
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- LDCYZAJDBXYCGN-VIFPVBQESA-N 5-hydroxy-L-tryptophan Chemical compound C1=C(O)C=C2C(C[C@H](N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-VIFPVBQESA-N 0.000 title claims abstract description 59
- 229940000681 5-hydroxytryptophan Drugs 0.000 title claims abstract description 58
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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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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/99—Other Carbon-Carbon Lyases (1.4.99)
- C12Y401/99001—Tryptophanase (4.1.99.1)
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Abstract
The invention provides a fermentation process for improving the yield of 5-hydroxytryptophan, which comprises the steps of adding metal ion chelated amino acid into a fermentation culture medium on one hand, and feeding composite auxiliary materials in a flowing manner during fermentation for 2 hours on the other hand, wherein the metal ion chelated amino acid is adopted in the method, so that the nutrient components of effective amino acid in the fermentation culture medium are supplemented, the absorption effect of thalli on trace elements is increased, and the growth activity and the production performance of the thalli are improved; simultaneously glutamine enhances the transamination effect of synthesizing 5-hydroxytryptophan, alpha-ketoglutaric acid meets the energy supply for synthesizing 5-hydroxytryptophan, and the precursor tryptophan promotes the synthesis capability of 5-hydroxytryptophan.
Description
Technical Field
The invention relates to the field of amino acid derivative production, in particular to a fermentation process for improving the yield of 5-hydroxytryptophan.
Background
5-hydroxytryptophan (5-HTP) with chemical name of 5-hydroxy-3-indolyl-a-aminopropionic acid, vitamin B in vivo6Is converted to 5-hydroxytryptamine (5-HT), also known as serotonin, by decarboxylation. 5-hydroxytryptophan is a precursor of the neurotransmitter 5-hydroxytryptamine (5-HT), and the product and metabolite thereof have biological activity, are involved in intercellular signal transmission, are involved in various activities in vivo, and are associated with depression, sleep, appetite and pain in humans. Studies have shown that 5-hydroxytryptophan can suppress appetite, reduce fat intake, reduce anxiety, control mood, and promote sleep. It can also be used as a food ingredient, found in many dietary proteins. Because serotonin is a precursor of melatonin, when the content of the serotonin is increased, the content of the melatonin is increased, so that the sleep is more fragrant and sweet.
At present, most of 5-hydroxytryptophan used in the pharmaceutical industry is extracted from the Gardner seeds by a hydrothermal method, an alcohol method and an ultrasonic method, and people such as Liudailin and the like adopt a resin adsorption method to separate the 5-hydroxytryptophan from the Gardner seeds, wherein the extraction rate is about 7 times of the unit content in raw materials. However, with the environmental over-development and pollution of human beings, the existence of African gardner trees is more and more rare, which is undoubtedly a difficult problem to solve for the 5-hydroxytryptophan production industry.
Disclosure of Invention
The invention aims to provide a fermentation process for improving the yield of 5-hydroxytryptophan.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a fermentation process for improving the yield of 5-hydroxytryptophan comprises the steps of adding metal ion chelated amino acid into a fermentation culture medium on one hand, and feeding composite auxiliary materials in a flow manner after 2 hours of fermentation on the other hand, wherein the metal ion chelated amino acid consists of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper, and the weight ratio of the methionine chelated iron to the alanine chelated manganese to the glutamic acid chelated copper is 4-5: 4-5: 1; the composite auxiliary material is a mixed solution of tryptophan, glutamine and alpha-ketoglutaric acid, and the weight ratio of the tryptophan to the glutamine to the alpha-ketoglutaric acid is 1-2: 4-6: 1-2.
