CN117363549A - Genetically engineered bacterium for producing 5-hydroxytryptamine and construction method and application thereof - Google Patents

Genetically engineered bacterium for producing 5-hydroxytryptamine and construction method and application thereof Download PDF

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CN117363549A
CN117363549A CN202311200115.0A CN202311200115A CN117363549A CN 117363549 A CN117363549 A CN 117363549A CN 202311200115 A CN202311200115 A CN 202311200115A CN 117363549 A CN117363549 A CN 117363549A
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gene
hydroxytryptamine
producing
genetically engineered
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徐庆阳
刘韪玮
时唐恩
余子辰
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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Abstract

The invention provides a genetic engineering bacterium for producing 5-hydroxytryptamine, a construction method and application thereof, wherein the genetic engineering bacterium strengthens the tryptophan synthesis pathway of escherichia coli by utilizing a metabolic engineering means on the basis of the existing bacterial strain HT03 (CN 116590210A), eliminates the feedback inhibition of key enzymes in the tryptophan synthesis pathway, introduces related exogenous genes for expression, knocks out essential genes for bacterial strain growth and supplements back to plasmids to improve plasmid stability; the strain can be used for efficiently producing 5-hydroxytryptamine by taking glucose as a substrate, and has good industrial application value.

Description

Genetically engineered bacterium for producing 5-hydroxytryptamine and construction method and application thereof
Technical Field
The invention relates to the field of biotechnology production, in particular to a genetically engineered bacterium for producing 5-hydroxytryptamine, and a construction method and application thereof.
Background
5-hydroxytryptamine (5-HT), also known as serotonin (serotonin), is an indole derivative metabolized by L-tryptophan, can be used as neurotransmitter for regulating emotion, treating insomnia, scavenging harmful free radicals, etc., and has wide application in industries such as medicine, health care products, etc.
At present, the 5-hydroxytryptamine is expensive to produce, the steps are complex, the extraction yield is low, and the market price of the 5-HT is extremely high, so that a method which is low in cost, convenient to operate, green and pollution-free is urgently needed for synthesizing the 5-HT. In recent years, the development of synthetic biology has been rapid, and the production of various organic substances by microbial fermentation has been largely used in industrial production, which also provides a new idea of synthesizing 5-HT by using microorganisms.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a genetically engineered bacterium for producing 5-hydroxytryptamine.
The invention aims to provide a construction method of the genetically engineered bacterium for producing 5-hydroxytryptamine.
The invention aims to provide the application of the genetically engineered bacterium for producing 5-hydroxytryptamine.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the genetically engineered bacterium for producing 5-hydroxytryptamine is a strain HT12, is obtained by modifying a chassis strain escherichia coli HT03 (being a strain HT03 in CN 116590210A) by a metabolic engineering modification method, and specifically comprises the following steps: knocking out trpR gene on chassis strain HT03 genome; knocking out the serA gene; use of P on trpLE Gene trc trpE with promoter over-expression to release feedback inhibition fbr A gene; in ycgH falseGene locus usage P trc aroG with over-expression of promoter to release feedback inhibition fbr A gene; use of P at ilvG site trc The promoter overexpresses the gdh gene from bacillus subtilis; the target strain HT12 was obtained by introducing the 5-hydroxytryptamine synthetic plasmid pSTV28-HT12.
