CN113493761B - Fermentation process for improving yield of 5-hydroxytryptophan - Google Patents
Fermentation process for improving yield of 5-hydroxytryptophan Download PDFInfo
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- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- 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
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- 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)
Abstract
The invention provides a fermentation process for improving the yield of 5-hydroxytryptophan, which comprises the steps of adding metal ion chelating amino acid into a fermentation culture medium, and feeding composite auxiliary materials into the fermentation culture medium for 2h, wherein the metal ion chelating amino acid is adopted in the method, so that the nutrition components of effective amino acid in the fermentation culture medium are supplemented, the absorption effect of thalli on trace elements is improved, and the growth vigor and the production performance of the thalli are improved; meanwhile, glutamine enhances the ammonia transfer effect of synthesizing 5-hydroxytryptophan, alpha-ketoglutarate meets the energy supply of synthesizing 5-hydroxytryptophan, and the precursor tryptophan promotes the synthesis capacity 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-hydroxytryptophan, 5-HTP), chemical name 5-hydroxy-3-indolyl-a-aminopropionic acid, which can be vitamin B in vivo 6 Is converted into 5-hydroxytryptamine (5-HT), also known as serotonin, by decarboxylation. 5-hydroxytryptophan is a precursor of the neurotransmitter 5-hydroxytryptamine (5-HT), which has biological activity and its metabolites, is involved in intercellular signaling, in various activities in the body, in depression and sleep in humansSleep, appetite and pain are all associated with it. Research shows that 5-hydroxytryptophan can inhibit appetite, reduce fat intake, reduce anxiety, control emotion and promote sleep. Can also be used as food ingredient, and can be found in proteins contained in many diets. Since serotonin is a precursor of melatonin, when the content of serotonin is increased, the content of melatonin is also increased, making sleep more fragrant and sweet.
At present, most of 5-hydroxytryptophan used in the pharmaceutical industry is extracted from the garna seeds, and heretofore, most of the 5-hydroxytryptophan is extracted by a hydrothermal method, an alcohol method and an ultrasonic method, liu Dailin and the like adopt a resin adsorption method to separate the 5-hydroxytryptophan from the garna seeds, and the extraction rate is about 7 times of the unit content in the raw materials. However, with the over development and pollution of the environment by human beings, the presence of african garna is becoming more and more rare, which is undoubtedly a difficult to solve for the production industry of 5-hydroxytryptophan.
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:
on one hand, metal ion chelated amino acid is added into a fermentation culture medium, and on the other hand, compound auxiliary materials are fed into the fermentation culture medium for 2 hours, 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, a step of; 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 improving the yield of 5-hydroxytryptophan comprises the following specific steps:
(1) And (3) activating and culturing: taking out 5-hydroxytryptophan producing strain E.coll HTP10 bacteria-preserving tube from a refrigerator at-80 ℃, transferring to two generations of inclined planes, and performing activation culture by adopting an inclined plane culture medium as follows: 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 pH=6.8-7.0;
(2) Seed culture: inoculating all the activated strains into a seed tank to obtain seed liquid, wherein the seed culture medium is as follows: glucose 20g/L, mgSO 4 0.5g/L，KH 2 PO 4 1.2g/L，(NH 4 ) 2 SO 4 5g/L, yeast extract 10g/L, V B1 0.3mg/L，V H 0.2mg/L of defoamer 1g/L, and adjusting and maintaining the pH of the culture medium to 6.7-7.0 with ammonia water;
(3) Fermentation culture: inoculating 12-16% of seed liquid into a fermentation tank, continuously culturing, fermenting for 2h, feeding composite auxiliary material mixed liquid, culturing strain for 40h to obtain fermentation liquid, wherein the adopted fermentation culture medium is as follows: methionine chelated iron 200mg/L, alanine chelated manganese 200mg/L, glutamic acid chelated copper 50mg/L (metal ion chelated amino acid formed by mixing methionine chelated iron 200mg/L, alanine chelated manganese 200mg/L, and glutamic acid chelated copper 50 mg/L), glucose 20g/L, yeast powder 4g/L, mgSO 4 1.2g/L，KH 2 PO 4 2g/L，(NH 4 ) 2 SO 4 5g/L, 20ml/L of corn steep liquor, V B Mix 2mg/L, and the pH of the fermentation tank is adjusted and maintained to 6.7-7.0 by ammonia water.