Preferably, the fermentation process for increasing the yield of 5-hydroxytryptophan comprises the following steps:
(1) activation culture: taking out a 5-hydroxytryptophan producing strain E.coli HTP10 bacteria-protecting tube from a refrigerator at the temperature of-80 ℃, carrying out slant culture for two generations, and carrying out activated culture by adopting a slant culture medium which comprises the following components: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 2.5g/L of NaCl, 25g/L of agar powder and 6.8-7.0 of pH;
(2) seed culture: inoculating all the activated strains into a seed tank to obtain a seed solution, wherein the seed culture medium is as follows: glucose 20g/L, MgSO4 0.5g/L，KH2PO4 1.2g/L，(NH4)2SO45g/L, 10g/L yeast extract powder, VB1 0.3mg/L，VH0.2mg/L, 1g/L of defoaming agent, and adjusting the culture medium by ammonia water and maintaining the pH value to 6.7-7.0;
(3) fermentation culture: inoculating 12-16% of seed liquid into a fermentation tank, continuously culturing, feeding a composite auxiliary material mixed liquid after 2h of fermentation, and culturing the strain for 40h to obtain a fermentation liquid, wherein the adopted fermentation medium is as follows: 200mg/L of methionine iron chelate, 200mg/L of alanine manganese chelate, 50mg/L of glutamic acid copper chelate (metal ion chelated amino acid is formed by mixing 200mg/L of methionine iron chelate, 200mg/L of alanine manganese chelate and 50mg/L of glutamic acid copper chelate), 20g/L of glucose, 4g/L of yeast powder, MgSO4 1.2g/L，KH2PO4 2g/L，(NH4)2SO45g/L, 20ml/L of corn steep liquor, VB Mix2mg/L, adjusting the culture in the fermentation tank with ammonia water and maintaining the pH to 6.7-7.0.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the metal ion chelated amino acid is prepared by adding a mixture of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper in proportion, grinding, mixing, sealing under a dry condition, and reserving for later use.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the metal ion chelated amino acid is a solid mixture and is prepared into a fermentation medium together with other culture media.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the compound auxiliary materials are fed in the early stage of fermentation, the three auxiliary materials are added with sterile water according to a proportion to prepare a compound auxiliary material liquid mixed solution with a certain concentration, the mixed solution is sterilized at 115 ℃ for 15 minutes, the mixed solution is slowly fed into the fermentation liquid through a peristaltic pump right before the fermentation, and the feeding rate is just finished when the fermentation is finished.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the contents of the iron methionine chelate, the manganese alanine chelate and the copper glutamate chelate in the fermentation medium are respectively 200-250mg/L, 200-250mg/L and 40-62.5 mg/L.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the contents of iron methionine chelate, manganese alanine chelate and copper glutamate chelate in the metal ion chelated amino acid mixture in the fermentation medium are 200mg/L, 200mg/L and 50mg/L respectively.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the contents of tryptophan, glutamine and alpha-ketoglutaric acid added in the mixed liquid of the composite auxiliary materials are 1-2g/L, 4-6g/L and 1-2g/L respectively.
Preferably, the fermentation process for increasing the yield of 5-hydroxytryptophan is a precursor of 5-hydroxytryptophan (see FIG. 1 for a specific synthetic route from tryptophan (L-Trp) to 5-hydroxytryptophan (5-HTP)); glutamine provides amino for the synthesis of anthranilic acid (the specific synthetic route is shown in figure 1, glutamine (Gln) is added from chorismic acid (CHO) to 5-hydroxytryptophan (ATN)); alpha-ketoglutarate strengthens TCA cycle pathway and promotes generation of coenzyme FADH2 and NADH.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the formula for producing coenzyme FADH2 and NADH from alpha-ketoglutaric acid (alpha-KG) is alpha-KG + H2O+FAD+2NAD+ADP＝OAA+FADH2+2NADH+CO2+ ATP, this reaction is in the tricarboxylic acid cycle (TCA).
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the fed-batch content of tryptophan, glutamine and alpha-ketoglutaric acid is 2g/L, 4g/L and 2g/L respectively.
Preferably, in the fermentation process for increasing the yield of 5-hydroxytryptophan, the mixed solution of the compound auxiliary materials which is fed back is dissolved before the sterile water extraction, and the pH value is not adjusted.
Preferably, the fermentation process for increasing the yield of 5-hydroxytryptophan described above, said VB MixIs a VB1、VB3、VB5、VB12Mixed solution of equal mass.
