CN117866866A - Recombinant escherichia coli for producing exendin by utilizing fumaric acid as well as construction method and application thereof - Google Patents
Recombinant escherichia coli for producing exendin by utilizing fumaric acid as well as construction method and application thereof Download PDFInfo
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 51
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 title claims abstract description 38
- 239000001530 fumaric acid Substances 0.000 title claims abstract description 19
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title description 7
- WQXNXVUDBPYKBA-YFKPBYRVSA-N ectoine Chemical compound CC1=[NH+][C@H](C([O-])=O)CCN1 WQXNXVUDBPYKBA-YFKPBYRVSA-N 0.000 claims abstract description 73
- 238000000855 fermentation Methods 0.000 claims abstract description 72
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention uses metabolic engineering and synthetic biology technology, and based on the halophilic bacteria ectoin biosynthesis way, uses E.coli BL21 (DE 3) as a chassis expression cell, expresses ectoin synthesis gene cluster ectA, ectB, ectC from halophilic bacteria, and realizes the heterologous biosynthesis of ectoin; the recombinant escherichia coli capable of efficiently biosynthesizing the ectoin from fumaric acid is finally obtained by strengthening and expressing an aspartokinase gene aspA, an aspartokinase gene ask and an aspartyl semialdehyde dehydrogenase gene asd which are derived from escherichia coli in the original recombinant ectoin. The genetically engineered bacterium has excellent ectoine production performance, glucose and ammonium fumarate are fed continuously for fermentation, the yield can reach 30.8g/L, the production efficiency is 0.616g/L/h, the specific yield is 0.6864g/g DCW, the glucose conversion rate is 0.201g/g, and the fumaric acid conversion rate is 23.7%. The gene engineering strain of the ectoin has good industrial application prospect.
Description
Technical Field
The invention particularly relates to recombinant escherichia coli for producing ectoin by utilizing fumaric acid, and a construction method and application thereof, belonging to the fields of genetic engineering and biotechnology.
Background
Excreta (Ectoine) chemical name is 1,4,5, 6-tetrahydro-2-methyl 4-pyrimidinecarboxylic acid. Trade name: ricketiness. The heterocyclic amino acid derivative has the characteristics of strong polarity and easy dissolution in water, and can stabilize the cell expansion pressure without affecting the normal physiological functions of cells, thereby maintaining the osmotic balance of cells and the environment. As the ectoin can form a hydration layer on the surface of biomacromolecules such as protein and the like, the ectoin can be used as a biological protective agent for cells and macromolecular substances, and can relieve and alleviate the toxic and harmful effects of harmful environments such as high permeability, high temperature, freeze thawing, drying, radiation, chemical reagents and the like on the protein, nucleic acid, biological film and whole cells. Since escitalopram is the best compound for protecting biological cells and macromolecular substances among all currently known compatible solutes, it has been widely used in cosmetics, biotechnology, pharmaceutical industry and other fields in recent years. How to efficiently produce and prepare the ectoin becomes a current research hot spot.
Ikeduo factor (Ectoine)
The main production methods of the ectoin mainly comprise a microbial fermentation method and a living cell transformation method. The traditional microbial fermentation method utilizes halophilic microorganisms to produce the ectoin by adopting a milking process under the high-salt culture condition, but a large amount of high-salt wastewater is generated in the production process of the method, so that the method has great influence on production equipment and environmental pollution, and is being phased out. With the development of genetic engineering technology, researchers have conducted intensive research on the biosynthetic metabolic pathway of escitalopram, and have constructed a series of genetic engineering strains by metabolic engineering means to produce escitalopram by microbial fermentation synthesis. The method is mainly used for synthesizing the ectoin by high-density fermentation culture of the constructed recombinant escherichia coli, corynebacterium glutamicum and the like. The method for producing the exendin has higher requirements on fermentation equipment, and byproducts with similar structures are easy to appear in the process, so that the subsequent separation cost is too high; the living cell transformation method is to use L-aspartate and glycerol (or glucose) as substrates to perform biocatalysis synthesis of the ectoin by using recombinant engineering bacteria cells, and enterprises currently carry out large-scale production of the ectoin through the production method. The catalytic process for preparing ectoin by transformation of living cells is as follows: l-aspartic acid (Asp) forms phosphorylated L-aspartic acid under the catalysis of L-aspartokinase (ash), and phosphorylated L-aspartic acid forms L-aspartic acid beta semialdehyde (ASA), a precursor of ectoin, under the catalysis of L-aspartic acid beta semialdehyde dehydrogenase (Asd). ASA gradually synthesizes the ectoin under the action of ectoin synthesis gene cluster ectABC. The first precursor ASA forms L-2, 4-diaminobutyric acid (DABA) under the catalysis of L-2, 4-diaminobutyric acid aminotransferase (EctB), then DABA forms N-gamma-acetyl-2, 4-diaminobutyric acid (ADABA) under the catalysis of L-2, 4-diaminobutyric acid gamma-acetyltransferase (EctA), and finally ADABA forms escin under the catalysis of escin synthase (EctC).
