CN114934062B - Engineering bacterium for efficiently expressing D-psicose 3-epimerase and application - Google Patents
Engineering bacterium for efficiently expressing D-psicose 3-epimerase and application Download PDFInfo
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- 108030002106 D-psicose 3-epimerases Proteins 0.000 title claims abstract description 53
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- 230000004186 co-expression Effects 0.000 claims abstract description 30
- 101150052825 dnaK gene Proteins 0.000 claims abstract description 21
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- 101150115114 dnaJ gene Proteins 0.000 claims abstract description 20
- 101150053330 grpE gene Proteins 0.000 claims abstract description 20
- 101100278084 Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) dnaK1 gene Proteins 0.000 claims abstract description 18
- 101100117145 Synechocystis sp. (strain PCC 6803 / Kazusa) dnaK2 gene Proteins 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 101100439426 Bradyrhizobium diazoefficiens (strain JCM 10833 / BCRC 13528 / IAM 13628 / NBRC 14792 / USDA 110) groEL4 gene Proteins 0.000 claims abstract description 16
- 101150077981 groEL gene Proteins 0.000 claims abstract description 16
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- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 1
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- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention constructs engineering bacteria for efficiently expressing D-psicose 3-epimerase, wherein the engineering bacteria comprise a constructed recombinant expression vector and a co-expression vector, the recombinant expression vector comprises a gene for expressing the D-psicose 3-epimerase and a constitutive promoter HCE gene, the co-expression vector comprises a gene of chaperone proteins, and the chaperone proteins comprise dnaJ, dnaK, groEL and grpE. According to the invention, the expression is carried out more efficiently by replacing the promoter, and the D-psicose 3-epimerase and the molecular chaperone are expressed together, so that the formation of inclusion body proteins is effectively reduced, the molecular chaperone is folded in an auxiliary way to form a tetramer higher structure, and the efficiency of expressing soluble proteins is improved, thereby improving the expression level of the D-psicose 3-epimerase and reducing the preparation cost.
Description
Technical field:
the invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for efficiently expressing D-psicose 3-epimerase and application thereof.
The background technology is as follows:
psicose is also called as D-psicose, is an epimer corresponding to the carbon number three of D-fructose, has a taste similar to sucrose, but has sweetness equivalent to 70% of sucrose, calories equivalent to 0.3% of sucrose, and is hardly metabolized in vivo. And because the glucose can be inhibited by inhibiting the absorption of the glucose, the compound has the efficacy of reducing the blood sugar, and is one of the most potential sucrose substitutes.
Psicose naturally exists in raisin, fig, kiwi fruit, brown sugar and other foods, is rare monosaccharide with extremely small content in the nature, has high cost by means of a natural product extraction method, and is difficult to realize mass production. Among the prior art methods for producing D-psicose are chemical and biological methods. For example, belik et al have developed a technique for producing psicose from fructose using the catalytic action of molybdic acid ions. Mecaner (mcdonald) produced psicose by a three-step chemical treatment of 1,2:4, 5-di-o-acetonide-beta-d-fructose.
However, chemical methods produce large amounts of byproducts and use large amounts of chemical reagents, and the production efficiency is not high. In recent years, the biological preparation of psicose has become a mainstream trend in the future due to the advantages of safety, green and environmental protection. Early days, ken-iz Mo Li (kenizumari) et al demonstrated that psicose was produced from galactitol, d-tagatose, or d-talitol by microbial cell reaction, but the raw materials were not readily available and were costly. Through continuous researches of the former, the biological conversion method at the present stage mainly takes fructose as a raw material, and D-psicose is synthesized by catalyzing with D-psicose 3-epimerase, and the method has higher catalytic synthesis efficiency and is a main mode for synthesizing psicose at present.
The use of a D-psicose 3-epimerase as disclosed in application No. 20171014243. X, wherein the D-psicose 3-epimerase is the D-psicose 3-epimerase of paenibacillus (Paenibacillus senegalensis), and the D-psicose 3-epimerase is used for industrial catalytic production of psicose or as a catalyst for industrial catalytic production of psicose.
However, the 3-epimerase of the D-psicose which is derived from a wild strain and is not modified has certain limitations in the aspects of heat stability, catalytic activity and the like, so that the application cost of the enzyme is high, and the industrial application range of the enzyme is limited. The prior art usually improves the catalytic activity of the D-psicose 3-epimerase by modifying thalli, such as a mutant of the D-psicose 3-epimerase disclosed in application No. 2016818847. X and application thereof, and the patent provides the mutant of the D-psicose 3-epimerase, which contains one or more mutation sites in G86D, D164E, W S mutation sites, the catalytic activity is obviously improved, the catalytic activity is more than 1.4 times of that of a wild type, and the enzyme dosage in the process of synthesizing the D-psicose is reduced.
Although the prior art improves the catalytic activity of the enzyme by mutation and the like, the problem of low efficiency of expressing soluble protein due to a large amount of inclusion body proteins when expressing the protein by using escherichia coli or bacillus still exists. And because the difference between psicose and fructose is low, the key factor in preparing psicose is the cost of preparing the enzyme. Based on the above, how to construct an engineering strain for efficiently and solubly expressing D-psicose 3-epimerase becomes a key point for preparing psicose at low cost.
The invention comprises the following steps:
in order to solve the technical problems, the invention constructs the engineering bacteria for efficiently expressing the D-psicose 3-epimerase, and the engineering bacteria comprise the constructed recombinant expression vector and the co-expression vector, so that the efficiency of expressing soluble protein is improved, and the expression level of the D-psicose 3-epimerase is improved.
The technical scheme for solving the technical problems is as follows:
one of the technical schemes provided by the invention is to provide a recombinant expression vector which comprises a gene for expressing D-psicose 3-epimerase and a constitutive promoter HCE gene.