The gene engineering bacteria for producing 5-hydroxytryptamine knock out the gene trpR for encoding tryptophan repressor protein on genome to prevent the gene trpR from repressing tryptophan synthesis; knocking out a gene serA encoding 3-phosphoglycerate dehydrogenase on a genome; genomic application of P trc Promoter-initiated anthranilate synthase trpE with feedback inhibition released fbr Replaces the original trpLE gene to accelerate tryptophan accumulation; integration of the P-amino acid sequence at the ycgH locus trc Promoter-initiated 3-deoxy-D-arabinoheptulonic acid-7-phosphate synthase mutant gene aroG for relieving feedback inhibition fbr Accelerating the accumulation of 3-deoxy-D-arabinoheptulonic acid-7-phosphate to provide a precursor for tryptophan synthesis; integration of P at genomic ilvG locus trc The promoter-initiated bacillus subtilis-derived glucose dehydrogenase gdh provides NADPH to maintain the cell redox balance; introducing a tryptophan hydroxylation-decarboxylation-tetrahydropterin synthetic regeneration plasmid pSTV28-HT12 carrying a serine supplementing pathway, tryptophan hydroxylation, decarboxylation pathway and synthetic and regeneration pathway of coenzyme tetrahydropterin with a E.coli-derived 3-phosphoglycerate dehydrogenase serA gene, a human-derived hydroxylase chopping mutant TM2 gene, an Aspergillus niger-derived TDC gene, a Bacillus subtilis-derived mtrA and human-derived PTPS, SPR, PCD and DHPR genes, wherein serA consists of P serA Promoter-directed expression, TM2 and TDC by P T7 Promoter-directed expression, mtrA, PTPS and SPR are expressed from P trc Promoter-directed expression, PCD and DHPR are expressed from P T7 The promoter directs expression.
Preferably, the genetically engineered bacterium for producing 5-hydroxytryptamine is disclosed as the chassis strain E.coli HT03, and the publication number is CN116590210A.
Preferably, the trpR gene (encoding tryptophan repressor protein) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from E.coli W3110, and the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO. 1.
Preferably, the serA gene (encoding 3-phosphoglycerate dehydrogenase) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from escherichia coli W3110, and the nucleotide sequence of the serA gene is shown as a sequence table SEQ ID NO. 9.
Preferably, the genetically engineered bacterium for producing 5-hydroxytryptamine, the P trc The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 4.
Preferably, the above genetically engineered bacterium for producing 5-hydroxytryptamine, wherein the trpLE gene (encoding tryptophan operon leader peptide and o-methylbenzoate synthase) is derived from E.coli W3110, wherein the nucleotide sequence of the tryptophan operon leader peptide trpL is shown in a sequence table SEQ ID NO.2, and the nucleotide sequence of the o-aminobenzoate synthase trpE is shown in a sequence table SEQ ID NO. 3.
Preferably, the above genetically engineered bacterium for producing 5-hydroxytryptamine, the trpE fbr The gene (encoding o-methylbenzoate synthase for releasing feedback inhibition, wherein arginine encoding 63 st amino acid residue of the original trpE gene is mutated into valine, and cysteine encoding 465 st amino acid residue is mutated into tyrosine) is derived from E.coli W3110, and the nucleotide sequence of the gene is shown as SEQ ID NO.5 of a sequence table.
Preferably, the above genetically engineered bacterium for producing 5-hydroxytryptamine, the aroG fbr The gene (encoding 3-deoxy-D-arabinoheptonic acid-7-phosphate synthase for releasing feedback inhibition, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 6) is obtained by mutating serine encoding 221 th amino acid residue into phenylalanine) from E.coliW3110, and the nucleotide sequence of which is shown in the sequence table SEQ ID NO. 7.
Preferably, the gene engineering bacteria for producing 5-hydroxytryptamine is derived from Bacillus subtilis, and the nucleotide sequence of the gene gdh (encoding glucose dehydrogenase) is shown as a sequence table SEQ ID NO. 8.
Preferably, the plasmid pSTV28-HT12 has a nucleotide sequence shown in a sequence table SEQ ID NO.17 of the genetically engineered bacterium for producing 5-hydroxytryptamine.