Many researchers have conducted extensive studies on the preparation of exendin by living cell transformation using aspartic acid as a substrate precursor. HeYZ heterologously expressed the ectopic synthetic gene cluster ectoABC of Salmonella elongata (Halomonas elongata) in E.coli BW25113, and the expression of ectA, ectB, ectC gene was induced with arabinose during the culture. After collecting the cells, aspartic acid was added to prepare ectoin, and the conversion level of ectoin was 25.1g/L for 24 hours (He et al high production of ectoine from aspartate and glycerol by use of whole-Cell biocatalysis in recombinant Escherichia coll. Microbial Cell industries.2015; 14 (1): 55). Chinese patent CN104560844B introduced a recombinant plasmid containing the synthetic gene cluster EctABC of Salmonella elongata Halomonas elongate ectoin into E.coli. The recombinant escherichia coli realizes the soluble expression of three key enzymes synthesized by the ectoin under the control of an arabinose promoter. The high-efficiency secretory synthesis of the ectoin is realized by using sodium aspartate as a precursor through a bioconversion method. 1.1 g of ectoin can be synthesized per gram of thallus. Chinese patent CN104593442B provides a method for producing the ectoin by high-density culture of recombinant escherichia coli, which comprises the steps of carrying out high-density culture on escherichia coli containing recombinant plasmid of extended halomonas Halomonas elongate ectoin synthetic gene cluster EctABC, collecting thalli, adding sodium L-aspartate, and carrying out bioconversion on the thalli to prepare the ectoin, wherein the synthetic efficiency reaches 11.67g/L.d.
The prior art uses L aspartic acid as a substrate and utilizes living cell bioconversion to prepare the ectoin, but the gene recombination of the patent is utilized to construct escherichia coli, so that the conversion rate of the ectoin synthesis is not high.
Disclosure of Invention
Aiming at the problems existing in the prior art, the patent opens up a metabolic pathway from fumaric acid to the exendin (figure 1), constructs a exendin biosynthesis pathway and constructs a novel exendin production strain, thereby simplifying the production process, improving the synthesis and conversion efficiency, further reducing the production cost, improving the market competitiveness of the product and having important practical significance for the application of the exendin.
The invention is realized by the following technical scheme:
the invention uses metabolic engineering and synthetic biology technology, uses E.coli BL21 (DE 3) as chassis expression cell, expresses halophilic bacteria source ectoin synthesis gene cluster ectA, ectB, ectC, realizes ectoin heterologous biosynthesis; and through a double plasmid expression system, the aspartase gene aspA, aspartokinase gene ask and aspartyl semialdehyde dehydrogenase gene asd which are derived from escherichia coli are enhanced and expressed in original bacteria of the ectoin, and finally the recombinant escherichia coli capable of efficiently biosynthesizing the ectoin is obtained.
As a first aspect of the present invention, there is provided a genetically engineered bacterium of Escherichia coli producing exendin using fumaric acid, which expresses an exendin synthesis gene cluster ectoABC derived from halophila, wherein ectB, ectA, ectC genes (sequences shown in sequence tables SEQ ID NO. 1-3) encode 2-aminobutyric acid transaminase, 2-aminobutyric acid acetyltransferase and exendin synthase, respectively, and which expresses any one or more of three genes (a), (b) and (c), wherein:
(a) The aspartase gene aspA is derived from escherichia coli (a sequence shown in a sequence table SEQ ID NO. 4);
(b) The aspartokinase gene ask is derived from escherichia coli (a sequence shown in a sequence table SEQ ID NO. 5);
(c) The aspartate semialdehyde dehydrogenase gene asd is derived from Escherichia coli (sequence shown in SEQ ID NO.6 of the sequence Listing).