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the preparation method of the metal ion chelating amino acid comprises the steps of adding a mixture of methionine chelating iron, alanine chelating manganese and glutamic acid chelating copper in proportion, grinding, uniformly mixing, sealing under a dry condition, and reserving for later use.
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the metal ion chelating amino acid is a solid mixture, and is prepared into a fermentation medium together with other culture mediums.
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the compound auxiliary materials are fed in the earlier stage of fermentation, 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 for 15 minutes at the temperature of 115 ℃, and the mixed solution is fed into the fermentation liquid slowly by a peristaltic pump just before fermentation, wherein the feeding rate is just the feeding is up to the end of fermentation.
Preferably, in the fermentation process for improving the yield of 5-hydroxytryptophan, the contents of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper in a fermentation culture medium are respectively 200-250mg/L, 200-250mg/L and 40-62.5mg/L.
Preferably, in the fermentation process for improving the yield of 5-hydroxytryptophan, the contents of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper in the metal ion chelated amino acid mixture in the fermentation medium are respectively 200mg/L, 200mg/L and 50mg/L.
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the fed-batch content of tryptophan, glutamine and alpha-ketoglutaric acid in the mixed solution of the compound auxiliary materials is 1-2g/L, 4-6g/L and 1-2g/L respectively.
Preferably, the fermentation process for increasing the yield of 5-hydroxytryptophan is described above, wherein the tryptophan is a 5-hydroxytryptophan precursor (the specific synthetic route is shown in FIG. 1, from tryptophan (L-Trp) to 5-hydroxytryptophan (5-HTP)); glutamine provides an amino group for the synthesis of anthranilic acid (a specific synthetic route is shown in fig. 1, with glutamine (gin) added from Chorismate (CHO) to 5-hydroxytryptophan (ATN)); alpha-ketoglutarate, which strengthens TCA circulatory path and promotes the generation of coenzyme FADH2 and NADH.
Preferably, the fermentation process for improving the yield of the 5-hydroxytryptophan comprises the steps of producing coenzyme FADH2 by alpha-ketoglutarate (alpha-KG) and producing NADH by alpha-KG+H 2 O+FAD+2NAD+ADP＝OAA+FADH 2 +2NADH+CO 2 +atp, this reaction is in the tricarboxylic acid cycle (TCA).
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the fed-batch contents of the tryptophan, the glutamine and the alpha-ketoglutaric acid are 2g/L, 4g/L and 2g/L respectively.
Preferably, in the fermentation process for improving the yield of the 5-hydroxytryptophan, the fed-batch mixed solution of the compound auxiliary materials is dissolved in sterile water in advance, and the pH is not regulated.
Preferably, the fermentation process for improving the yield of 5-hydroxytryptophan, wherein V B Mix Is V (V) B1 、V B3 、V B5 、V B12 Is a mixed liquid with equal quality.
The beneficial effects are that:
according to the fermentation process for improving the yield of the 5-hydroxytryptophan, the metal ions are adopted to chelate the amino acid, so that on one hand, the nutrient components of the effective amino acid in the fermentation medium are supplemented, on the other hand, the absorption effect of thalli on trace elements is improved, and the growth vigor and the production performance of the thalli are improved; meanwhile, the addition of glutamine provides amino for the synthesis of the 5-hydroxytryptophan intermediate anthranilic acid, so that the ammonia conversion efficiency of synthesizing the 5-hydroxytryptophan is improved; the alpha-ketoglutarate meets the energy supply of synthesizing 5-hydroxytryptophan, the precursor tryptophan promotes the synthesis capacity of 5-hydroxytryptophan, and the fed-batch alpha-ketoglutarate can strengthen TCA (ternary ammonium chloride) circulatory path and promote coenzyme FADH 2 NADH production provides a large amount of energy for the synthesis of 5-hydroxytryptophan. As 60 yuan of tryptophan per kg, 800 yuan of 5-hydroxytryptophan per kg, and cheap tryptophan is taken as a precursor of 5-hydroxytryptophan, and the synthesis rate of expensive 5-hydroxytryptophan can be improved by adding a small amount of the 5-hydroxytryptophan; at the same time, the synthesis of 5-hydroxytryptophan requires a large energy supply.
