CN113373103A - Method for improving yield of 5-hydroxytryptophan - Google Patents
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Abstract
The invention provides a method for improving the yield of 5-hydroxytryptophan, which reduces ventilation volume and dissolved oxygen in the acid production stage in the middle stage of fermentation, adds citric acid in a flow manner, ensures sufficient reducing force in the synthesis process of BH4, reduces sugar supplement rate after the consumption of base sugar, and reduces accumulation of tryptophan; according to the method, the dissolved oxygen is controlled to be in a sub-proper amount in the cells, citric acid is fed in to inhibit an EMP (electron cyclotron peptide) pathway, an HMP pathway is enhanced, the glucose feeding rate in the fermentation process is reduced to improve the yield of 5-HTP, the tryptophan as an intermediate product is reduced, and the problems that the tetrahydropterin is oxidized and consumed in the biological fermentation process, the reducing power in the synthesis process is insufficient, and the 5-HTP yield is influenced by excessive accumulation of the tryptophan are solved.
Description
Technical Field
The invention relates to the field of amino acid production by a fermentation method, in particular to a method for improving the yield of 5-hydroxytryptophan.
Background
Depression is a common mental disorder threatening millions of people worldwide. Serotonin deficiency of the neurotransmitter in the Central Nervous System (CNS) is considered to be an important physiological factor in depression.
5-hydroxytryptophan (5-HTP) is a direct biosynthetic precursor of serotonin in humans and animals. It has been shown to be clinically effective in the treatment of depression with relatively few side effects. 5-hydroxytryptophan (5-HTP) is a natural amino acid not involved in protein synthesis, and is produced by substituting the 5' -hydrogen atom on the phenyl ring of tryptophan with a hydroxyl group, and is chemically named 5-hydroxy-3-indolyl-alpha-aminopropionic acid. In mammals, 5-HTP is a precursor of the neurotransmitter Serotonin (Serotonin) and the amine hormone Melatonin (Melatonin), has a regulating effect on physiological functions such as sleep, pain sensation, body temperature, appetite and behavior, and has been successfully used for treating diseases such as depression, insomnia and migraine.
Due to the high medicinal health care and market value of 5-HTP, the production research of 5-HTP is rapidly developed in recent years. The existing 5-HTP production method mainly comprises the process technologies of natural product extraction, chemical synthesis, microbial fermentation and the like, wherein 5-HTP is extracted from seeds of African plant Gardner by a method such as Chenyi and the like (CN111978238A) and is subjected to procedures of oil pressing, extraction, coarse filtration, fine filtration, vacuum concentration, extraction impurity removal, solid-liquid separation, refining, water washing solvent removal, drying, crushing and the like to obtain 5-HTP. However, the method has low yield, is easily affected by seasons, has insufficient raw materials and is limited by regions, which becomes a main bottleneck limiting large-scale production. Chemical synthesis methods such as Huwenhui and the like (CN102351775B) use L-tryptophan methyl ester/ethyl ester to obtain L-tryptophan methyl ester/ethyl ester hydrochloride, desalt acid under alkaline conditions to obtain L-tryptophan methyl ester/ethyl ester, perform acetylation to obtain N-acetyl-L-tryptophan methyl ester/ethyl ester, reduce indole ring under a triethylsilane-trifluoroacetic acid reduction system, oxidize 1-bit hydrogen peroxide nitrogen of the indole ring under a sodium tungstate-30% hydrogen peroxide system, and finally perform deacetylation of a protecting group under acidic conditions to obtain 5-hydroxytryptophan. But generally speaking, the chemical synthesis reaction system is complex and the steps are complicated, so the microbial synthesis method has the advantages of low cost, simple operation, environmental protection and mild reaction, and can be used for mass production to become a 5-HTP production mode with huge potential. However, the prior microbiological method still has the problems of unstable enzyme activity, high cost, low catalytic efficiency and the like because expensive cofactors are required to be added externally.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for improving the yield of 5-hydroxytryptophan.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for improving the yield of 5-hydroxytryptophan features that the ventilation volume is decreased in the acid-producing stage of middle stage of fermentation, the dissolved oxygen is reduced, citric acid is added to ensure sufficient reducing power in the synthesis process of BH4, the sugar supplementing rate is decreased after the consumption of bottom sugar, and the accumulation of tryptophan is reduced.