In one embodiment, the genetically engineered bacterium expresses three genes (a), (b), and (c) simultaneously.
In one embodiment, the E.coli genetically engineered bacterium has a two plasmid expression system, including pET24a (+) and pACYCDuet-1.
In one embodiment, the E.coli genetically engineered bacterium, the ectoin synthetic gene cluster ectA, ectB, ectC is expressed in the pET24a (+) plasmid and the aspA, ask, asd gene is expressed in the pACYCDuet-1 plasmid.
In one embodiment, the E.coli genetically engineered bacterium regulates the expression of the ectA, ectB, ectC, aspA, ask, asd gene via the T7 promoter and the lac operator, respectively. There is an RBS sequence between the lac operon and the gene sequence.
In one embodiment, the host of the genetically engineered bacterium is e.collbl21, e.collbl 21 (DE 3), e.colljm 109, e.colldh5α, or e.colltop 10.
As a second aspect of the present invention, the present invention also provides a method for constructing a genetically engineered bacterium of Escherichia coli, comprising the steps of:
step 1, constructing gene clusters ectoABC which are derived from halophiles and respectively encode 2-aminobutyric acid aminotransferase, 2-aminobutyric acid acetyltransferase and ectoin synthase into a pET24a (+) expression vector to obtain a recombinant expression vector E;
step 2, constructing aspartase genes aspA, aspartokinase genes ask and aspartyl semialdehyde dehydrogenase genes asd into pACYCDuet-1 expression vectors to obtain recombinant expression vectors AAA;
and step 3, transferring the constructed E, AAA recombinant expression vector into escherichia coli BL21 (DE 3) by a calcium chloride method to obtain recombinant escherichia coli genetic engineering bacteria E-AAA/BL21.
As a third aspect of the present invention, the present invention also provides a method for producing ectoin by fermenting the recombinant escherichia coli genetically engineered bacterium, the method comprising: and fermenting and culturing the escherichia coli engineering bacteria in a continuous feed fermentation medium for at least 46 hours.
In one embodiment, the fermentation culture comprises the following specific steps:
in one embodiment, the fermentation culture comprises the following specific steps:
step (1), seed culture: inoculating recombinant escherichia coli into a seed culture medium for seed culture;
and (2) glucose supplementing fermentation culture: inoculating the cultured seeds into a fermentation culture medium for culturing, and controlling pH7.0 in the fermentation process; when the glucose in the culture medium is exhausted, 80% glucose solution is fed in; when the concentration of the cells in the fermentation broth (OD 600 ) Reaching above 30, adding 0.2mM IPTG inducer.
In one embodiment, the fermentation culture comprises the following specific steps:
step 1, seed culture: inoculating recombinant escherichia coli into a seed culture medium for seed culture;
step 2, glucose and fumaric acid are supplemented for fermentation culture: inoculating the cultured seeds to a fermentation medium for culturing, and controlling pH7.0 in the fermentation process; when the glucose in the culture medium is exhausted, 80% glucose solution is fed in; when the concentration of the cells in the fermentation broth (OD 600 ) Over 30 mM IPTG inducer (0.2 mM) was added, and after 12 hours of fermentation induction, 20% sodium aspartate solution was added and after 12 hours of fermentation induction, 30% ammonium fumarate solution was added.
In a preferred embodiment, the fermentation culture comprises the following specific steps:
step 1, seed culture: 0.1ml of recombinant E.coli glycerol tube seeds were inoculated into 10ml LB shake flasks (50 ppm kanamycin, 25ppm chloramphenicol added), and incubated at 37℃for 12h at 250 revolutions. 7ml of primary seed bottle seeds are inoculated into 700ml LB shake flask and cultured for 12h at 37 ℃ and 250 revolutions.
Step 2, glucose supplementing fermentation culture: the 700ml secondary seeds after cultivation are inoculated into 7L sterilized fermentation culture medium (pH 7.0 is regulated by ammonia before inoculation) according to 10 percent of inoculum size, and are cultivated under the conditions of 37 ℃ and 300 revolutions and air flow rate of 400L/h, wherein the pH7.0 is controlled by ammonia during fermentation, and the rotation speed and the air flow rate are regulated to keep the dissolved oxygen above 30 percent. When the medium had been depleted of glucose, 80% strength dextrose solution was added. When the concentration of the cells in the fermentation broth (OD 600 ) Over 30 m of MIPTG inducer is added, and the same as that of the mixtureThe pH was controlled at 8.0.