The plasmid pSTV28-HT12 was expressed using ClonExpressMultiS One Step Cloning Kit the kit is constructed by taking a plasmid pSTV28 as a linear vector, wherein the nucleotide of the plasmid pSTV28 is shown as a sequence table SEQ ID NO.12, the plasmid pSTV28 is a medium copy plasmid and has a P15A replication initiation site (ori), the toxicity of exogenous gene expression to thalli can be reduced, and the plasmid pSTV28 is cloned from P respectively T7 The promoter guides the expressed human source 2 tryptophan hydroxylase short cut mutant TM2 gene and aspergillus niger source tryptophan decarboxylase TDC gene, which are composed of P serA Promoter-directed Escherichia coli-derived 3-phosphoglycerate dehydrogenase serA gene, consisting of P trc Promoter-directed expression of bacillus subtilis-derived GTP cyclohydrolase mtrA gene, human-derived 6-pyruvoyl tetrahydrobiopterin synthase PTPS gene and guanterin reductase SPR gene and methods of using the same T7 The promoter directs the expression of the human pterin-4. Alpha. -methanol ammonia dehydratase PCD gene and dihydropterin reductase DHPR gene to construct plasmid pSTV28-HT12.
In conclusion, the TM2 and TDC, serA, mtrA, PTPS, SPR, PCD, DHPR genes are cloned on a plasmid vector pSTV28 for replication and expression, and the recombinant plasmid finally constructed and obtained carries genes consisting of tryptophan hydroxylase mutant TM2, tryptophan decarboxylase TDC, 3-phosphoglycerate dehydrogenase serA, GTP cyclohydrolase mtrA, 6-pyruvoyl tetrahydrobiopterin synthase PTPS, guanine pterin reductase SPR, pterin-4 alpha-methanol ammonia dehydratase PCD and dihydropterin reductase DHPR, wherein the genes TM2 and TDC consist of P T7 Promoter-driven expression, serA is expressed by P serA Promoter driven expression, mtrA, PTPS and SPR are expressed by P trc Promoter driven expression, PCD and DHPR are expressed from P T7 The promoter drives expression.
Preferably, the genetically engineered bacterium for producing 5-hydroxytryptamine, the P T7 The promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 18.
Preferably, the nucleotide sequence of the TM2 gene for encoding the humanized 2 tryptophan hydroxylase short-cut mutant of the genetically engineered bacterium for producing 5-hydroxytryptamine is shown as a sequence table SEQ ID NO. 10; the tryptophan hydroxylase mutant gene TM2 is derived from homo sapiens and human-derived tryptophan hydroxylase type 2 TPH2 (humanTPH 2, EC: 1.14.16.4), and is obtained by deleting 145 amino acid residues at the N-terminal end and 30 amino acid residues at the C-terminal end of the human-derived tryptophan hydroxylase type 2 TPH2, mutating the amino acid residue at the 2 nd position from glutamic acid to lysine, mutating the amino acid residue at the 97 th position from asparagine to isoleucine, and mutating the amino acid residue at the 99 th position from proline to cysteine.
Preferably, the TDC gene (encoding tryptophan decarboxylase) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from Aspergillus niger, and the nucleotide sequence of the TDC gene is shown as a nucleotide sequence shown as SEQ ID NO.11 of a sequence table.
Preferably, the mtrA gene (GTP cyclohydrolase I gene) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from bacillus subtilis and is responsible for encoding GTP cyclohydrolase I (GCHI), and the nucleotide sequence of the mtrA gene is shown as a sequence table SEQ ID NO. 12.
Preferably, the PTPS gene (6-pyruvoyl tetrahydrobiopterin synthase gene) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from Chinesemese and is responsible for encoding 6-pyruvoyl tetrahydrobiopterin synthase (PTPS), and the nucleotide sequence of the PTPS gene is shown as SEQ ID No.13 of a sequence table.
Preferably, the above genetically engineered bacterium for producing 5-hydroxytryptamine, wherein the SPR gene (the pterin reductase gene) is derived from homo sapiens and is responsible for encoding the pterin reductase (SPR), and the nucleotide sequence of the SPR gene is shown in a sequence table SEQ ID No. 14.
Preferably, the PCD gene (pterin-4 alpha-methanol ammonia dehydratase gene) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from Chinesemese and is responsible for encoding the pterin-4 alpha-methanol ammonia dehydratase (PCD), and the nucleotide sequence of the PCD gene is shown in a sequence table SEQ ID NO. 15.