Further, the D-psicose 3-epimerase is the following a) or b) protein:
a) Has the sequence of SEQ NO:1, a protein having an amino acid sequence shown in the specification;
b) Consists of SEQ NO:1 and (a) is derived from the protein which has D-psicose 3-epimerase activity and is obtained by substitution and/or deletion and/or addition of one or more amino acid residues.
Further, the constitutive promoter HCE is a DNA fragment of c) or d) as follows:
c) Has the sequence of SEQ NO:2, a DNA fragment of the nucleotide sequence shown in fig. 2;
d) And SEQ NO:2, and has a DNA fragment with more than 90% homology and promoter function.
Preferably, the vector is any one of pET21a, pET20b, pET22b and pET28 a.
The second technical scheme provided by the invention is to provide a co-expression vector, wherein the co-expression vector comprises genes of chaperone proteins, and the chaperone proteins comprise dnaJ, dnaK, groEL and grpE.
Further, the nucleotide sequence of the dnaJ gene is shown in a sequence table SEQ ID NO. 3.
Further, the nucleotide sequence of the dnaK gene is shown in a sequence table SEQ ID NO. 4.
Further, the nucleotide sequence of the GroEL gene is shown in a sequence table SEQ ID NO. 5.
Further, the nucleotide sequence of the grpE gene is shown in a sequence table SEQ ID NO. 6.
In one embodiment, the method for constructing the co-expression vector comprises the following steps:
the gene sequences of dnaJ, dnaK, groEL and grpE are fully synthesized according to the sequence of dnaJ-dnaK-GroEL-grpE, and the fully synthesized sequences are inserted into the multiple cloning sites of the vector to construct the co-expression vector.
In another embodiment, the method for constructing the co-expression vector comprises the following steps:
the gene sequences of dnaJ, dnaK, groEL and grpE were synthesized in the order of dnaJ-dnaK-grpE-GroEL, and the total synthesized sequences were inserted into the multiple cloning sites of the vector to construct a co-expression vector.
The third technical scheme provided by the invention is an engineering bacterium for efficiently expressing the D-psicose 3-epimerase, and the chassis strain of the engineering bacterium comprises the recombinant expression vector and the co-expression vector.
Preferably, the chassis strain is selected from one of E.coli K12, E.coli BL21, E.coli JM109, E.coli Rosetta, E.coli BL21 plysS, E.coli Top 10 and E.coli DH5 alpha.
In a specific embodiment, the construction method of the engineering bacteria for efficiently expressing the D-psicose 3-epimerase comprises the following steps: transferring the recombinant expression vector and the co-expression vector into a chassis strain to obtain engineering bacteria.
The invention provides an application of engineering bacteria for efficiently expressing D-psicose 3-epimerase, which comprises the following steps: is used for producing D-psicose 3-epimerase in cells and catalyzing D-fructose to produce D-psicose.
The fourth technical scheme provided by the invention is a method for preparing D-psicose, comprising the following steps:
1) Inoculating the genetically engineered strain of claim 7 into a fermentation medium for fermentation culture;
2) Adding an inducer into a fermentation medium for induced expression to obtain fermentation liquor;
3) Separating the fermentation liquor to obtain thalli, and adding a buffer solution for re-suspension to obtain a bacterial solution;
4) And adding bacterial liquid into the D-fructose solution to carry out bioconversion, so as to obtain the D-psicose.
Compared with the prior art, the invention has the following advantages:
in one aspect of the invention, the promoter for expressing the target gene in the vector for expressing the D-psicose 3-epimerase is replaced by a constitutive promoter HCE, and the expression is more efficiently carried out by replacing the promoter. On the other hand, the invention converts the carrier containing the molecular chaperone protein dnaJ, dnaK, groEL and grpE in engineering bacteria for expressing the D-psicose 3-epimerase simultaneously, so that the D-psicose 3-epimerase and the molecular chaperone are expressed together, thereby effectively reducing the formation of inclusion body protein, and the molecular chaperone is folded in an auxiliary way to form a tetramer higher structure, thereby improving the efficiency of expressing soluble protein, further improving the expression level of the D-psicose 3-epimerase and reducing the preparation cost. And the constitutive promoter HCE and the molecular chaperone gene are derived from the same donor organism, so that the homology is good, the stability of target gene expression is facilitated, and the expression efficiency is further improved.
Description of the drawings:
FIG. 1 is a diagram showing the electrophoresis verification of nucleic acid according to example 1.
FIG. 2 is a plasmid map of the recombinant expression vector pHCE-DPE constructed in example 1.
FIG. 3 is a plasmid map of the co-expression vector pUC19-JKLE constructed in example 2.
FIG. 4 is a plasmid map of the co-expression vector pUC19-JKEL constructed in example 3.
FIG. 5 is a diagram showing the verification of protein electrophoresis of crude enzyme solutions of D-psicose-3-epimerase prepared from three engineering bacteria in example 5.
Fig. 6 is a sample HPLC chromatogram of example 6.
The specific embodiment is as follows:
the recombinant expression vector provided by the invention comprises a gene for expressing D-psicose 3-epimerase and a constitutive promoter HCE gene.
Wherein the D-psicose 3-epimerase is derived from Ruminococcus sp.
The D-psicose 3-epimerase is the following a) or b) protein:
a) Has the sequence of SEQ NO:1, a protein having an amino acid sequence shown in the specification;
b) Consists of SEQ NO:1 and (a) is derived from the protein which has D-psicose 3-epimerase activity and is obtained by substitution and/or deletion and/or addition of one or more amino acid residues.
The constitutive promoter HCE is derived from bacillus (Geobacillus sp. WCH 70), one of thermophilic organisms isolated from hot compost of Middleton, wisconsin, deposited at NCBI (CP 001638) at 12 months 2009.