In one embodiment, step 2 is a fermentation culture of glucose and fumaric acid: the 700ml secondary seeds after cultivation are inoculated into 7L sterilized fermentation culture medium (pH 7.0 is regulated by ammonia before inoculation) according to 10 percent of inoculum size, and are cultivated under the conditions of 37 ℃ and 300 revolutions and air flow rate of 400L/h, wherein the pH7.0 is controlled by ammonia during fermentation, and the rotation speed and the air flow rate are regulated to keep the dissolved oxygen above 30 percent. When the medium had been depleted of glucose, 80% strength dextrose solution was added. When the concentration of the cells in the fermentation broth (OD 600 ) Over 30 m of MIPTG inducer (0.2 m) is added, and after 12h of fermentation induction, 30% ammonium fumarate solution is added, and pH is controlled at 8.0.
In one embodiment, the seed medium composition used in step (1) is 1-10g/L peptone, 1-5g/L yeast extract, 1-10g/L sodium chloride.
In one embodiment, the fermentation medium used in step (2) is 2-10g/L glucose, 4-20g/L (NH) 4 ) 2 SO 4 2-10g/L yeast extract powder, 4-20g/L corn steep liquor, 0.4-2g/L K 2 HPO 4 ，0.4-2g/L KH 2 PO 4 ，0.2-1g/L MgSO 4 .7H 2 O，0.04-0.02g/L FeSO 4 .7H 2 O，0.004-0.02g/L MnSO 4 .H 2 O。
As a fourth aspect of the invention, the invention also provides the application of the escherichia coli genetically engineered bacterium in the production of food, medicine, health care product or cosmetic containing the ectoin.
The invention has the beneficial effects that:
the invention uses T7 promoter and lac operon expression system to express the gene cluster ectA, ectB, ectC of the ectoin in the escherichia coli for the first time, and simultaneously enhances the expression of aspA, ask and asd genes, and optimizes the fermentation process of the ectoin genetic engineering bacteria, thereby obtaining the following effects:
(1) On the basis of the synthetic route of the ectoin in halophiles, the invention constructs a precursor metabolic route in the biosynthesis process of the ectoin in escherichia coli, simultaneously strengthens the metabolic route from fumaric acid to aspartic acid, and finally constructs escherichia coli genetic engineering bacteria with a complete ectoin biosynthetic route.
(2) The invention carries out batch feeding high-density fermentation culture on the engineering bacteria, and the engineering bacteria show excellent escin production performance. The yield of fermentation for 50 hours can reach 30.8g/L, the production efficiency is 0.616g/L/h, the specific yield is 0.6864g/g DCW, the glucose conversion rate is 0.201g/g, and the fumaric acid conversion rate is 23.7%. The gene engineering strain of the ectoin has good industrial application prospect.
(3) He YZ, chinese patent CN104560844B and Chinese patent CN104593442B are all literature reports of the same unit, and the research content is that ectopic induction expression is carried out on ectopic induction expression of ectopic gene cluster ectopic gene is carried out on escherichia coli BW25113, and the induced thalli are collected and added with aspartic acid, potassium chloride and glycerol for carrying out bioconversion of ectopic gene.
Compared with the report of the above document, the invention has the following remarkable progress: (1) The genetically engineered bacterium E-AAA/BL21 constructed in the invention can prepare the ectoine without adding a substrate, can directly prepare the ectoine by glucose fermentation, has the yield of the ectoine reaching 11.4g/L after 46h fermentation, can prepare the ectoine without adding the substrate, has high purity of the ectoine in fermentation liquor and less impurities, and can avoid the influence of the substrate on the separation and the extraction of the ectoine in the subsequent extraction process. (2) The genetically engineered bacteria constructed in the invention can directly prepare the ectoine by adding fumaric acid in the fermentation process without centrifugally collecting the bacteria, the step of centrifugally collecting the bacteria is omitted, and the ectoine preparation process is simple. (3) The genetic engineering bacteria constructed in the invention use fumaric acid substrate for fermentation conversion, and the price of fumaric acid is cheaper than that of aspartic acid, so that the cost can be reduced. (4) The genetic engineering bacteria constructed in the invention use fumaric acid substrate for fermentation, 0.1M potassium chloride is not added in the conversion process, and the pollution of high-concentration salt to the environment is reduced. (5) The yield of the genetically engineered bacterium fumaric acid can reach 30.8g/L after 50 hours of fermentation, the synthesis efficiency reaches 14.78g/L.d, and the synthesis efficiency is higher than 11.67g/L.d of the ectoin reported in Chinese patent CN 104593442B.