Preferably, the DHPR gene (dihydropterin reductase gene) of the genetically engineered bacterium for producing 5-hydroxytryptamine is derived from Chinesemese and is responsible for encoding the dihydropterin reductase (DHPR), and the nucleotide sequence of the DHPR gene is shown as a sequence table SEQ ID NO. 16.
The gene engineering bacteria for producing 5-hydroxytryptamine are artificially synthesized after codon optimization of TM2 gene, aspergillus niger TDC gene, bacillus subtilis mtrA gene, human PTPS, SPR, PCD and DHPR gene for encoding human tryptophan hydroxylase type 2 short-cut mutant.
The construction method of the genetically engineered bacterium for producing 5-hydroxytryptamine is characterized by comprising the steps of preparing the existing bacterial strain
The specific steps of the directional transformation based on HT03 (strain HT03 in CN 116590210A) are as follows:
(1) Knocking out trpR genes on genome of chassis strain HT03 to obtain strain HT04;
(2) Using strain HT04 as chassis strain, P was used on trpLE gene trc trpE with promoter over-expression to release feedback inhibition fbr Genes to obtain a strain HT05;
(3) Using strain HT05 as chassis strain, P was used at the ycgH pseudogene locus trc aroG with over-expression of promoter to release feedback inhibition fbr Genes give strain HT06;
(4) Using strain HT06 as chassis strain and P at ilvG site trc The promoter overexpresses the gdh gene from bacillus subtilis to obtain a strain HT07;
(5) Taking the strain HT07 as a chassis strain, and knocking out a serA gene to obtain a strain HT08;
(6) Taking the strain HT08 as a chassis strain, and introducing a 5-hydroxytryptamine synthetic plasmid pSTV28-HT12 to obtain a strain HT12, wherein the strain HT12 is the target strain after the transformation is successful.
The genetically engineered bacterium for producing 5-hydroxytryptamine is applied to fermenting and producing 5-hydroxytryptamine.
Preferably, the application of the genetically engineered bacteria for producing 5-hydroxytryptamine takes glucose as a substrate, and the 5-hydroxytryptamine is synthesized by fermentation (using a mechanical stirring type fermentation tank), and the specific fermentation steps of the fermentation tank are as follows:
(1) Seed culture: the culture temperature is 37 ℃, the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring rotation speed or ventilation quantity, and the OD is considered 600nm Up to 20 is a seed culture solution maturation mark;
(2) Fermentation culture: the inoculation amount is 20%, the culture temperature is 37 ℃, the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the fermentation dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring speed or ventilation amount, the glucose concentration in the tank is controlled to be less than or equal to 1g/L by feeding 80% glucose solution, and the fermentation period is 24 hours.
Preferably, the application of the genetically engineered bacterium for producing 5-hydroxytryptamine is that a seed culture medium adopted in the seed culture is as follows: 30g/L of glucose, 15mg/L of chloramphenicol, 4g/L of yeast extract, 2g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 3g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 2g/L of glutamic acid, 0.5g/L of methionine and the balance of water.
Preferably, the application of the genetically engineered bacteria for producing 5-hydroxytryptamine is that a fermentation medium adopted in the fermentation culture is as follows: 20g/L of glucose, 10g/L of xylose, 15mg/L of chloramphenicol, 2g/L of yeast extract, 1g/L of peptone, 1g/L of citric acid, 1g/L of ammonium sulfate, 4g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 40mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, 2g/L of glutamine, 1g/L of choline chloride and the balance of water.
The above culture medium can be prepared by standard method.
The beneficial effects are that:
the genetically engineered bacterium for producing 5-hydroxytryptamine is genetically engineered bacterium obtained by modifying a metabolic synthesis way of 5-hydroxytryptamine, can efficiently produce 5-hydroxytryptamine by taking glucose as a carbon source, is the highest yield of producing 5-hydroxytryptamine from the head by taking glucose as a current report, and can produce 5-hydroxytryptamine for 24 hours to reach 3.67g/L at most; the strain has high production rate, short fermentation period and higher industrial application value; the construction method is a directional and rational strain construction method, is more efficient and convenient compared with the traditional mutagenesis method, has strong operability and has good application prospect.