Has the advantages that:
according to the fermentation process for improving the yield of the 5-hydroxytryptophan, metal ions are adopted to chelate amino acid, so that on one hand, the nutrient components of effective amino acid in a fermentation culture medium are supplemented, on the other hand, the absorption effect of thalli on trace elements is increased, and the growth activity and the production performance of the thalli are improved; meanwhile, the addition of glutamine provides amino for the synthesis of 5-hydroxytryptophan intermediate anthranilic acid, so that the transamination efficiency of the synthesized 5-hydroxytryptophan is improved; alpha-ketoglutaric acid satisfies energy supply for synthesizing 5-hydroxytryptophan, the precursor tryptophan promotes the synthesis capability of 5-hydroxytryptophan, and the fed alpha-ketoglutaric acid can strengthen TCA cycle path and promote coenzyme FADH2Generation of NADH provides a large amount of energy for synthesis of 5-hydroxytryptophan. Since the tryptophan comprises 60 per kg and the 5-hydroxytryptophan comprises 800 per kg, the cheap tryptophan is used as a precursor of the 5-hydroxytryptophan, and the synthesis rate of the expensive 5-hydroxytryptophan can be improved by adding a small amount; meanwhile, the synthesis of 5-hydroxytryptophan requires a large energy supply.
The E.coli HTP10 of the process disclosed by the invention is added with the tryptophan carboxylase gene, can catalyze tryptophan to generate 5-HTP, and has the advantages of short production period, continuous production, mild reaction conditions and the like. Coli HTP10 is a model strain of prokaryotes, has clear genetic information and mature fermentation conditions, and is suitable for research make internal disorder or usurp work for 5-HTP biosynthesis as a host cell.
Drawings
FIG. 1 is a scheme for the synthesis of 5-hydroxytryptophan.
Detailed Description
Example 1
A fermentation process for improving the yield of 5-hydroxytryptophan comprises the following specific steps:
(1) activation culture: taking out 5-hydroxytryptophan producing strain E.coli HTP10 (obtained by artificial modification of E.coli W3110(ATCC 27325) and purchased from Tianjin science and technology university) from a refrigerator at-80 ℃, carrying out two-generation slant culture, and performing activated culture by using a slant culture medium comprising: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 2.5g/L of NaCl, 25g/L of agar powder and 6.8-7.0 of pH;
(2) seed culture: inoculating all the activated strains into a seed tank to obtain a seed solution, wherein the seed culture medium is as follows: glucose 20g/L, MgSO4 0.5g/L，KH2PO4 1.2g/L，(NH4)2SO45g/L, 10g/L yeast extract powder, VB1 0.3mg/L，VH0.2mg/L, 1g/L of defoaming agent, and adjusting the culture medium by ammonia water and maintaining the pH value to 6.7-7.0;
(3) fermentation culture: inoculating 15% of seed liquid to a fermentation tank, continuously culturing, adding 100ml of composite auxiliary material mixed liquid in a flowing manner for 2h, adding the composite auxiliary material mixed liquid with the components of 2g of tryptophan, 4g of glutamine and 2g of alpha-ketoglutaric acid in each liter of fermentation liquid, and fermenting for 34h to obtain the fermentation liquid. The adopted fermentation medium is as follows: glucose 20g/L, yeast powder 4g/L, MgSO4 1.2g/L，KH2PO42g/L，(NH4)2SO45g/L, 20ml/L of corn steep liquor, VB Mix2mg/L, adjusting the culture of the fermentation tank by ammonia water and maintaining the pH value to be between 6.7 and 7.0.