Drawings
FIG. 1 is a diagram of the biosynthesis and metabolism of exedoline;
FIG. 2 is a diagram of recombinant expression vector construction;
FIG. 3 recombinant bacterium glucose fed fermentation to produce exendin;
FIG. 4 recombinant bacterium supplemented glucose and ammonium fumarate fermentation to produce escidodine.
Detailed Description
The present invention is further described below by way of specific examples, which are not intended to limit the scope of the invention. Modifications, combinations, or substitutions of the present invention within the scope of the invention or without departing from the spirit and scope of the invention will be apparent to those skilled in the art and are included within the scope of the invention.
EXAMPLE 1 construction of recombinant expression vectors
The biosynthetic pathway of ectoin is mainly controlled by the ectoABC gene cluster, wherein ectA, ectB, ectC genes (sequences shown in sequence table SEQ ID NO. 1-3) respectively encode 2-aminobutyric acid aminotransferase, 2-aminobutyric acid acetyltransferase and ectoin synthetase. The sequence of the halophilic bacteria ectoABC gene cluster reported in the literature publication is subjected to codon optimization according to the codon preference of escherichia coli. The ectoABC gene cluster was synthesized by gene synthesis company. P is added in front of the upstream primer of the ectoB and ectoC genes T7 The synthesized ectABC gene cluster fragments are respectively subjected to PCR amplification by a promoter, a lac operon and an RBS sequence, thereby obtaining ectA gene fragments and containing P T7 P of promoter T7 ectB、P T7 An electric gene fragment. Fusion PCR is carried out on the 3 fragments to obtain the ectA-P T7 ectB-P T7 The ecto fragment is cloned and recombined with the pET-24a vector linearized by restriction enzymes BamHI and XholI in one step to obtain a recombinant expression vector E (pET 24 a-P) T7 ectA-P T7 ectB-P T7 electric) (fig. 2).
If necessary, P is added before the upstream primer of each gene T7 The promoter, lac operon and RBS sequence, and the genome of colibacillus is used as template to PCR amplify aspartokinase gene ask (sequence shown in SEQ ID No. 5) and aspartokinase halfAldehyde dehydrogenase gene asd (sequence shown in sequence table SEQ ID NO. 6) and aspartase gene aspA (sequence shown in sequence table SEQ ID NO. 4) to obtain ask and P T7 asd, aspA gene fragment. Linearizing the pACYCDuet-1 vector by using restriction enzymes EcoRV and XhoI (second multiple cloning site), adding 20bp nucleic acid sequences homologous to the two ends of the linearized pACYCDuet-1 vector at the two ends of the primer, and performing ask and P T7 Fusion PCR is carried out on the asd fragment to obtain ask-P with homologous arms T7 asd fragment, recombinant expression vector AA (pACYCDuet-P) is obtained by recombining the fragment into pACYCDuet-1 expression vector by using a one-step cloning kit T7 ask-P T7 asd)。
Linearizing the AA vector by restriction enzymes BamHI and HindIII (a first multiple cloning site), adding 20bp nucleic acid sequences homologous to both ends of the linearized AA vector at both ends of aspA primer, performing PCR amplification to obtain aspA fragment with homology arms, cloning the aspA fragment into an AA expression vector in one step to obtain a recombinant expression vector AAA (pACYCDuet-P) T7 aspA-P T7 ask-P T7 asd) (fig. 2).
Transferring E, AAA recombinant expression vector into escherichia coli BL21 (DE 3) by a calcium chloride method, and finally obtaining recombinant escherichia coli E-AAA/BL21 capable of producing the ectoin.
The primers used in the construction of the recombinant expression vector are shown in Table 1.
TABLE 1 list of primers used in construction of recombinant expression vectors
The underlined part is a one-step cloning or fusion PCR homology arm sequence.