The constitutive promoter HCE is a DNA fragment of c) or d) as follows:
c) Has the sequence of SEQ NO:2, a DNA fragment of the nucleotide sequence shown in fig. 2;
d) And SEQ NO:2, and has a DNA fragment with more than 90% homology and promoter function.
The vectors used in common are suitable for the recombinant expression vector, and are not particularly limited. Preferably, the vector is a plasmid, selected from but not limited to any one of pET21a, pET20b, pET22b, pET28 a.
Further preferably, the vector used in the present invention is pET28a plasmid.
The invention also provides co-expression vectors comprising genes for chaperones including dnaJ, dnaK, groEL and grpE.
The chaperones dnaJ, dnaK, groEL and grpE are derived from bacillus geobacilus sp.
Wherein,,dnaJthe nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 3;
the nucleotide sequence of the dnaK gene is shown in a sequence table SEQ ID NO. 4;
the nucleotide sequence of GroEL gene is shown in sequence table SEQ ID NO. 5;
the nucleotide sequence of the grpE gene is shown in a sequence table SEQ ID NO. 6.
The vectors used are all suitable for the co-expression vector, and are not particularly limited. Preferably, the vector is a plasmid, selected from but not limited to one of pUC18 and pUC 19.
dnaJ, dnaK, groEL and grpE genes are assembled in different sequences in the vector, and have a certain influence on expression.
Preferably, in a specific embodiment, the method for constructing the co-expression vector includes:
the gene sequences of dnaJ, dnaK, groEL and grpE are fully synthesized according to the sequence of dnaJ-dnaK-GroEL-grpE, and the fully synthesized sequences are inserted into the multiple cloning sites of the vector to construct the co-expression vector.
In another embodiment, the method for constructing the co-expression vector comprises the following steps:
the gene sequences of dnaJ, dnaK, groEL and grpE were synthesized in the order of dnaJ-dnaK-grpE-GroEL, and the total synthesized sequences were inserted into the multiple cloning sites of the vector to construct a co-expression vector.
On the basis of the recombinant expression vector and the co-expression vector, the invention also provides an engineering bacterium for efficiently expressing the D-psicose 3-epimerase, and the chassis strain of the engineering bacterium comprises the recombinant expression vector and the co-expression vector.
Preferably, the chassis strain is selected from one of E.coli K12, E.coli BL21, E.coli JM109, E.coli Rosetta, E.coli BL21 plysS, E.coli Top 10 and E.coli DH5 alpha.
In a specific embodiment, the construction method of the engineering bacteria for efficiently expressing the D-psicose 3-epimerase comprises the following steps: transferring the recombinant expression vector and the co-expression vector into a chassis strain to obtain engineering bacteria.
The invention provides an application of engineering bacteria for efficiently expressing D-psicose 3-epimerase, which comprises the following steps: is used for producing D-psicose 3-epimerase in cells and catalyzing D-fructose to produce D-psicose.
The fourth technical scheme provided by the invention is a method for preparing D-psicose, comprising the following steps:
1) Inoculating the genetically engineered strain of claim 7 into a fermentation medium for fermentation culture;
2) Adding an inducer into a fermentation medium for induced expression to obtain fermentation liquor;
3) Separating the fermentation liquor to obtain thalli, and adding a buffer solution for re-suspension to obtain a bacterial solution;
4) And adding bacterial liquid into the D-fructose solution to carry out bioconversion, so as to obtain the D-psicose.
Further, in step 1), the fermentation medium is: 20-25 g/L yeast extract, 5-10 g/L soybean peptone, 4-6 g/L glycerol, 2-3 g/L potassium dihydrogen phosphate and 12-16 g/L dipotassium hydrogen phosphate.
The conditions of fermentation culture are as follows: the initial pH value is 6-7, the culture temperature is 22-37 ℃, the culture speed is 200-600 rpm, the culture time is 20-28 h, and the dissolved oxygen is 20-30%.
Further, in the step 2), after fermentation culture until OD600 is more than 20, the temperature is reduced to 22-25 ℃, and an inducer is added at a final concentration of 0.21-0.25 mM.
Preferably, the inducer is IPTG.
Further, tris-HCl buffer is selected as the buffer in step 3, ph=8.0, and the ratio of buffer to cell is 5:1.
in the step 4), the D-fructose solution is 500-800 g/L, and the bacterial liquid addition amount is 6-12g/L.
The biological transformation condition is pH6-7, 50-60 deg.C, transformation 16-18 h.
In order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments.
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention. The reagents or raw materials used in the present invention are commercially available unless otherwise specified. The experimental method without specific conditions noted in the examples of the present invention is generally carried out according to conventional conditions, such as the guidelines for molecular cloning experiments: the conditions described in 4 th edition, or according to the instructions of the kit and the conditions recommended by the reagent manufacturers.
EXAMPLE 1 construction of recombinant expression vectors
In this example, the gene for D-psicose 3-epimerase was derived from Ruminococcus sp.and the gene for the constitutive promoter HCE was derived from Bacillus Geobacillus sp.WCH 70.
The nucleotide sequence of the D-psicose 3-epimerase gene is optimized by Genescript company, a constitutive promoter HCE and a Ribosome Binding Site (RBS) sequence are added at the 5' end, xbaI and XhoI enzyme cutting sites are respectively introduced at the two ends, and total synthesis is carried out to obtain a total synthetic gene fragment. The nucleotide sequence of the total synthetic gene fragment is shown in a sequence table SEQ ID NO. 7.
And respectively carrying out XbaI and XhoI double digestion on the total synthetic gene fragment and the pET28a vector, recovering and purifying the target fragment through a gel recovery kit, and connecting the two products by using T4 ligase to obtain the recombinant plasmid.
This recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistant plates and incubated overnight at 37 ℃. And (3) selecting the positive clones which grow out, extracting plasmids, and carrying out sequencing verification to obtain a recombinant expression vector pHCE-DPE.