The E.coli HTP10 of the technology has the advantages of short production period, continuous production, mild reaction conditions and the like, and can catalyze tryptophan to generate 5-HTP. Meanwhile, the E.coli HTP10 is a model strain of prokaryote, has clear genetic information and mature fermentation conditions, and is suitable for being used as a host cell for the research of 5-HTP biosynthesis.
Drawings
FIG. 1 shows the synthesis pathway of 5-hydroxytryptophan.
Detailed Description
Example 1
A fermentation process for improving the yield of 5-hydroxytryptophan comprises the following specific steps:
(1) And (3) activating and culturing: taking out 5-hydroxytryptophan producing strain E.coll HTP10 (obtained by artificial modification of E.coll W3110 (ATCC 27325) from the university of Tianjin science and technology) from a refrigerator at-80 ℃, transferring to two generations of inclined planes, and performing activation culture, wherein the adopted inclined plane culture medium comprises: 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 pH=6.8-7.0;
(2) Seed culture: inoculating all the activated strains into a seed tank to obtain seed liquid, wherein the seed culture medium is as follows: glucose 20g/L, mgSO 4 0.5g/L，KH 2 PO 4 1.2g/L，(NH 4 ) 2 SO 4 5g/L, yeast extract 10g/L, V B1 0.3mg/L，V H 0.2mg/L of defoamer 1g/L, and adjusting and maintaining the pH of the culture medium to 6.7-7.0 with ammonia water;
(3) Fermentation culture: inoculating 15% of seed liquid to a fermentation tank, continuously culturing, feeding 100ml of composite auxiliary material mixed liquid into 2h, feeding 2g of tryptophan, 4g of glutamine and 2g of alpha-ketoglutaric acid into each liter of fermentation liquid, and fermenting for 34h to obtain fermentation liquid. The fermentation medium adopted is: glucose 20g/L, yeast powder 4g/L, mgSO 4 1.2g/L，KH 2 PO 4 2g/L，(NH 4 ) 2 SO 4 5g/L, 20ml/L of corn steep liquor, V B Mix 2mg/L, and the pH of the fermentation tank is adjusted and maintained to be between 6.7 and 7.0 by ammonia water.
The E.coli HTP10 is obtained by artificial modification of E.coli W3110 (ATCC 27325) by the following specific method: is obtained by modifying wild escherichia coli by the following method: knocking out the tnaA gene to prevent catabolism of tryptophan and 5-hydroxytryptophan; integration of the xylose promoter P at the lacIZ site of its genome xylF The controlled T7RNAP gene, while inactivating lacI protein, allows the cell to utilize xylose to induce the production of RNA polymerase T7RNAP; with mutant trpE releasing feedback inhibition fbr Gene replacement of the original trpE gene of E.coli and P trc The promoter directs expression of the tryptophan operon to enhance the chorismate pathway; mutant aroG that will release feedback inhibition fbr Gene serA fbr The gene was integrated in tandem into the yjiV locus of the E.coli genome and P was used trc The promoter directs expression to enhance shikimate pathway and serine synthesis pathway; knocking out tyrR gene and trpR gene to realize deletion of negative transcription regulatory proteins TyrR and TrpR; the coding human-derived type 2 color is integrated at the mbhA locus of the genome thereof and expressed under the guidance of the T7 promoterThe amino acid hydroxylase truncates the mutant TPH150 gene to construct an intracellular tryptophan hydroxylation pathway; the mtrA gene derived from bacillus subtilis and the PTPS gene, SPR gene, PCD gene and DHPR gene derived from human are integrated in series at yghX locus of escherichia coli genome to introduce synthetic pathway and regeneration pathway of coenzyme tetrahydropterin, wherein the mtrA gene, the PTPS gene and the SPR gene are formed by the same P trc The promoter guides the expression, PCD gene and DHPR gene are formed by the same P trc The promoter directs expression; wherein, the liquid crystal display device comprises a liquid crystal display device,
the wild type E.coli is E.coli W3110, accession number ATCC 27325;
the xylose promoter P xylF Has 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 trpE fbr The gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3;
the P is trc A promoter having a nucleotide sequence shown in a sequence table SEQ ID NO. 4;
the aroG fbr The gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5;
the serA fbr The 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 encoding the humanized 2 tryptophan hydroxylase short-cut mutant has a nucleotide sequence shown in a sequence table SEQ ID NO. 8;
the mtrA gene is derived from bacillus subtilis and is responsible for coding 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 humanized SPR gene is responsible for encoding the pterin 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 humanized DHPR gene is responsible for encoding 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, with reference to example 1, differs in that: the mixed solution of the compound auxiliary materials is not fed, and metal ion chelated amino acid is added into the fermentation culture medium, wherein the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are respectively 200mg/L, 200mg/L and 50mg/L.