Example 2 recombinant bacterium glucose-supplementing fermentation production of Excreta-Producin
E-AAA/BL21 recombinant strain integrated with the biosynthesis path of ectoin is subjected to high-density fermentation culture in 15L fermentation tank, and the ectoin is verifiedFermentation production level of keduoyin. 0.1ml of recombinant E.coli glycerol tube seeds were inoculated into 10ml LB shake flasks (peptone 1%, yeast extract 0.5%, sodium chloride 1%, 50ppm kanamycin, 25ppm chloramphenicol) and cultured at 37℃for 12h at 250 revolutions. 7ml of primary seed bottle seeds are inoculated into 700ml LB shake flask, and are cultured for 12h at 37 ℃ and 250 revolutions. The cultured 700ml secondary seeds were inoculated in an inoculum size of 10% to 7L sterilized fermentation medium (10 g/L glucose, 20g/L (NH) 4 ) 2 SO 4 10g/L yeast extract powder, 20g/L corn steep liquor, 2g/L K 2 HPO4，2g/L KH 2 PO 4 ，1g/L MgSO 4 .7H 2 O，0.02g/L FeSO 4 .7H 2 O，0.02g/L MnSO 4 .H 2 O, pH7.0 was adjusted with ammonia before inoculation. ) Culturing at 37deg.C and 300 rpm at air flow rate of 400L/h, controlling pH to 7.0 with ammonia water during fermentation, and regulating rotation speed and air flow rate to maintain dissolved oxygen at above 30%. When the medium had been depleted of glucose, 80% strength dextrose solution was added. When the concentration of the cells in the fermentation broth (OD 600 ) Over 30, 0.2mM IPTG inducer was added while controlling pH to 8.0. Sampling and detecting the concentration OD of the bacterial cells at intervals 600 The content of the ectoin and the glucose concentration. After 46h fermentation, the cell concentration OD 600 Up to 80, the residual glucose in the fermentation process is lower than 1g/L. The yield of the ectoin is 11.4g/L, the production efficiency is 0.247g/L/h, the specific yield is 0.3149g/g DCW, and the glucose conversion rate is 0.094g/g. (FIG. 3)
EXAMPLE 3 recombinant bacterium glucose supplementing and ammonium fumarate fermentation production of Excreta
E-AAA/BL21 recombinant strain integrated with the biosynthesis path of the ectoin is subjected to high-density fermentation culture in a 15L fermenter, and the fermentation production level of the ectoin is verified. 0.1ml of recombinant E.coli glycerol tube seeds were inoculated into 10ml LB shake flasks (peptone 1%, yeast extract 0.5%, sodium chloride 1%, 50ppm kanamycin, 25ppm chloramphenicol) and cultured at 37℃for 12h at 250 revolutions. 7ml of primary seed bottle seeds are inoculated into 700ml LB shake flask, and are cultured for 12h at 37 ℃ and 250 revolutions. Inoculating 700ml of the cultured secondary seeds into 7L of sterilized fermentation medium (10 g/L glucose, 20g/L (NH 4) 2SO4, 10g/L yeast) according to 10% inoculum sizeSoaking powder, 20g/L corn steep liquor, 2g/L K 2 HPO4，2g/L KH 2 PO 4 ，1g/L MgSO 4 .7H 2 O，0.02g/L FeSO 4 .7H 2 O，0.02g/L MnSO 4 .H 2 O, pH7.0 was adjusted with ammonia before inoculation. ) Culturing at 37deg.C and 300 rpm at air flow rate of 400L/h, controlling pH to 7.0 with ammonia water during fermentation, and regulating rotation speed and air flow rate to maintain dissolved oxygen at above 30%. When the medium had been depleted of glucose, 80% strength dextrose solution was added. When the concentration of the cells in the fermentation broth (OD 600 ) Reaching above 30, adding 0.2mM IPTG inducer. After 12h of fermentation induction, a 30% ammonium fumarate solution was fed in while controlling the pH at 8.0. Sampling and detecting the concentration OD of the bacterial cells at intervals 600 The content of the ectoin and the glucose concentration. After 50h fermentation, the cell concentration OD 600 Up to 101, the glucose residue in the fermentation process is lower than 1g/L. The yield of the ectoin is 30.8g/L, the production efficiency is 0.616g/L/h, the specific yield is 0.6864g/g DCW, the glucose conversion rate is 0.201g/g, and the fumaric acid conversion rate is 23.7%. (FIG. 4).