Further, plasmids were extracted, target gene fragments were obtained by PCR, and subjected to nucleic acid electrophoresis.
The PCR primers were as follows:
p1：ATGAAGTACGGTATCTACTATGCTTATTGGG
p2：CCTCGAAAACGTGTTTAACAAAATGCAAC
the size of the target gene is 873bp, and the size of the target gene is consistent with that of the target gene, which shows that the recombinant expression vector is successfully constructed.
EXAMPLE 2 construction of the dnaJ-dnaK-GroEL-grpE Co-expression vector
In this example, the genes for 4 chaperones dnaJ, dnaK, groEL, grpE are derived from Bacillus geobacilus sp.WCH 70.
Chaperones dnaJ, dnaK, grpE, groEL are assembled in the order dnaJ-dnaK-GroEL-grpE, with RBS sequences inserted after dnaJ and GroEL, and lac promoters and RBS sequences inserted after dnaK. The XbaI and KpnI enzyme cutting sites are respectively introduced at two ends of the whole sequence, and the total synthesis is carried out by Genescript company to obtain a total synthetic gene fragment, and the nucleotide sequence of the gene fragment is shown as a sequence table SEQ ID NO. 8.
The total synthetic gene fragment and pUC19 vector are subjected to double digestion of XbaI and KpnI respectively, the target fragment is recovered and purified by a gel recovery kit, and the two products are connected by T4 ligase to obtain the recombinant plasmid.
This recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells, coated with ampicillin resistant plates and incubated overnight at 37 ℃. And selecting the positive clones, extracting plasmids, and carrying out sequencing verification to obtain a co-expression vector pUCMOD-JKLE.
EXAMPLE 3 construction of dnaJ-dnaK-grpE-GroEL Co-expression vector
In this example, the genes for 4 chaperones dnaJ, dnaK, groEL, grpE are derived from Bacillus geobacilus sp.WCH 70.
Chaperones dnaJ, dnaK, grpE, groEL are assembled in the order dnaJ-dnaK-grpE-GroEL, with RBS sequences inserted after dnaJ and grpE, respectively, and lac promoter and RBS sequences inserted after dnaK. XbaI and KpnI enzyme cutting sites are respectively introduced at two ends of the sequence, and the total synthesis is carried out by Genescript company to obtain a total synthetic gene fragment, and the nucleotide sequence of the gene fragment is shown as a sequence table SEQ ID NO. 9.
The total synthetic gene fragment and pUC19 vector are subjected to double digestion of XbaI and KpnI respectively, the target fragment is recovered and purified by a gel recovery kit, and the two products are connected by T4 ligase to obtain the recombinant plasmid.
This recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells, coated with ampicillin resistant plates and incubated overnight at 37 ℃. And selecting the positive clones, extracting plasmids, and carrying out sequencing verification to obtain a co-expression vector pUCMOD-JKEL.
EXAMPLE 4 construction of engineering bacteria
The recombinant expression plasmid pHCE-DPE prepared in example 1 is transformed into E.coli BL21 (DE 3) competent cells, and the competent cells are cultured by a plate containing kanamycin, positive clones are selected, plasmids are extracted, and after sequencing verification is correct, the strain DPE-A is obtained.
Competent cells are prepared by using DPE-A strain, molecular chaperone plasmid pUCMOD-JKLE prepared in example 2 is transformed into DPE-1 competent cells, ampicillin-containing plates are used for culturing, positive clones are selected, plasmids are extracted, and engineering bacteria DPE-B1 is obtained after sequencing verification is correct.
Competent cells are prepared by using DPE-A strain, molecular chaperone plasmid pUCMOD-JKEL prepared in example 3 is transformed into DPE-1 competent cells, ampicillin-containing plates are used for culturing, positive clones are selected, plasmids are extracted, and engineering bacteria DPE-B2 is obtained after sequencing verification is correct.
Comparative example 1
In this example, the gene for D-psicose 3-epimerase was derived from Ruminococcus sp.and the gene for the constitutive promoter HCE was derived from Bacillus Geobacillus sp.WCH 70.
The nucleotide sequence of the D-psicose 3-epimerase gene is optimized by Genescript company, and XbaI and XhoI restriction sites are respectively introduced at two ends for total synthesis to obtain a total synthesis gene fragment. And respectively carrying out XbaI and XhoI double digestion on the total synthetic gene fragment and the pET28a vector, recovering and purifying the target fragment through a gel recovery kit, and connecting the two products by using T4 ligase to obtain the recombinant plasmid.
This recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistant plates and incubated overnight at 37 ℃. And (3) picking out the positive clones, extracting plasmids, and carrying out sequencing verification to obtain a recombinant expression vector pET28a-DPE.
And (3) transforming the recombinant expression vector pET28a-DPE into E.coli BL21 (DE 3) competent cells, culturing by using a plate containing kanamycin, picking positive clones, extracting plasmids, and obtaining engineering bacteria DPE-C after sequencing and verifying correctness.
Example 5 Strain culture and expression
Experimental strains:
engineering bacteria DPE-B1 constructed in the embodiment 4;
engineering bacteria DPE-B2 constructed in the embodiment 4;
engineering bacteria DPE-C constructed in comparative example 1.
Picking the engineering bacteria DPE-B1, engineering bacteria DPE-B2 and engineering bacteria DPE-C, respectively inoculating into LB liquid culture medium with corresponding resistance, and culturing at 37deg.C to OD 600 Reaching 0.7-0.8, adding 0.5mM IPTG, and inducing expression at 22deg.C and 150rpm overnight. 4 ℃ and 6000r/min separationThe cells were collected from the heart for 10-15min, and the cells were incubated with buffer (20 mmol/L Tris-HCl, ph 8.0) according to a ratio of 5:1 (5 mL buffer solution is added into 1 gram of wet thalli), the thalli are resuspended, the thalli are crushed by ultrasonic, the thalli are centrifuged for 20min at 10000r/min, and the supernatant is collected, thus obtaining the crude enzyme solution of the D-psicose-3-epimerase.