Example 3
A fermentation process for improving the yield of 5-hydroxytryptophan, with reference to example 1, differs in that: and adding metal ion chelated amino acid into the fermentation culture medium, wherein the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are respectively 200mg/L, 200mg/L and 50mg/L.
Example 4
A fermentation process for improving the yield of 5-hydroxytryptophan, with reference to example 1, differs in that: 100ml of composite auxiliary material mixed solution is fed in a flowing way for 2 hours, and each liter of fermentation liquid is fed in a flowing way, wherein the components of the composite auxiliary material mixed solution are 1g tryptophan, 4g glutamine and 1g alpha-ketoglutaric acid; and adding metal ion chelated amino acid into the fermentation culture medium, wherein the components of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper are respectively 250mg/L, 250mg/L and 60mg/L.
The analysis of the examples 1-4 shows that in the fermentation of the-hydroxytryptophan, the optimal addition amount of methionine chelated iron, alanine chelated manganese and glutamic acid chelated copper in the metal ion chelated amino acid is 200mg/L, 200mg/L and 50mg/L, the optimal components of the mixed solution of the fed-batch composite auxiliary materials are 2g/L tryptophan, 4g/L glutamine and 2g/L alpha-ketoglutaric acid,finally, in a 5L fermentation tank, the biomass OD of the thallus 600 The yield of 126,5-hydroxytryptophan is 2.6g/L.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> environmental medical and working medical institute of military medical institute of academy of military science
Tianjin University of Science and Technology
<120> fermentation process for increasing yield of 5-hydroxytryptophan
<160> 13
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aaatcaagaa ataaaccaaa aatcgtaatc gaaagataaa aatctgtaat tgttttcccc 180
tgtttagttg ctaaaaattg gttacgttta tcgcggtgat tgttacttat taaaactgtc 240
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<210> 2
<211> 2652
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<213> Bacteriophage T7
<220>
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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
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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
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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
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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 (4)
1. A fermentation process for improving the yield of 5-hydroxytryptophan is characterized by comprising the following steps: the method comprises the following specific steps:
(1) And (3) activating and culturing: extracting 5-hydroxytryptophan producing strain from refrigerator at-80deg.CE.coli HTP10The bacteria-retaining tube is transmitted to two generations of inclined planes for activation culture, and the adopted inclined plane culture medium is as follows: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, naCl 2.5g/L,25g/L of agar powder and pH=6.8-7.0; the saidE.coli HTP10From deposit number ATCC 27325E.coli W3110The method is characterized by comprising the following steps of: knock-out tnaAGenes to prevent catabolism of tryptophan and 5-hydroxytryptophan; in its genomelacIZAt the site is integrated by xylose promoter P xylF Controlled byT7RNAPGenes that allow cells to utilize xylose to induce the production of RNA polymerase T7RNAP while inactivating the lacI protein; with mutants releasing feedback inhibitiontrpE fbr Gene replacement of E.colitrpEGene, and P trc The promoter directs expression of the tryptophan operon to enhance the chorismate pathway; mutants that will release feedback inhibitionaroG fbr GeneserA fbr Integration of genes into the E.coli genome in tandemyjiVSites and use P trc The promoter directs expression to enhance shikimate pathway and serine synthesis pathway; knock-outtyrRGene and genetrpRGenes to achieve deletion of negative transcription regulatory proteins TyrR and TrpR; in its genomembhASite integration