Drawings
FIG. 1 shows a map of plasmid pSTV28-HT12.
FIG. 2 is a flow chart of the metabolic pathway for glucose de novo synthesis of 5-HT.
FIG. 3 is a graph showing the fermentation process curve and yield histogram of strain HT12 in a 5L fermenter.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.
The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.
Strain e.coli w3110, accession No. ATCC 273250, referred to in the examples; the chassis strain E.coli HT03 was engineered as strain HT03 described in CN116590210A.
Example 1
1. Method for gene editing
Gene editing methods employed are described in the literature (Li Y, lin Z, huang C, et al, metabolic Engineering of Escherichia coli using CRISPR-Cas9 mediated genome editing.Metabolic Engineering,2015, 31:13-21.). Engineering plasmids pREDCas9 and pGRB related by the method, wherein pREDCas9 carries an elimination system of a gRNA expression plasmid pGRB, a Red recombination system of lambda phage, a Cas9 protein expression system and the resistance of Qamycin (working concentration: 100 mg/L); pGRB takes pUC18 as a framework and comprises the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, and ampicillin resistance (working concentration: 100 mg/L). The terms referred to in the following examples 2-4 are all explained in this article.
2. The primers used in the strain construction process are shown in Table 1
TABLE 1 primers used in the construction of strains
Example 2
This example is intended to illustrate the procedure for knocking out the trpR gene in the genome.
The method comprises the following specific steps:
(1) taking the E.coli W3110 genome as a template, and respectively carrying out PCR amplification by using primers trpR-1 and trpR-2, trpR-3 and trpR-4 to obtain upstream and downstream homology arms; overlapping PCR (polymerase chain reaction) is carried out by using the recovered upstream and downstream homology arms as templates and using primers trpR-1 and trpR-4 to obtain a patch segment required by knocking out the gene trpR;
(2) and (3) carrying out ligation on the DNA fragments obtained by annealing the primers PGRB-trpR-1 and PGRB-trpR-2 with a plasmid pGRB to construct pGRB-trpR. Transferring the plasmid into DH5 alpha conversion competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-trpR;
(3) and (3) electrotransferring the DeltatrpR integrated fragment obtained in the step (1) and the plasmid pGRB-trpR gene fragment into electrotransferring competence of HT03/pRed-Cas9, and screening positive transformants to obtain the strain E.coli HT04.
Example 3
This example is intended to illustrate the procedure in which the genomic serA gene is knocked out.
The method comprises the following specific steps:
(1) taking E.coli W3110 genome as a template, and respectively carrying out PCR amplification by primers serA-1 and serA-2, serA-3 and serA-4 to obtain upstream and downstream homology arms; overlapping PCR (polymerase chain reaction) is carried out by using the recycled upstream and downstream homology arms as templates and primers serA-1 and serA-4 to obtain a patch segment required by knocking out the gene serA;
(2) and (3) carrying out ligation on the DNA fragments prepared by annealing the primers PGRB-serA-1 and PGRB-serA-2 with a plasmid pGRB line, so as to construct pGRB-serA. Transferring the cells into DH5 alpha conversion competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-serA;
(3) and (3) electrotransferring the delta serA integration fragment obtained in the step (1) and the plasmid pGRB-serA gene fragment into electrotransferring competence of HT04/pRed-Cas9, and screening positive transformants to obtain the strain E.coli HT05.
Example 4
This example is intended to illustrate the use of P at the trpLE gene locus trc The promoter controls trpE fbr And (3) a step of gene overexpression.