Preferably, the method 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 comprising: 1-3g/L of glucose, 5-10g/L of peptone, 5-10g/L of beef extract, 2-5g/L of yeast extract powder, 2-5g/L of sodium chloride and 15-30g/L of agar powder;
(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: 20-40g/L of glucose, 2-5g/L of yeast extract powder, 1-5g/L of ammonium sulfate, 1-5g/L of monopotassium phosphate, 0.5-2g/L of anhydrous magnesium sulfate, 10-30mg/L of ferrous sulfate heptahydrate, 10-30mg/L of manganese sulfate monohydrate, and VH 0.1-0.5mg/L，VB10.5-1mg/L, 1-2ml of mixed solution of trace elements and 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, and supplementing materials to obtain fermentation liquid, wherein the adopted fermentation medium is as follows: 20-40g/L of glucose, 2-5g/L of yeast extract powder, 1-5g/L of ammonium sulfate, 1-5g/L of monopotassium phosphate, 0.5-2g/L of anhydrous magnesium sulfate, 30-60mg/L of ferrous sulfate heptahydrate, 20-40mg/L of manganese sulfate monohydrate, and VH 0.1-0.5mg/L，VB10.5-1mg/L, 1-2ml of mixed solution of trace elements, and culturing and adjusting the fermentation tank by ammonia water and maintaining the pH value to be 6.7-7.0; wherein, after fermenting for 6h, the dissolved oxygen is reduced by 0-50% in a gradient way by reducing the ventilation; after fermenting for 8h, feeding 0.4-1% citric acid aqueous solution; after the depletion of the sugar, the sugar-supplementing rate is reduced by 1 g/L.multidot.h.
Preferably, in the method for increasing the yield of 5-hydroxytryptophan, the component content of the mixed solution of trace elements is molybdic acidAmmonium 0.28mg/L, boric acid 5mg/L, CoCl2·6H2O 1.4mg/L，MnSO4·H2O 0.5mg/L，CuSO4·7H2O 0.5mg/L，ZnSO4·7H2O0.6 mg/L, the above components were weighed to be solid, dissolved in 1L of water, and stored at 4 ℃.
Preferably, in the method for increasing the yield of 5-hydroxytryptophan, the gradient test is performed by the oxygen dissolving reduction strategy, and when the fermentation time is 6-32 h, the oxygen dissolving intervals are respectively controlled to be 30-50%, 20-30%, 10-20% and 0-10%; the rotation speed is 800 rpm.
Preferably, in the method for increasing the yield of 5-hydroxytryptophan, the intermediate feed is 0.4-1% citric acid aqueous solution, the flow addition strategy is 2.7L of fermentation tank preparation culture medium, 300ml of 0.4-1% citric acid aqueous solution is fed from 8 hours of fermentation, and the feeding is completed.
Preferably, the method for improving the yield of 5-hydroxytryptophan is based on the dissolved oxygen gradient experiment, and the fed-batch sugar rate is 5-6 g/L.multidot.h from the logarithmic growth phase (6-8h) of the thalli; 8-10h, 7-8 g/L.h; 10-12h, 9-10 g/L.h; 12-18h, 11-12 g/L.h; 18-24h, 10-11 g/L.h; 24-32, 9-10 g/L.h.
Preferably, the method for increasing the yield of 5-hydroxytryptophan reduces the sugar supplementing rate by 1 g/L.multidot.h based on the sugar feeding strategy.
Preferably, the above method for increasing the production of 5-hydroxytryptophan comprises the following steps: the inoculation amount is 600ml, the fermentation volume is 3L, the culture temperature is 32 ℃, the pH value is 6.8-7.0, the rotation speed is linked with dissolved oxygen, and the fermentation time is 32 h.
Has the advantages that:
the method for improving the yield of the 5-hydroxytryptophan comprises three parts of regulating and controlling the dissolved oxygen sub-amount, feeding citric acid and reducing the sugar supplement rate, performing a gradient test on the optimal mass fraction of the citric acid after determining the optimal dissolved oxygen sub-amount, and finally reducing the sugar supplement rate to reduce the accumulation amount of the tryptophan; the accumulation of tryptophan as an intermediate product is reduced by the oxidative protection of hydroxylase coenzyme tetrahydropterin (BH4) and the enhancement of the reducing power of the catalytic process of the hydroxylase through the regulation of metabolic equilibrium (citrate feeding strategy), and the reduction of the sugar supplement amount.
According to the method, the dissolved oxygen is controlled to be in a sub-proper amount in cells, citric acid is fed to inhibit an EMP (electron brain protein) pathway, an HMP pathway is enhanced, the glucose feeding rate in the fermentation process is reduced to improve the yield of 5-HTP (tetrahydropterin) (BH4), the tryptophan as an intermediate product is reduced, and the problems that the tetrahydropterin (BH4) is oxidized and consumed in the biological fermentation process, the reducing power in the synthesis process is insufficient, and the 5-hydroxytryptophan (5-HTP) yield is influenced by excessive accumulation of tryptophan are solved.