Carrying out protein electrophoresis verification on the collected crude enzyme solutions of the three D-psicose-3-epimerases, wherein the results are shown in a figure 5, and 1 the crude enzyme solution prepared by engineering bacteria DPE-B1; 2 is crude enzyme liquid prepared by engineering bacteria DPE-B2; and 3, preparing a crude enzyme solution by engineering bacteria DPE-C. The result of protein electrophoresis verification is consistent with the size of the target gene, which shows that the three engineering bacteria successfully express the target gene.
D-psicose-3-epimerase conversion assay:
the reaction system is as follows: the reaction was carried out at 60℃for 2, 4, 8, 12 and 16 hours with a reaction volume of 10mL, 100. Mu.L of crude enzyme solution, 50mM Tris-HCl buffer (pH=8.0), 700mg/mL D-fructose, and the samples were taken, and the contents of fructose and psicose were measured by HPLC to calculate the conversion rate.
HPLC chromatographic conditions are as follows:
analytical column: chromCore Sugar-10Ca;
mobile phase: deionized water
Column temperature: 80 ℃;
differential detector temperature: 50 ℃;
flow rate: 0.5mL/min;
sample injection amount: 10 mu L.
The reaction results are shown in Table 1 below, and it can be seen from the data shown in Table 1 that the maximum conversion rate of more than 33% was achieved by only about 18 hours when the D-psicose 3-epimerase of the engineering bacterium constructed in example 4 was reacted with fructose at a high concentration:
TABLE 1
| Engineering bacteria | 2 hours psicose conversion,% | Psicose conversion at 4 hours,% | 8 hours psicose conversion,% | 12 hours psicose conversion,% | 16 hours psicose conversion,% |
| DPE-B1 | 11.57 | 17.35 | 25.99 | 27.34 | 29.25 |
| DPE-B2 | 11.31 | 16.45 | 24.25 | 26.94 | 28.82 |
| DPE-C | 8.70 | 14.51 | 19.53 | 22.20 | 24.38 |
EXAMPLE 6 preparation of D-psicose
The engineering bacterium DPE-B1 with the best conversion rate in the example 5 is selected as a bacterial strain for preparing D-psicose by biosynthesis, and the genetic engineering bacterial strain is inoculated into a fermentation culture medium for fermentation culture, wherein the fermentation culture medium comprises 25g/L yeast extract, 8g/L soybean peptone, 6g/L glycerol, 2g/L monopotassium phosphate and 15g/L dipotassium phosphate. The initial pH of fermentation culture is 7, the culture temperature is 26 ℃, the dissolved oxygen is controlled to be 20-30%, and the culture is carried out for 24h. When fermentation is carried out to OD 600 And after the temperature is more than 20, cooling to 22 ℃, and starting to add an inducer, wherein the inducer adopts IPTG, and the addition amount of the IPTG is 0.25mM of final concentration. After fermentation, obtaining fermentation liquor, and centrifuging at 6000r/min for 10-15min to obtain thalli.
Buffer (20 mmol/L Tris-HCl, ph 8.0) was used according to 5:1 to obtain bacterial liquid, adding 12g/L bacterial liquid into 700g/L D-fructose solution, performing bioconversion at pH of 6 and 60 ℃ for 18 hours, sampling after conversion is finished, measuring the content of D-psicose by HPLC, and calculating the conversion rate, wherein the result is shown in figure 6. The result showed that the peak area of D-psicose was 29.19.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that while the above describes exemplifying embodiments of the invention, there are several different embodiments of the invention, which are intended to be used as alternatives to the above described embodiments, and that the invention should be construed as limited only by the appended claims.
Sequence listing
<110> Sichuan-Jia Hesheng technology Co., ltd
<120> engineering bacterium for efficiently expressing D-psicose 3-epimerase and application thereof
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gaaggattga aaaacgttac agctggcgct aacccaatgg gcatccgtaa aggtattgaa 360