The specific method for obtaining the E.coli HTP10 by artificially modifying E.coli W3110(ATCC 27325) comprises the following steps: the wild type Escherichia coli is obtained by modifying the wild type Escherichia coli by the following method: knocking out tnaA gene to prevent catabolism of tryptophan and 5-hydroxytryptophan; the lacIZ site of its genome integrates the xylose promoter PxylFControlled T7RNAP gene, inactivation of lacI protein and cell induction of RN production by xyloseA polymerase T7 RNAP; with feedback-inhibiting releasing mutant trpEfbrReplacing the original trpE gene of Escherichia coli with a gene, and using PtrcThe promoter directs expression of the tryptophan operon to enhance the chorismate pathway; mutant aroG that will relieve feedback inhibitionfbrGene serAfbrThe gene is integrated into the yjiV site of the Escherichia coli genome in tandem and is expressed by PtrcThe promoter guides expression to strengthen shikimic acid pathway and serine synthesis pathway; knocking out tyrR gene and trpR gene to realize deletion of negative transcription regulatory protein TyrR and TrpR; the TPH150 gene which is guided by a T7 promoter to express and codes the human type 2 tryptophan hydroxylase truncation mutant is integrated at the genome mbhA site so as to construct an intracellular tryptophan hydroxylation path; the mtrA gene from bacillus subtilis and the PTPS gene, SPR gene, PCD gene and DHPR gene from human being are serially integrated to the yghX site of colibacillus genome to introduce the synthesis path and regeneration path of coenzyme tetrahydropterin, wherein the mtrA gene, PTPS gene and SPR gene are composed of the same PtrcThe promoter directs the expression, the PCD gene and the DHPR gene are from the same PtrcThe promoter directs the expression; wherein,
coli W3110, accession number ATCC 27325;
the xylose promoter PxylFHas a nucleotide sequence shown in a sequence table SEQ ID NO. 1;
the RNA polymerase T7RNAP has a nucleotide sequence shown in a sequence table SEQ ID NO. 2;
the trpEfbrThe gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3;
the P istrcThe promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 4;
the aroGfbrThe gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5;
the serAfbrThe gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 6;
the strong promoter PT7 promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 7;
the TPH150 gene for coding the human 2-type tryptophan hydroxylase truncation mutant has a nucleotide sequence shown in a sequence table SEQ ID NO. 8;
the mtrA gene is derived from bacillus subtilis, is responsible for encoding GTP cyclohydrolase I and has a nucleotide sequence shown in a sequence table SEQ ID NO. 9;
the humanized PTPS gene is responsible for encoding 6-pyruvoyl tetrahydrobiopterin synthetase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 10;
the human SPR gene is responsible for coding the guanine reductase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 11;
the human PCD gene is responsible for encoding pterin-4 alpha-methanol ammonia dehydratase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 12;
the human DHPR gene is responsible for encoding the dihydropterin reductase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 13.
Example 2
A fermentation process for improving the yield of 5-hydroxytryptophan, which is as shown in example 1, and comprises the following steps: the mixed liquid of the composite auxiliary materials is not added, and simultaneously the metal ion chelated amino acid is added into the fermentation medium, and the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are respectively 200mg/L, 200mg/L and 50 mg/L.
Example 3
A fermentation process for improving the yield of 5-hydroxytryptophan, which is as shown in example 1, and comprises the following steps: and adding metal ion chelated amino acid into the fermentation medium, wherein the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are respectively 200mg/L, 200mg/L and 50 mg/L.
Example 4
A fermentation process for improving the yield of 5-hydroxytryptophan, which is as shown in example 1, and comprises the following steps: adding 100ml of composite auxiliary material mixed liquor in a flowing manner for 2 hours, wherein the components of the composite auxiliary material mixed liquor added in each liter of fermentation liquor are 1g of tryptophan, 4g of glutamine and 1g of alpha-ketoglutaric acid; and adding metal ion chelated amino acid into the fermentation medium, wherein the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are 250mg/L, 250mg/L and 60mg/L respectively.