Detailed Description
Example 1
A method 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 pH 6.8-7.0;
(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: 20g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 1.5g/L anhydrous magnesium sulfate, 20mg/L ferrous sulfate heptahydrate, 20mg/L manganese sulfate monohydrate, VH 0.4mg/L，VB10.5mg/L, 2ml of mixed solution of trace elements and 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% seed solution into a fermentation tank, continuously culturing at dissolved oxygen of 40%, rotating speed of 800rpm, and adding no citric acid, 3L system. The fermentation medium is as follows: 25g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 2g/L anhydrous magnesium sulfate, 40mg/L ferrous sulfate heptahydrate, 30mg/L manganese sulfate monohydrate, VH 0.2mg/L，VB10.5mg/L and 2ml of mixed solution of trace elements. In the logarithmic growth phase (6-8h) of the thalli, the fed-batch rate is 5-6 g/L.h; 8-10h, 7-8 g/L.h; 10-12h, 9-10 g/L.h; 12-18h, 11-12 g/L.h; 18-24h, 10-11 g/L.h; 24-32, 9-10 g/L.h.
Coli HTP10 from EThe specific method of artificially modifying coli W3110(ATCC 27325) is as follows: 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 PxylFThe controlled T7RNAP gene can make the cells to generate RNA polymerase T7RNAP by utilizing xylose induction while inactivating lacI protein; 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 the content of the first and second substances,
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 method for improving the yield of 5-hydroxytryptophan is disclosed, which is characterized in that the dissolved oxygen content in the fermentation culture process in example 1 is reduced to 25% based on the method in example 1.
Example 3
A method for improving the yield of 5-hydroxytryptophan is disclosed, which is characterized in that the dissolved oxygen content is reduced to 16% in the fermentation culture process in example 2 based on the method in example 2.
Example 4
A method for increasing the yield of 5-hydroxytryptophan, which is characterized in that the dissolved oxygen content in the fermentation culture process in example 3 is reduced to 5% based on that in example 3.
TABLE 1 comparison of dissolved oxygen with the yields of the examples
As is clear from Table 1, in example 4, the acid production was the best, and the dissolved oxygen was 5%, so that the dissolved oxygen in the subsequent examples was controlled to be about 5%.
Example 5
A method for improving the yield of 5-hydroxytryptophan 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 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 pH 6.8-7.0;
(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: seed culture: inoculating all the activated strains into a seed tank to obtain a seed solution, wherein the seed culture medium is as follows: 20g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 1.5g/L anhydrous magnesium sulfate, 20mg/L ferrous sulfate heptahydrate, 20mg/L manganese sulfate monohydrate, VH 0.4mg/L，VB10.5mg/L, 2ml of mixed solution of trace elements and 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 300mL of citric acid in a flowing manner at the rotating speed of 800rpm from 8h of fermentation time, wherein the mass fraction of the citric acid is 0.4%, adding in the flowing manner in the whole process, and the volume of the base material and the flowing material is fixed to be 3L. The fermentation medium is as follows: 25g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 2g/L anhydrous magnesium sulfate, 40mg/L ferrous sulfate heptahydrate, 30mg/L manganese sulfate monohydrate, VH 0.2mg/L，VB10.5mg/L and 2ml of mixed solution of trace elements. Fermenting for 6-8h, wherein the feeding rate of sugar is 5-6 g/L.h; 8-10h, 7-8 g/L.h；10-12h，9-10g/L·h；12-18h，11-12g/L·h；18-24h，10-11g/L·h；24-32h，9-10g/L·h。
Example 6
A method for improving the yield of 5-hydroxytryptophan is disclosed, wherein the mass fraction of 0.4% of citric acid aqueous solution in example 5 is increased to 0.6% in reference to example 5.
Example 7
A method for improving the yield of 5-hydroxytryptophan is disclosed, wherein the mass fraction of the citric acid aqueous solution in example 6 is increased to 0.8% based on that in example 6.
Example 8
A method for improving the yield of 5-hydroxytryptophan is disclosed, which is characterized in that the mass fraction of 0.8% of the citric acid aqueous solution in example 7 is increased to 1.0% in reference to example 7.
TABLE 2 citric acid mass fraction vs. yield
| Example No.2 | 5 | 6 | 7 | 8 |
| Yield (g/L) | 1.39 | 1.51 | 1.43 | 1.37 |
As can be seen from Table 2, the yield was the highest at a citric acid mass fraction of 0.6%, and the other gradients were not very different.