aaagcggtcg ctgtggcagt agaagaatta aaagcaatct ccaaaccaat caaaggaaaa 420
gaatcgattg ctcaagttgc ggctatctct gcagctgacg aagaagttgg ccaattaatc 480
gcagaagcaa tggaacgcgt tggtaacgac ggtgttatca cattagaaga atcaaaaggc 540
ttcacaacag aattagatgt tgtggaaggt atgcaatttg accgtggtta tgtatctcca 600
tacatgatca cagatacaga aaaaatggaa gcagtgcttg aaaatccata tatcttaatc 660
acagacaaaa aaatctctaa catccaagac atcttgccta tcttagaaca agtagttcaa 720
caaggcaaac cattattaat catcgcagaa gatgtcgaag gcgaagcact tgcgacatta 780
gtagtgaaca aacttcgcgg tacattcact gccgtagcag ttaaagctcc tggcttcggt 840
gatcgtcgta aagcaatgct tgaagacatc gcaatcttaa ctggcggtga agttatctcc 900
gaagaactag gacgtgactt aaaatctaca acaatcgcat cacttggccg cgcttcgaaa 960
gtagtcgtaa caaaagaaaa tacaacaatt gtagatggcg ctggcgactc tgaacgcatt 1020
aaagctcgca tcaaccaaat tcgtgcgcaa ttagaagaaa caacttctga attcgatcgt 1080
gaaaaattgc aagaacgtct agcaaaatta gctggcggcg tagcggtaat caaagttggt 1140
gcagctacag aaacagaatt gaaagaacgc aaattacgca tcgaagacgc gctcaactct 1200
actcgtgccg ctgtggaaga aggtatcgta gccggcggtg gtacagcatt aatgaacgta 1260
tacaataaag ttgctgcgat tgaagcagaa ggcgatgaag caactggtgt gaaaatcgtt 1320
cttcgcgcaa ttgaagagcc agttcgccaa atcgcgcaaa acgctggttt ggaaggctct 1380
gtcattgttg aacgcttaaa aacagaaaaa cctggcatcg gcttcaacgc ggctactggt 1440
gaatgggtag acatgattga agctggtatc gtagacccaa cgaaagtaac tcgttccgca 1500
cttcaaaacg cagcttctgt tgccgctatg ttcttaacaa cagaagcagt tgtcgctgac 1560
aaaccagaag aaaacaaagg cggcaaccca ggcatgcctg acatgggcgg aatgatgtaa 1620
<210> 6
<211> 627
<212> DNA
<213> Geobacillus sp. WCH70
<400> 6
atggaaaaag agcagaaagc agcacaagaa caggctacat acgaacagga accgttaaat 60
acggaaccgc aagaggaaaa agtagagcaa catgaagtaa atgaacatca agaggaaatc 120
gaaatagaag ggcaagaaaa agcacaagaa gagcaaaacg atgaattggc ggcggcaaac 180
gcaaaaattg cggaactaga agcgaaaata aaagaaatgg agaaccgcta tcttcgttta 240
tacgccgatt ttgaaaattt ccgccgtcgt acaaaaatgg aaatggaagc agctgaaaaa 300
tatcgcgccc aaagcttggt tagcgatctt ttgcctgctt tggacaactt tgagcgtgcg 360
ttaaagatag aggctgataa cgaacaagca aaatcgattc tgcaaggaat ggaaatggtg 420
tatcgctccg tgttggatgc gctgaaaaaa gaaggagtag aagcgatcga agcggttggc 480
aaaccgtttg atccgaactt gcatcaagcc gtgatgcaag tagaagacag caattatgag 540
ccgaatacag ttgtggaaga atttcagaaa ggttataaac tgaaagatcg tgtcattcgt 600
ccagcaatgg tgaaagtaag ccaataa 627
<210> 7
<211> 1144
<212> DNA
<213> Artificial Sequence
<400> 7
tctagagatc tctccttcac agattcccaa tctcttgtta aataacgaaa aagcatcaat 60
taaaacggcg gcatgtcttt ctatattcca gcaatgtttt ataggggaca tattgatgaa 120
gatgggtatc accttagtaa aaaaaaagaa ttgctataag ctgctctttt ttgttcgtga 180
tatactgata ataaattgaa ttttcacact tctggaaaaa ggagaataat tttgtttaac 240
tttaagaagg agatatacca tgggcatgaa gtacggtatc tactatgctt attgggaaaa 300
ggagtggaac ggtgactaca agtattacat cgataagatt agcaaattgg gttttgacat 360
tctggaaatc agctgtggtg ctttcagcga ctattacacc aaagatcagg aactgatcga 420
cattggtaaa tacgcgaagg aaaagggtgt taccttgacc gctggttacg gtccgcattt 480
caacgagagc ttgagcagca gcgaaccgaa cacccaaaag caggctatta gcttctggaa 540
ggaaaccttg cgtaaactga agttgatgga cattcatatc gttggtggtg ctctgtacgg 600
ttattggccg gttgattaca gcaaaccgtt cgataagaag cgtgacctgg aaaacagcat 660
caagaacatg aaaattatca gccagtatgc ggaagagtac gacatcatga tgggtatgga 720
agttttgaac cgtttcgaag gttacatgct gaacacctgc gatgaagctc tggcgtacgt 780
tgaggaagtt ggtagcagca acgttggtgt tatgctggac acctttcaca tgaacattga 840
ggaagacaac attgctgcgg ctattcgtaa ggctggtgac cgtctgtacc acttccacat 900
cggtgaaggt aaccgtaaag ttccgggtaa aggtatgctg ccgtggaacg aaatcggtca 960
ggctttgcgt gatatcaact accagcatgc tgctgttatg gagccgtttg ttatgcaggg 1020
tggtaccgtt ggtcacgaca tcaaaatttg gcgtgacatc attggtaact gcagcgaagt 1080
taccctggat atggacgctc aaagcgcgtt gcattttgtt aaacacgttt tcgaggttct 1140
cgag 1144
<210> 8
<211> 5405
<212> DNA
<213> Artificial Sequence
<400> 8
tctagagatg gcgaaacgag attattacga aattctcgga gtgagcaaaa acgcgacaaa 60
agaagagatt aaaaaagcgt accgaaaact ttcgaaaaag tatcatccag atattaacaa 120
agaaccagat gcggcagaaa aattcaaaga aattaaagaa gcgtatgaag tattaagcga 180