As can be seen from the analyses of examples 1 to 4, in the fermentation of hydroxytryptophan, the optimum amounts of iron methionine chelate, manganese alanine chelate and copper glutamate chelate in the metal ion chelated amino acids were 200mg/L, 200mg/L and 50mg/L, the optimum amounts of the fed-batch composite adjuvant mixture were 2g/L tryptophan, 4g/L glutamine and 2g/L alpha-ketoglutaric acid, and finally the OD biomass of the cells was in a 5L fermentation tank600The yield of 126, 5-hydroxytryptophan is 2.6 g/L.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of environmental and operational medicine of military medical research institute of military science institute
Tianjin University of Science and Technology
<120> fermentation process for improving yield of 5-hydroxytryptophan
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> DNA
<213> promoter
<220>
<221> promoter
<222> (1)..(268)
<400> 1
gagataattc acaagtgtgc gctcgctcgc aaaataaaat ggaatgatga aactgggtaa 60
ttcctcgaag agaaaaatgc aataagtaca attgcgcaac aaaagtaaga tctcggtcat 120
aaatcaagaa ataaaccaaa aatcgtaatc gaaagataaa aatctgtaat tgttttcccc 180
tgtttagttg ctaaaaattg gttacgttta tcgcggtgat tgttacttat taaaactgtc 240
ctctaactac agaaggccct acaccatg 268
<210> 2
<211> 2652
<212> DNA
<213> Bacteriophage T7
<220>
<221> tRNA
<222> (1)..(2652)
<400> 2
atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60
ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120
catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180
gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240
atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300
acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360
accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420
atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480
cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540
gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600
tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660
attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720
tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780
ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840
attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900
agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960
aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020
atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080
ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140
gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200
atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260
gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320
aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380
aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440
ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500
tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560
gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620
tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680
ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740
attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800
aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860
ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920
tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980
tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040
atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100
tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160
aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220
aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280
attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340
aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400
aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460
gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520
gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580
atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640
gcgttcgcgt aa 2652
<210> 3
<211> 1563
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(1563)
<400> 3