Example 9
A method for improving the yield of 5-hydroxytryptophan 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 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 pH 6.8-7.0;
(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: 20g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 1.5g/L anhydrous magnesium sulfate, 20mg/L ferrous sulfate heptahydrate, 20mg/L manganese sulfate monohydrate, VH 0.4mg/L，VB10.5mg/L, 2ml of mixed solution of trace elements and 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 300mL of citric acid in a flowing manner at the rotating speed of 800rpm from 8h of fermentation time, wherein the mass fraction of the citric acid is 0.6%, adding in the flowing manner in the whole process, and the volume of the base material and the flowing material is fixed to be 3L. The fermentation medium is as follows: 25g/L glucose, 3g/L yeast extract powder, 3g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 2g/L anhydrous magnesium sulfate, 40mg/L ferrous sulfate heptahydrate, 30mg/L manganese sulfate monohydrate, VH 0.2mg/L，VB10.5mg/L and 2ml of mixed solution of trace elements. After fermentation for 6-8h, the feeding rate of sugar is 5-6 g/L.h; 8-10h, 7-8 g/L.h; 10-12h, 9-10 g/L.h; 12-18h, 11-12 g/L.h; 18-24h, 10-11 g/L.h; 24-32, 9-10 g/L.h.
Example 10
A method for improving the yield of 5-hydroxytryptophan, which is characterized by referring to example 9, wherein the difference is only in the feeding rate, and in example 10, the feeding rate is as follows: 6-8h, 4-5 g/L.h; 8-10h, 6-7 g/L.h; 10-12h, 8-9 g/L.h; 12-18h, 10-11 g/L.h; 18-24h, 9-10 g/L.h; 24-32h, 8-9 g/L.h.
TABLE 3 Feedthrough sugar Rate vs. Tryptophan accumulation and 5-hydroxytryptophan production
| Example No.2 | 9 | 10 |
| Accumulation amount of Tryptophan (g/L) | 2.81 | 1.72 |
| Yield (g/L) | 1.75 | 1.93 |
As can be seen from Table 3, the accumulation of tryptophan can be reduced by controlling the addition of glucose, the conversion efficiency can be improved, and the material can be saved.
In conclusion, the cell reaches a proper dissolved oxygen content by regulating and controlling the dissolved oxygen, the oxidation of BH4 can be effectively avoided, BH4 can participate in the synthesis of hydroxylase more efficiently, meanwhile, the sufficient reducing power in the catalysis process of tryptophan hydroxylase can be ensured by using a metabolic regulation and control technology and adding a proper amount of citric acid in a flow mode, the glucose flow rate is properly reduced, the metabolic pathway balance can be promoted, the sugar-acid conversion rate is improved, and the yield is further improved.
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
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atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480
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gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600
tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660
attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720
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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
<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 (8)
1. A method for improving the yield of 5-hydroxytryptophan, which is characterized by comprising the following steps: the ventilation quantity is reduced in the acid production stage in the middle stage of fermentation, the dissolved oxygen is reduced, citric acid is added in a flowing mode, the sufficient reducing force in the synthesis process of BH4 is guaranteed, the sugar supplementing rate is reduced after the consumption of the base sugar is completed, and the accumulation of tryptophan is reduced.
2. The method for increasing the production 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 comprising: 1-3g/L of glucose, 5-10g/L of peptone, 5-10g/L of beef extract, 2-5g/L of yeast extract powder, 2-5g/L of sodium chloride and 15-30g/L of agar powder;
(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 20-40g/L, yeast extract powder 2-5g/L, ammonium sulfate 1-5g/L, potassium dihydrogen phosphate 1-5g/L, and anhydrous sulfuric acid0.5-2g/L of magnesium, 10-30mg/L of ferrous sulfate heptahydrate, 10-30mg/L of manganese sulfate monohydrate, VH 0.1-0.5mg/L，VB10.5-1mg/L, 1-2ml of mixed solution of trace elements and 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, and supplementing materials to obtain fermentation liquid, wherein the adopted fermentation medium is as follows: 20-40g/L of glucose, 2-5g/L of yeast extract powder, 1-5g/L of ammonium sulfate, 1-5g/L of monopotassium phosphate, 0.5-2g/L of anhydrous magnesium sulfate, 30-60mg/L of ferrous sulfate heptahydrate, 20-40mg/L of manganese sulfate monohydrate, and VH 0.1-0.5mg/L，VB10.5-1mg/L, 1-2ml of mixed solution of trace elements, and culturing and adjusting the fermentation tank by ammonia water and maintaining the pH value to 6.7-7.0; wherein, after fermenting for 6h, the dissolved oxygen is reduced by 0-50% in a gradient way by reducing the ventilation; after fermenting for 8h, feeding 0.4-1% citric acid aqueous solution; after the depletion of the sugar, the sugar-supplementing rate is reduced by 1 g/L.multidot.h.