tgatcaaaaa cgtgcgcatt acgatcagtt tgggcatgcg gatccaaatc aagggttcgg 240
cgggttccgc agcgatgatt ttgactttgg cggtttcagc ggcttcggtg gtttcgagga 300
tattttcagc accttttttg gcggcggccg ccggcgcgat ccaaatgcgc caagagctgg 360
tgccgattta caatatacga tgacattaac atttgaagag gcggcattcg gaaaagaaac 420
ggatattgaa attccacggg aagaaacgtg tgacacatgc catggcacgg gagcaaaacc 480
gggaacgaaa aaagaaacat gttcatattg tcatggaaca ggtcaaatca gcacagagca 540
gtccacaccg tttggccgca tcgttaatcg ccgcacatgc ccatattgcg gcgggacagg 600
gcaatatatc aaagaaaaat gcacaacatg cggcgggact ggccgcgtga aaaaacggaa 660
gaaaatccat gtcaaaatcc cagctggaat tgatgatgga caacaattgc gggtcgctgg 720
ccaaggagaa ccgggaatta acggcggacc tccgggagat ttatatatcg ttttccatgt 780
ggagccacat gagttttttg agcgtgatgg cgatgatatt tactgtgaaa taccgcttac 840
atttgctcag gctgcgctcg gtgacgaaat cgaagtgccg acgcttcatg gcaaagtgaa 900
gctgaaaata ccagcaggca ctcaaacagg aacaaaattc cgcttaaaag ggaaaggggt 960
gccgaatgta cgcggctatg gctatggcga ccaacatgtg attgtccgcg ttgtgacgcc 1020
gacaaaactg acagaaaaac aaaagcaatt attgcgcgaa tttgatcaat taggcggttc 1080
aagcatgcat caaggaccat acggccgctt ttttgacaag gtaaaaaaag cgtttaaagg 1140
ggaatcatga aataattttg tttaacttta agaaggagat ataccatggg cagcagcatg 1200
agtaaaatta tcggtattga cttaggtaca accaactcat gcgtcgctgt gctagaaggc 1260
ggcgaaccaa aagtaattcc aaacccagaa gggaaccgaa ccactccttc tgttgtggcg 1320
tttaaaaacg gcgaacgttt agtcggagaa gtcgccaaac gtcaggcaat taccaaccca 1380
aacaccatca tttcgattaa acgccatatg ggaacggatt ataaggtaga aattgaaggg 1440
aaaaaatata caccacaaga aatttcggca attattctac aatacttaaa atcatatgca 1500
gaagactatt taggcgaacc agtgacaaga gcggtaatta ccgttccggc gtactttaac 1560
gatgcgcaac gccaagcgac aaaagacgca gggcgcatcg ctggattgga agtagaacgg 1620
attattaacg agccgacagc agcggcgctt gcgtacggtt tggataaaga agaagatcaa 1680
acgatcctcg tttacgactt aggcggcggt acgtttgacg tatcgatcct tgaacttggc 1740
gacggggtat ttgaagtaaa agcgactgcc ggcgacaacc atcttggcgg cgacgatttt 1800
gaccaagtca tcatcgatta cttagtagag caattcaaac aagaacacgg catcgattta 1860
tccaaagaca aaatggcgtt gcaacgcctt aaagatgctg cggaaaaagc gaaaaaagaa 1920
ctttccggcg taacgcaaac gcaaatttca ttgccgttca ttagcgcaaa tgaaacgggg 1980
ccattacact tagaaacgac attaacaaga gcgaaatttg aagagctatc cgcccatcta 2040
gttgaacgga caatgggacc ggtacgtcaa gcgttgcaag atgcgggatt gacgccttcg 2100
gatatcgata aagtgatcct tgtcggcggt tcgacgcgca ttccagcggt acaagaagcg 2160
attaaacgcg agcttggaaa agagccgcat aaaggcgtta acccagacga agtggtagcg 2220
atcggcgcgg cgattcaagg cggtgtcatt gccggtgaag taaaagacgt cgttcttctt 2280
gacgtaacgc cgctatcgct tggaattgaa acaatgggtg gtgtgtttac aaaattaatt 2340
gaacgcaaca caacgattcc aacaagtaaa tcacaaattt tcactacggc agccgataac 2400
caaacaacgg ttgacattca tgtgctgcaa ggcgaacgtc cgatggctgc tgacaacaaa 2460
acgctcggcc gtttccaatt aacagatatc ccacctgcac cacgcggcgt accgcaaatc 2520
gaagtaacgt ttgacatcga cgccaacggt attgtccatg ttcgcgcgaa agatttgggc 2580
acaaacaaag aacaatccat tacgattaaa tcttcatccg gtctttctga agaagagatt 2640
caacgaatga ttaaagaagc agaagaaaac gcggaagcgg acagaaaacg taaagaagcg 2700
gcagaacttc gcaacgaagc cgaccaatta gtattcacga ccgaaaaaac attaaaagag 2760
gtagaaggaa aagtagacga agcagaagtg aaaaaagcgc aagaagcaaa agacgcgtta 2820
aaagctgcgc ttgagaaaaa cgacatcgat gacattcgca aaaagaaaga agcgcttcaa 2880
gaaatcgtac agcagctttc catcaagcta tacgaacaag cggcaaaaca agcacaagct 2940
cagcagcagg caggagccgg cggcgctgcg aaaaaagacg aaaatgttgt cgatgcagaa 3000
tttgaagaag taaaagacga caaataattt acactttatg cttccggctc gtatgttgaa 3060
taattttgtt taactttaag aaggagatat accatgggca gcagcatggc aaaagaaatt 3120
aaattcagcg aagaagctcg tcgtgcgatg ttacgtggtg tggacaaatt agctgatgcg 3180
gtaaaagtaa cgttaggtcc aaaaggccgt aacgtcgtat tagagaaaaa attcggttct 3240
ccattaatta cgaatgacgg tgtaacgatc gcgaaagaaa tcgaattaga agatccattt 3300
gaaaacatgg gtgcgaagct tgttgctgaa gttgcaagca aaacaaacga tgttgctggg 3360
gacggtacaa caacggcaac agttttagcg caagcaatga tccgcgaagg attgaaaaac 3420
gttacagctg gcgctaaccc aatgggcatc cgtaaaggta ttgaaaaagc ggtcgctgtg 3480