The method comprises the following specific steps:
(1) PCR is carried out by taking the E.coli W3110 genome as a template and primers trc-trpE-1 and trpE-2, trpE-3 and trc-trpE-4 respectively to obtain upper and lower gene fragments of the trpE gene with the feedback inhibition released; the recovered upper and lower gene fragments are used as templates, and the primers trc-trpE-1 and trc-trpE-4 are used for carrying out overlapped PCR to obtain the gene trpE fbr Fragments;
(2) and (3) connecting DNA fragments prepared by annealing the primers PGRB-trpE-1 and PGRB-trpE-2 with a plasmid pGRB in a linear manner, and constructing pGRB-trpE. Transferring the cells into DH5 alpha transformation competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-trpE;
(3) the gene trpE obtained in the steps (1) and (2) is subjected to a gene amplification fbr The fragment and the plasmid pGRB-trpE gene fragment are electrically transferred into the electrotransfer competence of HT05/pRed-Cas9 together, positive transformants are screened, and the strain E.coli HT06 is obtained.
Example 5
This example is intended to illustrate the use of P at the ycgH pseudogene locus trc Promoter control aroG fbr And (3) a step of gene overexpression.
The method comprises the following specific steps:
(1) the E.coli W3110 genome is used as a template, and the upstream homology arm, the downstream homology arm and the upper and lower gene fragments of the target gene are obtained by PCR amplification respectively using ycgH-1, ycgH-trc-2, ycgH-trc-7, ycgH-8 and trc-aroG-3, aroG-4, aroG-5 and trc-aroG-6 as primers; and then using the PCR primer as a template to obtain delta ycgH-P through overlapping PCR trc -aroG fbr Gene fragment consisting of ycgH upstream homology arm, P trc -aroG fbr The target gene and the downstream homology arm of ycgH.
(2) Constructing a DNA fragment containing a target sequence used by pGRB-ycgH through a PCR annealing program by taking pGRB-ycgH-S and pGRB-ycgH-A as primers, transforming the DNA fragment into DH5 alpha transformation competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-ycgH.
(3) The delta ycgH-P obtained in the steps (1) and (2) is reacted with trc -aroG fbr The gene fragment and pGRB-ycgH plasmid are simultaneously transferred into competent cells of the strain E.coli HT06/Cas9 in an electrotransformation mode, and the strain E.coli is finally obtained HT07。
Example 6
This example is intended to illustrate the use of P at the ilvG pseudogene locus trc And a step of controlling overexpression of the gdh gene by the promoter.
The method comprises the following specific steps:
(1) PCR amplification is carried out by taking E.coli W3110 genome as a template and respectively taking ilvG-1, ilvG-trc-2, ilvG-trc-5 and ilvG-6 as primers to obtain an upstream homology arm and a downstream homology arm; PCR (polymerase chain reaction) amplification is carried out by taking a bacillus subtilis genome as a template and trc-gdh-3 and trc-gdh-4 as primers to obtain a target gene fragment; then using the three as templates to obtain delta ilvG-P through overlapping PCR trc -a gdh gene fragment consisting of an ilvG upstream homology arm, a Ptrc-gdh gene of interest and an ilvG downstream homology arm.
(2) Ext> constructingext> aext> DNAext> fragmentext> containingext> aext> targetext> sequenceext> forext> pGRBext> -ext> ilvGext> byext> usingext> pGRBext> -ext> ilvGext> -ext> Sext> andext> pGRBext> -ext> ilvGext> -ext> Aext> asext> primersext> throughext> aext> PCRext> annealingext> programext>,ext> transformingext> theext> DNAext> fragmentext> intoext> DHext> 5ext> alphaext> transformedext> competentext> cellsext>,ext> screeningext> toext> obtainext> positiveext> transformantsext>,ext> andext> extractingext> plasmidext> pGRBext> -ext> ilvGext>.ext>
(3) And (3) transferring the delta ilvG-Ptrc-gdh gene fragment obtained in the step (1) and the step (2) and pGRB-ilvG plasmid into competent cells of the strain E.coli HT07/Cas9 in an electric conversion mode, and finally obtaining the strain E.coli HT08.
Example 7
This example is directed to the construction and expression procedure of the 5-hydroxytryptamine synthetic plasmid pSTV28-HT12.