3. The method for increasing the production of 5-hydroxytryptophan according to claim 2, wherein: the component contents of the mixed solution of the trace elements are 0.28mg/L of ammonium molybdate, 5mg/L of boric acid and CoCl2·6H2O 1.4mg/L，MnSO4·H2O 0.5mg/L，CuSO4·7H2O 0.5mg/L，ZnSO4·7H2O0.6 mg/L, the above components were weighed to be solid, dissolved in 1L of water, and stored at 4 ℃.
4. The method for increasing the production of 5-hydroxytryptophan according to claims 1 and 2, characterized in that: the strategy for reducing the dissolved oxygen is to perform a gradient test, and when the fermentation time is from 6h to the end of fermentation (32h), the dissolved oxygen intervals are respectively regulated to 30-50%, 20-30%, 10-20% and 0-10%; the rotation speed is 800 rpm.
5. The method for increasing the production of 5-hydroxytryptophan according to claims 1 and 2, characterized in that: the intermediate feeding is 0.4-1% citric acid aqueous solution, the feeding strategy is 2.7L fermentation tank preparation culture medium, 300ml of 0.4-1% citric acid aqueous solution is fed from 8h of fermentation, and the feeding is completed after the fermentation.
6. The method for increasing the production of 5-hydroxytryptophan according to claims 1 and 2, characterized in that: on the basis of the gradient experiment, a fed-batch strategy is carried out from the logarithmic growth phase of the thalli, and the fed-batch speed is 5-6 g/L.h; 8-10h, 7-8 g/L.h; 10-12h, 9-10 g/L.h; 12-18h, 11-12 g/L.h; 18-24h, 10-11 g/L.h; 24-32, 9-10 g/L.h.
7. The method for increasing the production of 5-hydroxytryptophan according to claims 1 and 2, characterized in that: the rate of sugar supplementation was reduced by 1 g/L.multidot.h on the basis of the fed-sugar strategy.
8. The method for increasing the production of 5-hydroxytryptophan according to claim 2, wherein: the basic culture conditions of the fermentation tank are as follows: the inoculation amount is 600ml, the fermentation volume is 3L, the culture temperature is 32 ℃, the pH value is 6.8-7.0, the rotation speed is linked with dissolved oxygen, and the fermentation time is 32 h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| CN113981015A (en) * | 2021-12-17 | 2022-01-28 | 四川龙蟒福生科技有限责任公司 | Process method for reducing S-ABA impurities in fermentation production |
| CN114134186A (en) * | 2021-11-04 | 2022-03-04 | 河北维达康生物科技有限公司 | Method for synthesizing 5-hydroxy beta-indolyl alanine by using glucose as substrate through biological method |
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|---|---|---|---|---|
| US20150037849A1 (en) * | 2012-02-29 | 2015-02-05 | Danmarks Tekniske Universitet | Microorganisms for the production of 5-hydroxytryptophan |
| US20170240939A1 (en) * | 2014-05-16 | 2017-08-24 | University Of Georgia Research Foundation, Inc. | Microbial approach for the production of 5-hydroxytryptophan |
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| US20150037849A1 (en) * | 2012-02-29 | 2015-02-05 | Danmarks Tekniske Universitet | Microorganisms for the production of 5-hydroxytryptophan |
| US20170240939A1 (en) * | 2014-05-16 | 2017-08-24 | University Of Georgia Research Foundation, Inc. | Microbial approach for the production of 5-hydroxytryptophan |
| CN112522222A (en) * | 2020-12-24 | 2021-03-19 | 保定保利瑞合生物科技有限公司 | Novel tryptophan hydroxylase mutant and application thereof |
| CN112708589A (en) * | 2021-01-19 | 2021-04-27 | 南京工业大学 | Genetically engineered bacterium, construction method thereof and application of genetically engineered bacterium in fermentation production of 5-hydroxytryptophan |
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| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| CN114134186A (en) * | 2021-11-04 | 2022-03-04 | 河北维达康生物科技有限公司 | Method for synthesizing 5-hydroxy beta-indolyl alanine by using glucose as substrate through biological method |
| CN113981015A (en) * | 2021-12-17 | 2022-01-28 | 四川龙蟒福生科技有限责任公司 | Process method for reducing S-ABA impurities in fermentation production |
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