Claims (10)
1. An escherichia coli genetic engineering bacterium for producing ectoin by using fumaric acid, which is characterized in that the genetic engineering bacterium expresses ectoin synthesis gene cluster ectABC from halophilic bacteria, wherein ectB, ectA, ectC genes respectively encode 2-aminobutyric acid transaminase, 2-aminobutyric acid acetyltransferase and ectoin synthase, and the genetic engineering bacterium further expresses any one or more of three genes (a), (b) and (c), wherein:
(a) The aspartase gene aspA is derived from Escherichia coli;
(b) The aspartokinase gene ask is derived from escherichia coli;
(c) The aspartate semialdehyde dehydrogenase gene asd is derived from E.coli.
2. The genetically engineered escherichia coli of claim 1, wherein the genetically engineered bacteria have a two-plasmid expression system, the plasmids comprising pET24a (+) and pacycguet-1.
3. The genetically engineered escherichia coli of claim 2, wherein the ectoin biosynthetic gene cluster ectA, ectB, ectC is expressed in pET24a (+) plasmid and the aspA, ask, asd gene is expressed in pACYCDuet-1 plasmid.
4. The genetically engineered escherichia coli of claim 2, wherein the expression of the ectA, ectB, ectC, aspA, ask, asd gene is regulated by a T7 promoter and a lac operator, respectively. There is an RBS sequence between the lac operon and the gene sequence.
5. The genetically engineered escherichia coli of any one of claims 1-4, wherein the host of the genetically engineered bacterium is e.coll BL21, e.coll BL21 (DE 3), e.coll JM109, e.coll DH5 a, or e.coll TOP10.
6. The method for constructing a genetically engineered escherichia coli strain as set forth in any one of claims 1 to 4, wherein the method comprises the steps of:
step 1, constructing gene clusters ectoABC which are derived from halophiles and respectively encode 2-aminobutyric acid aminotransferase, 2-aminobutyric acid acetyltransferase and ectoin synthase into a pET24a (+) expression vector to obtain a recombinant expression vector E;
step 2, constructing aspartase genes aspA, aspartokinase genes ask and aspartyl semialdehyde dehydrogenase genes asd into pACYCDuet-1 expression vectors to obtain recombinant expression vectors AAA;
and step 3, transferring the constructed E, AAA recombinant expression vector into escherichia coli BL21 (DE 3) sequentially through a calcium chloride method to obtain recombinant escherichia coli genetic engineering bacteria.
7. A method for producing ectoin by fermentation using the escherichia coli genetically engineered bacterium of any one of claims 1 to 4, the method comprising the steps of:
step 1, step (1), seed culture: inoculating recombinant escherichia coli into a seed culture medium for seed culture;
and (2) glucose supplementing fermentation culture: inoculating the cultured seeds into a fermentation culture medium for culturing, and controlling pH7.0 in the fermentation process; when the glucose in the culture medium is exhausted, 80% glucose solution is fed in; when the concentration of the cells in the fermentation broth (OD 600 ) Reaching above 30, adding 0.2mM IPTG inducer.
8. The method of claim 7, wherein step 2 is a fermentation culture of glucose and fumaric acid: inoculating the cultured seeds to a fermentation medium for culturing, and controlling pH7.0 in the fermentation process; when the glucose in the culture medium is exhausted, 80% glucose solution is fed in; when the concentration of the cells in the fermentation broth (OD 600 ) Over 30 mM IPTG inducer (0.2 mM) was added, and after 12 hours of fermentation induction, 20% sodium aspartate solution was added and after 12 hours of fermentation induction, 30% ammonium fumarate solution was added.
9. The method according to claim 7 or 8, wherein the seed medium composition used in step (1) is 1-10g/L peptone, 1-5g/L yeast extract, 1-10g/L sodium chloride; the fermentation medium used in the step (2) is 2-10g/L glucose, 4-20g/L (NH) 4 ) 2 SO 4 2-10g/L yeast extract powder, 4-20g/L corn steep liquor, 0.4-2g/L K 2 HPO 4 ，0.4-2g/L KH 2 PO 4 ，0.2-1g/LMgSO 4 .7H 2 O，0.04-0.02g/L FeSO 4 .7H 2 O，0.004-0.02g/L MnSO 4 .H 2 O。
10. Use of the genetically engineered escherichia coli as defined in any one of claims 1-4 for the production of a food, a pharmaceutical product, a health product or a cosmetic product comprising escitalopram.
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