The method comprises the following specific steps:
(1) the plasmid pSTV28-HT11 (see CN 116590210A) was used as a template and the primers line-1 and line-2 were used for PCR amplification to obtain pSTV28-HT11 linear vector; the Escherichia coli W3110 genome is used as a template, and primer pairs PserA-serA-1 and PserA-serA-2 are used for PCR amplification to obtain a serA gene fragment;
(2) the plasmid pSTV28-HT12 was constructed by ligating pSTV28-HT11 linear vector with serA upper gene fragment and serA lower gene fragment by homologous recombination using ClonExpress MultiS One Step Cloning Kit kit (Vazyme Biotech, nanjing, china), transforming it into DH 5. Alpha. Transformed competent cells, screening to obtain positive transformants, and extracting the plasmid pSTV28-HT12 (see FIG. 1).
(3) The pSTV28-HT12 plasmid obtained in the step (2) is electrotransformed into E.coli HT08 electrotransformation competence, positive transformant is obtained through screening, and the transformant is named as E.coli HT12.
Example 8
The E.coli HT12 strain is used as a 5-hydroxytryptamine production strain (shown in figure 2), and the embodiment aims at describing a production method of the 5-hydroxytryptamine by using the strain, and the specific culture steps are as follows:
inoculating preserved strain at-80deg.C onto chloramphenicol resistance activating slant, culturing at 37deg.C for 15 hr, and passaging for 2 times; eluting activated thallus on the inclined plane with sterilized distilled water, transferring to 5L mechanical stirring fermenter, culturing at 37deg.C and initial stirring speed of 200rpm, maintaining culture pH at 6.7+ -0.2 by automatic feeding 25% ammonia water solution, maintaining dissolved oxygen value of culture medium at 30% by adjusting stirring speed or ventilation, and viewing OD 600nm And 20 is reached as a seed culture solution maturation mark. After the seed solution is mature, 400mL of seed culture is reserved, and a fresh fermentation culture medium is immediately added, so that the final volume of fermentation is 2L, fermentation culture is started, the culture temperature is 37 ℃, the pH value of the culture is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, and the fermentation dissolved oxygen value of the culture medium is maintained at 30% by adjusting the stirring rotation speed or ventilation quantity. The concentration of glucose in the tank is controlled to be less than or equal to 1g/L by feeding 80% glucose solution, and the fermentation period is 24 hours.
The adopted slant culture medium comprises the following components: glucose 1g/L, peptone 5g/L, potassium dihydrogen phosphate 0.5g/L, yeast extract 5g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, agar powder 25g/L, and water balance, sterilizing at 121deg.C for 20min, and packaging into test tubes.
The components of the seed culture medium are as follows: glucose 30g/L, chloramphenicol 15mg/L, yeast extract 4g/L, peptone 2g/L, citric acid 2g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate heptahydrate 2g/L, glutamic acid 2g/L, and methionine 0.5g/L.
The components of the fermentation medium are as follows: 20g/L of glucose, 10g/L of xylose, 15mg/L of chloramphenicol, 2g/L of yeast extract, 1g/L of peptone, 1g/L of citric acid, 1g/L of ammonium sulfate, 4g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 40mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, 2g/L of glutamine, 1g/L of choline chloride and the balance of water.
Example 9
This example was designed to verify the 5-hydroxytryptamine productivity of strain E.coli HT12 using the culture method and medium of example 6, the results are shown in Table 2 below:
TABLE 2
As shown in FIG. 3, strain E.coli HT12 produced 5-hydroxytryptamine in a yield of up to 3.67g/L after 24 hours in a 5L fermenter.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.