atgcaaacac aaaaaccgac tctcgaactg ctaacctgcg aaggcgctta tcgcgacaat 60
cccaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaatcc 120
gcagatatcg acagcaaaga tgatttaaaa agcctgctgc tggtagacag tgcgctgcgc 180
attacagttt taggtgacac tgtcacaatc caggcacttt ccggcaacgg cgaagccctc 240
ctggcactac tggataacgc cctgcctgcg ggtgtggaaa gtgaacaatc accaaactgc 300
cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgctcg cttatgctcc 360
ctttcggttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420
cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480
ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540
ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600
aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660
gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720
agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780
attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840
tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900
ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960
attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020
gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080
gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140
agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200
tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260
atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320
gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380
gatctcgaca cctacattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440
caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500
aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560
tga 1563
<210> 4
<211> 74
<212> DNA
<213> promoter
<220>
<221> promoter
<222> (1)..(74)
<400> 4
ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac 60
acaggaaaca gacc 74
<210> 5
<211> 1053
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(1053)
<400> 5
atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcgatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg ttcgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210> 6
<211> 1233
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(1233)
<400> 6
atggcaaagg tatcgctgga gaaagacaag attaagtttc tgctggtaga aggcgtgcac 60
caaaaggcgc tggaaagcct tcgtgcagct ggttacacca acatcgaatt tcacaaaggc 120
gcgctggatg atgaacaatt aaaagaatcc atccgcgatg cccacttcat cggcctgcga 180
tcccgtaccc atctgactga agacgtgatc aacgccgcag aaaaactggt cgctattggc 240
tgtttctgta tcggaacaaa ccaggttgat ctggatgcgg cggcaaagcg cgggatcccg 300
gtatttaacg caccgttctc aaatacgcgc tctgttgcgg agctggtgat tggcgaactg 360
ctgctgctat tgcgcggcgt gccggaagcc aatgctaaag cgcaccgtgg cgtgtggaac 420
aaactggcgg cgggttcttt tgaagcgcgc ggcaaaaagc tgggtatcat cggctacggt 480
catattggta cgcaattggg cattctggct gaatcgctgg gaatgtatgt ttacttttat 540
gatattgaaa ataaactgcc gctgggcaac gccactcagg tacagcatct ttctgacctg 600
ctgaatatga gcgatgtggt gagtctgcat gtaccagaga atccgtccac caaaaatatg 660
atgggcgcga aagaaatttc actaatgaag cccggctcgc tgctgattaa tgcttcgcgc 720
ggtactgtgg tggatattcc ggcgctgtgt gatgcgctgg cgagcaaaca tctggcgggg 780
gcggcaatcg acgtattccc gacggaaccg gcgaccaata gcgatccatt tacctctccg 840
ctgtgtgaat tcgacaacgt ccttctgacg ccacacattg gcggttcgac tcaggaagcg 900
caggagaata tcggcctgga agttgcgggt aaattgatca agtattctga caatggctca 960
acgctctctg cggtgaactt cccggaagtc tcgctgccac tgcacggtgg gcgtcgtctg 1020
atgcacatcg ccgaaaaccg tccgggcgtg ctaactgcgc tgaacaaaat cttcgccgag 1080
cagggcgtca acatcgccgc gcaatatctg caaacttccg cccagatggg ttatgtggtt 1140
attgatattg aagccgacga agacgttgcc gaaaaagcgc tgcaggcaat gaaagctatt 1200
ccgggtacca ttcgcgcccg tctgctgtac taa 1233
<210> 7
<211> 61
<212> DNA
<213> promoter
<220>
<221> promoter
<222> (1)..(61)
<400> 7
taatacgact cactataggg tctagaaata attttgttta actttaagaa ggagatatac 60
c 61
<210> 8
<211> 951
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(951)
<400> 8
atggttccgt ggtttcctcg caaaatcagc gagctggaca aatgcagcca ccgcgtcctg 60
atgtacggtt ccgaactgga cgccgatcac cctggtttca aagacaacgt ttaccgtcag 120
cgccgtaaat acttcgtaga cgtggccatg ggttacaaat acggtcagcc gatcccgcgc 180