of mutant encoding human tryptophan hydroxylase type 2 truncated by T7 promoter-directed expressionTPH150Genes to construct an intracellular tryptophan hydroxylation pathway; derived from bacillus subtilismtrAGene and human derivedPTPSGenes (gene),SPRGenes (gene),PCDGene and geneDHPRGenes, tandem integration into E.coli genomeyghXAt the site to introduce the synthetic and regenerative pathways of coenzyme tetrahydropterin, whereinmtrAGenes (gene),PTPSGenes (gene),SPRThe genes are composed of the same P trc The promoter directs the expression of the polypeptide,PCDgenes (gene),DHPRThe genes are composed of the same P trc The promoter directs expression; wherein, the liquid crystal display device comprises a liquid crystal display device,
the xylose promoter P xylF The nucleotide sequence of (2) is shown as SEQ ID NO. 1;
the nucleotide sequence of the RNA polymerase T7RNAP is shown as SEQ ID NO. 2;
the saidtrpE fbr The nucleotide sequence of the gene is shown as SEQ ID NO. 3;
the P is trc The nucleotide sequence of the promoter is shown as SEQ ID NO. 4;
the saidaroG fbr The nucleotide sequence of the gene is shown as SEQ ID NO. 5;
the saidserA fbr The nucleotide sequence of the gene is shown as SEQ ID NO. 6;
the nucleotide sequence of the T7 promoter is shown in SEQ ID NO. 7;
the mutant codes for human tryptophan type 2 hydroxylaseTPH150The nucleotide sequence of the gene is shown as SEQ ID NO. 8;
the saidmtrAThe nucleotide sequence of the gene is shown as SEQ ID NO. 9;
the saidPTPSThe nucleotide sequence of the gene is shown as SEQ ID NO. 10;
the saidSPRThe nucleotide sequence of the gene is shown as SEQ ID NO. 11;
the saidPCDThe nucleotide sequence of the gene is shown as SEQ ID NO. 12;
the saidDHPRThe nucleotide sequence of the gene is shown as SEQ ID NO. 13;
(2) Seed culture: inoculating all the activated strains into a seed tank to obtain seed liquid, wherein the seed culture medium is as follows: glucose 20g/L MgSO 4 0.5 g/L，KH 2 PO 4 1.2 g/L，（NH 4 ） 2 SO 4 5g/L, yeast extract 10g/L, V B1 0.3 mg/L，V H 0.2mg/L, defoamer 1g/L, and ammonia water to adjust and maintain the pH of the culture medium to 6.7-7.0;
(3) Fermentation culture: inoculating 12-16% of seed liquid to a fermentation tank, continuously culturing, and fermenting 2h by feeding mixed liquid of composite auxiliary materials, wherein the mixed liquid of tryptophan, glutamine and alpha-ketoglutaric acid is adopted as the composite auxiliary materials, and the weight ratio of tryptophan, glutamine and alpha-ketoglutaric acid is 1-2:4-6:1-2, culturing the strain 40h to obtain a fermentation broth, wherein the fermentation medium is as follows: methionine chelated iron 200mg/L, alanine chelated manganese 200mg/L, glutamic acid chelated copper 50mg/L, glucose 20g/L, yeast powder 4g/L, mgSO 4 1.2 g/L，KH 2 PO 4 2 g/L，（NH 4 ） 2 SO 4 5g/L, corn steep liquor 20ml/L, V B Mix 2. 2mg/L, the fermenter was adjusted and maintained at pH 6.7-7.0 with ammonia.
2. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: the fed-batch contents of tryptophan, glutamine and a-ketoglutarate are respectively 2g/L, 4g/L and 2g/L.
3. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: and the added mixed solution of the composite auxiliary materials is dissolved in advance by using sterile water, and the pH is not regulated.
4. The fermentation process for increasing the yield of 5-hydroxytryptophan according to claim 1, wherein: the V is B Mix Is V (V) B1 、V B3 、V B5 、V B12 Is a mixed liquid with equal quality.
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| CN105385646A (en) * | 2014-09-02 | 2016-03-09 | 乔治亚大学研究基金公司 | Precursor-directed biosynthesis of 5-hydroxytryptophan |
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| CN112877270A (en) * | 2021-02-03 | 2021-06-01 | 天津科技大学 | Genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine and application thereof |
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| WO2013127914A1 (en) * | 2012-02-29 | 2013-09-06 | Danmarks Tekniske Universitet | Microorganisms for the production of 5-hydroxytryptophan |
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