Claims (7)

1. A genetically engineered bacterium for producing 5-hydroxytryptamine is characterized in that: the strain HT12 is obtained by modifying chassis strain escherichia coli HT03 by using a metabolic engineering modification method, and specifically comprises the following steps: knocking out trpR gene on chassis strain HT03 genome; knocking out the serA gene; use of P on trpLE Gene trc trpE with promoter over-expression to release feedback inhibition fbr A gene; use of P at the ycgH pseudogene locus trc aroG with over-expression of promoter to release feedback inhibition fbr A gene; use of P at ilvG site trc The promoter overexpresses the gdh gene from bacillus subtilis; the target strain HT12 was obtained by introducing the 5-hydroxytryptamine synthetic plasmid pSTV28-HT12.
2. The genetically engineered bacterium for producing 5-hydroxytryptamine of claim 1, wherein: the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO.1
The nucleotide sequence of the serA gene is shown in a sequence table SEQ ID NO. 9;
the P is trc The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 4;
the nucleotide sequence of tryptophan operon leader peptide trpL in the trpLE gene is shown in a sequence table SEQ ID NO.2, and the nucleotide sequence of anthranilate synthase trpE is shown in a sequence table SEQ ID NO. 3;
the trpE fbr The nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 5;
the aroG fbr The nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 7;
the nucleotide sequence of the gdh gene is shown in a sequence table SEQ ID NO. 8;
the nucleotide sequence of the plasmid pSTV28-HT12 is shown in a sequence table SEQ ID NO. 17.
3. The method for constructing genetically engineered bacteria for producing 5-hydroxytryptamine according to claim 1, wherein the method is characterized in that: the method comprises the following specific steps:
(1) Knocking out trpR genes on genome of chassis strain HT03 to obtain strain HT04;
(2) Using strain HT04 as chassis strain, P was used on trpLE gene trc trpE with promoter over-expression to release feedback inhibition fbr Genes to obtain a strain HT05;
(3) Using strain HT05 as chassis strain, P was used at the ycgH pseudogene locus trc aroG with over-expression of promoter to release feedback inhibition fbr Genes give strain HT06;
(4) Using strain HT06 as chassis strain and P at ilvG site trc The promoter overexpresses the gdh gene from bacillus subtilis to obtain a strain HT07;
(5) Taking the strain HT07 as a chassis strain, and knocking out a serA gene to obtain a strain HT08;
(6) Taking the strain HT08 as a chassis strain, and introducing a 5-hydroxytryptamine synthetic plasmid pSTV28-HT12 to obtain a strain HT12, wherein the strain HT12 is the target strain after the transformation is successful.
4. The use of the genetically engineered bacterium for producing 5-hydroxytryptamine of claim 1 in the fermentative production of 5-hydroxytryptamine.
5. The use of the genetically engineered bacterium for producing 5-hydroxytryptamine of claim 4, wherein: glucose is used as a substrate, and 5-hydroxytryptamine is synthesized by fermentation, and the fermentation steps of a specific fermentation tank are as follows:
(1) Seed culture: the culture temperature is 37 ℃, the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring rotation speed or ventilation quantity, and the OD is considered 600nm Up to 20 is a seed culture solution maturation mark;
(2) Fermentation culture: the inoculation amount is 20%, the culture temperature is 37 ℃, the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the fermentation dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring speed or ventilation amount, the glucose concentration in the tank is controlled to be less than or equal to 1g/L by feeding 80% glucose solution, and the fermentation period is 24 hours.
6. The use of the genetically engineered bacterium for producing 5-hydroxytryptamine of claim 5, wherein: the seed culture medium adopted in the seed culture is as follows: 30g/L of glucose, 15mg/L of chloramphenicol, 4g/L of yeast extract, 2g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 3g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 2g/L of glutamic acid, 0.5g/L of methionine and the balance of water.
7. The use of the genetically engineered bacterium for producing 5-hydroxytryptamine of claim 5, wherein: the fermentation culture medium adopted in the fermentation culture is as follows: 20g/L of glucose, 10g/L of xylose, 15mg/L of chloramphenicol, 2g/L of yeast extract, 1g/L of peptone, 1g/L of citric acid, 1g/L of ammonium sulfate, 4g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 40mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, 2g/L of glutamine, 1g/L of choline chloride and the balance of water.
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