gtcgaataca ctgaagaaga aaccaaaacg tggggcgtag tattccgtga actgtccaaa 240
ctgtacccga cccacgcttg ccgtgaatat ctgaaaaact ttccgctgct gaccaaatac 300
tgcggttacc gtgaagataa cgttccgcag ctggaagatg tttctatgtt cctgaaagag 360
cgttccggtt tcacggttcg tccagttgca ggttacctgt ctccgcgcga ttttctggcg 420
ggcctggctt accgtgtgtt ccactgtacc caatacatcc gtcacggcag cgatccgctg 480
tataccccgg aaccggacac ttgtcatgag ctgctgggcc acgttccact gctggctgac 540
ccaaaattcg cgcagttctc tcaggaaatt ggtctggcat ctctgggcgc gtctgacgaa 600
gacgtccaga aactggcaac ttgctacttc tttactatcg aatttggcct gtgcaagcaa 660
gaaggtcagc tgcgcgcgta tggtgcaggt ctgctgtcta gcatcggtga gctgaaacac 720
gcgctgtctg acaaggcctg cgtgaaggct tttgatccga aaaccacttg cctgcaggaa 780
tgcctgatca ccaccttcca ggaagcctac ttcgtaagcg agtccttcga agaagcgaaa 840
gagaaaatgc gtgatttcgc gaaaagcatt acccgtccgt tctctgtata cttcaacccg 900
tacacccagt ccatcgaaat cctgaaagat actcgttcca tcgaaaacgt t 951
<210> 9
<211> 573
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(573)
<400> 9
atgaaagaag ttaataaaga gcaaatcgaa caagctgttc gtcaaatttt agaagcgatc 60
ggagaagacc cgaatagaga agggcttctt gatactccga aaagagtcgc aaagatgtat 120
gccgaagtat tctccggctt gaatgaagat ccaaaagaac atttccagac tatcttcggt 180
gaaaaccatg aggagcttgt tcttgtaaaa gatatagcgt ttcattctat gtgtgagcat 240
caccttgttc ccttttatgg aaaagcacat gttgcatata tcccgcgagg cggaaaggtc 300
acaggactca gcaaactggc acgtgccgtt gaagccgttg caaagcgccc gcagcttcag 360
gaacgcatca cttctacaat tgcagaaagc atcgtagaaa cgcttgatcc gcatggcgta 420
atggtagtgg ttgaagcgga acacatgtgc atgacgatgc gcggtgtaag aaaaccgggt 480
gcgaaaactg tgacttcagc agtcagaggc gtttttaaag atgatgccgc tgcccgtgca 540
gaagtattgg aacatattaa acgccaggac taa 573
<210> 10
<211> 438
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(438)
<400> 10
atgagcacgg aaggtggtgg ccgtcgctgc caggcacaag tgtcccgccg catctccttc 60
agcgcgagcc accgattgta cagtaaattt ctaagtgatg aagaaaactt gaaactgttt 120
gggaaatgca acaatccaaa tggccatggg cacaattata aagttgtggt gacagtacat 180
ggagagattg accctgctac gggaatggtt atgaatctgg ctgatctcaa aaaatatatg 240
gaggaggcga ttatgcagcc ccttgatcat aagaatctgg atatggatgt gccatacttt 300
gcagatgtgg tgagcacgac tgaaaatgta gctgtttata tctgggacaa cctccagaaa 360
gttcttcctg taggagttct ttataaagta aaagtatacg aaactgacaa taatattgtg 420
gtttataaag gagaatag 438
<210> 11
<211> 783
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(783)
<400> 11
atggaaggtg gtctgggtcg tgctgtttgt ctgctgactg gtgctagccg tggtttcggc 60
cgtaccctgg cacctctgct ggcatctctg ctgtctccag gcagcgtgct ggtactgagc 120
gctcgtaacg atgaagcact gcgtcagctg gaagccgaac tgggtgctga acgttctggt 180
ctgcgtgtcg ttcgtgtacc tgcagatctg ggtgctgaag ctggcctgca acagctgctg 240
ggtgctctgc gtgaactgcc gcgtccaaaa ggcctgcaac gtctgctgct gatcaacaac 300
gctggttccc tgggtgacgt ttccaaaggt ttcgtagacc tgtccgattc cactcaggtt 360
aacaattact gggctctgaa cctgaccagc atgctgtgtc tgaccagctc cgttctgaaa 420
gcattcccag attctccggg cctgaaccgt accgttgtga acatttccag cctgtgcgcg 480
ctgcagccgt tcaagggttg ggcactgtat tgcgcgggta aagcagcccg tgacatgctg 540
ttccaggttc tggcgctgga agaaccaaac gttcgtgttc tgaactatgc tccgggtcct 600
ctggacaccg atatgcagca gctggcgcgt gaaacctccg ttgatccgga catgcgcaag 660
ggtctgcaag aactgaaagc taaaggtaaa ctggttgatt gcaaagtatc tgctcagaaa 720
ctgctgagcc tgctggaaaa agacgaattc aagagcggtg ctcacgtgga cttttacgac 780
aag 783
<210> 12
<211> 315
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(315)
<400> 12
atggctggta aagctcatcg tctgagcgcg gaagaacgtg atcagctgct gccaaacctg 60
cgtgcggttg gttggaacga actggaaggt cgtgatgcga tctttaaaca gtttcacttc 120
aaggatttta accgtgcttt cggtttcatg acccgtgtag cactgcaggc tgagaaactg 180
gaccaccacc cggaatggtt caacgtgtat aacaaagttc acatcactct gagcacccac 240
gaatgtgcag gcctgtctga acgtgacatc aacctggctt ctttcatcga acaggttgca 300
gtgtctatga cctag 315
<210> 13
<211> 735
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(735)
<400> 13
atggcagcag ctgcagcagc aggtgaagct cgtcgtgttc tggtttacgg tggtcgtggt 60
gcgctgggtt ctcgttgtgt tcaggctttc cgcgctcgta actggtgggt agcttccgtg 120
gatgttgtag agaacgaaga ggcgtctgct tccatcatcg ttaaaatgac cgactctttc 180
acggaacaag cagatcaggt taccgcagaa gttggcaaac tgctgggcga agaaaaagtt 240
gacgctatcc tgtgtgttgc gggtggctgg gctggtggta acgcaaaatc taagtctctg 300
ttcaaaaact gcgatctgat gtggaaacag agcatctgga cttccacgat ctcctcccac 360
ctggcgacta aacacctgaa agaaggcggt ctgctgaccc tggctggtgc aaaagctgct 420
ctggacggca ctccgggtat gattggctat ggtatggcca aaggcgcagt acatcagctg 480
tgccaaagcc tggctggcaa aaactccggt atgccaccgg gtgcagccgc aattgcagtt 540
ctgccagtga ccctggatac cccgatgaac cgtaaaagca tgccggaagc tgatttctct 600
tcttggaccc cgctggaatt cctggttgaa actttccatg actggatcac cggcaaaaat 660
cgcccgtctt ccggttccct gattcaggtt gttactaccg aaggtcgtac tgaactgacc 720
ccggcatact tctag 735
Claims (6)
1. A fermentation process for improving the yield of 5-hydroxytryptophan is characterized by comprising the following steps: on one hand, adding metal ion chelated amino acid into a fermentation culture medium, and on the other hand, feeding composite auxiliary materials in 2h of fermentation, wherein the metal ion chelated amino acid consists of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper, and the weight ratio of the methionine chelated iron to the alanine chelated manganese to the glutamic acid chelated copper is 4-5: 4-5: 1; the composite auxiliary material is a mixed solution of tryptophan, glutamine and alpha-ketoglutaric acid, and the weight ratio of the tryptophan to the glutamine to the alpha-ketoglutaric acid is 1-2: 4-6: 1-2.
2. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: the method comprises the following specific steps:
(1) activation culture: taking out a 5-hydroxytryptophan producing strain E.coli HTP10 bacteria-protecting tube from a refrigerator at the temperature of-80 ℃, carrying out slant culture for two generations, and carrying out activated culture by adopting a slant culture medium which comprises the following components: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 2.5g/L of NaCl, 25g/L of agar powder and 6.8-7.0 of pH;
(2) seed culture: inoculating all the activated strains into a seed tank to obtain a seed solution, wherein the seed culture medium is as follows: glucose 20g/L, MgSO4 0.5g/L，KH2PO4 1.2g/L，(NH4)2SO45g/L, 10g/L yeast extract powder, VB1 0.3mg/L，VH0.2mg/L, 1g/L of defoaming agent, and adjusting the culture medium by ammonia water and maintaining the pH value to 6.7-7.0;
(3) fermentation culture: inoculating 12-16% of seed liquid into a fermentation tank, continuously culturing, feeding a composite auxiliary material mixed liquid after 2h of fermentation, and culturing the strain for 40h to obtain a fermentation liquid, wherein the adopted fermentation medium is as follows: 200mg/L of methionine chelated iron, 200mg/L of alanine chelated manganese, 50mg/L of glutamic acid chelated copper, 20g/L of glucose, 4g/L of yeast powder and MgSO4 1.2g/L，KH2PO4 2g/L，(NH4)2SO45g/L, 20ml/L of corn steep liquor, VBMix2mg/L, adjusting the culture in the fermentation tank with ammonia water and maintaining the pH to 6.7-7.0.
3. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: tryptophan in the composite auxiliary material is a precursor of 5-hydroxytryptophan; glutamine provides amino for the synthesis of anthranilic acid; alpha-ketoglutarate strengthens TCA cycle pathway and promotes generation of coenzyme FADH2 and NADH.
4. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: the contents of tryptophan, glutamine and alpha-ketoglutaric acid fed-batch are respectively 2g/L, 4g/L and 2 g/L.
5. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: dissolving the mixed solution of the compound auxiliary materials added in the flowing way before using sterile water, and adjusting the pH value.
6. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 2, wherein: the V isBMixIs a VB1、VB3、VB5、VB12Mixed solution of equal mass.
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| CN116103347A (en) * | 2023-03-23 | 2023-05-12 | 天津科技大学 | Method for improving fermentation yield of L-tyrosine and sugar acid conversion rate |
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