gcagtagaag aattaaaagc aatctccaaa ccaatcaaag gaaaagaatc gattgctcaa 3540
gttgcggcta tctctgcagc tgacgaagaa gttggccaat taatcgcaga agcaatggaa 3600
cgcgttggta acgacggtgt tatcacatta gaagaatcaa aaggcttcac aacagaatta 3660
gatgttgtgg aaggtatgca atttgaccgt ggttatgtat ctccatacat gatcacagat 3720
acagaaaaaa tggaagcagt gcttgaaaat ccatatatct taatcacaga caaaaaaatc 3780
tctaacatcc aagacatctt gcctatctta gaacaagtag ttcaacaagg caaaccatta 3840
ttaatcatcg cagaagatgt cgaaggcgaa gcacttgcga cattagtagt gaacaaactt 3900
cgcggtacat tcactgccgt agcagttaaa gctcctggct tcggtgatcg tcgtaaagca 3960
atgcttgaag acatcgcaat cttaactggc ggtgaagtta tctccgaaga actaggacgt 4020
gacttaaaat ctacaacaat cgcatcactt ggccgcgctt cgaaagtagt cgtaacaaaa 4080
gaaaatacaa caattgtaga tggcgctggc gactctgaac gcattaaagc tcgcatcaac 4140
caaattcgtg cgcaattaga agaaacaact tctgaattcg atcgtgaaaa attgcaagaa 4200
cgtctagcaa aattagctgg cggcgtagcg gtaatcaaag ttggtgcagc tacagaaaca 4260
gaattgaaag aacgcaaatt acgcatcgaa gacgcgctca actctactcg tgccgctgtg 4320
gaagaaggta tcgtagccgg cggtggtaca gcattaatga acgtatacaa taaagttgct 4380
gcgattgaag cagaaggcga tgaagcaact ggtgtgaaaa tcgttcttcg cgcaattgaa 4440
gagccagttc gccaaatcgc gcaaaacgct ggtttggaag gctctgtcat tgttgaacgc 4500
ttaaaaacag aaaaacctgg catcggcttc aacgcggcta ctggtgaatg ggtagacatg 4560
attgaagctg gtatcgtaga cccaacgaaa gtaactcgtt ccgcacttca aaacgcagct 4620
tctgttgccg ctatgttctt aacaacagaa gcagttgtcg ctgacaaacc agaagaaaac 4680
aaaggcggca acccaggcat gcctgacatg ggcggaatga tgtaaaataa ttttgtttaa 4740
ctttaagaag gagatatacc atgggcagca gcatggaaaa agagcagaaa gcagcacaag 4800
aacaggctac atacgaacag gaaccgttaa atacggaacc gcaagaggaa aaagtagagc 4860
aacatgaagt aaatgaacat caagaggaaa tcgaaataga agggcaagaa aaagcacaag 4920
aagagcaaaa cgatgaattg gcggcggcaa acgcaaaaat tgcggaacta gaagcgaaaa 4980
taaaagaaat ggagaaccgc tatcttcgtt tatacgccga ttttgaaaat ttccgccgtc 5040
gtacaaaaat ggaaatggaa gcagctgaaa aatatcgcgc ccaaagcttg gttagcgatc 5100
ttttgcctgc tttggacaac tttgagcgtg cgttaaagat agaggctgat aacgaacaag 5160
caaaatcgat tctgcaagga atggaaatgg tgtatcgctc cgtgttggat gcgctgaaaa 5220
aagaaggagt agaagcgatc gaagcggttg gcaaaccgtt tgatccgaac ttgcatcaag 5280
ccgtgatgca agtagaagac agcaattatg agccgaatac agttgtggaa gaatttcaga 5340
aaggttataa actgaaagat cgtgtcattc gtccagcaat ggtgaaagta agccaataag 5400
gtacc 5405
<210> 9
<211> 5405
<212> DNA
<213> Artificial Sequence
<400> 9
tctagagatg gcgaaacgag attattacga aattctcgga gtgagcaaaa acgcgacaaa 60
agaagagatt aaaaaagcgt accgaaaact ttcgaaaaag tatcatccag atattaacaa 120
agaaccagat gcggcagaaa aattcaaaga aattaaagaa gcgtatgaag tattaagcga 180
tgatcaaaaa cgtgcgcatt acgatcagtt tgggcatgcg gatccaaatc aagggttcgg 240
cgggttccgc agcgatgatt ttgactttgg cggtttcagc ggcttcggtg gtttcgagga 300
tattttcagc accttttttg gcggcggccg ccggcgcgat ccaaatgcgc caagagctgg 360
tgccgattta caatatacga tgacattaac atttgaagag gcggcattcg gaaaagaaac 420
ggatattgaa attccacggg aagaaacgtg tgacacatgc catggcacgg gagcaaaacc 480
gggaacgaaa aaagaaacat gttcatattg tcatggaaca ggtcaaatca gcacagagca 540
gtccacaccg tttggccgca tcgttaatcg ccgcacatgc ccatattgcg gcgggacagg 600
gcaatatatc aaagaaaaat gcacaacatg cggcgggact ggccgcgtga aaaaacggaa 660
gaaaatccat gtcaaaatcc cagctggaat tgatgatgga caacaattgc gggtcgctgg 720
ccaaggagaa ccgggaatta acggcggacc tccgggagat ttatatatcg ttttccatgt 780
ggagccacat gagttttttg agcgtgatgg cgatgatatt tactgtgaaa taccgcttac 840
atttgctcag gctgcgctcg gtgacgaaat cgaagtgccg acgcttcatg gcaaagtgaa 900
gctgaaaata ccagcaggca ctcaaacagg aacaaaattc cgcttaaaag ggaaaggggt 960
gccgaatgta cgcggctatg gctatggcga ccaacatgtg attgtccgcg ttgtgacgcc 1020
gacaaaactg acagaaaaac aaaagcaatt attgcgcgaa tttgatcaat taggcggttc 1080
aagcatgcat caaggaccat acggccgctt ttttgacaag gtaaaaaaag cgtttaaagg 1140
ggaatcatga aataattttg tttaacttta agaaggagat ataccatggg cagcagcatg 1200
agtaaaatta tcggtattga cttaggtaca accaactcat gcgtcgctgt gctagaaggc 1260
ggcgaaccaa aagtaattcc aaacccagaa gggaaccgaa ccactccttc tgttgtggcg 1320
tttaaaaacg gcgaacgttt agtcggagaa gtcgccaaac gtcaggcaat taccaaccca 1380
aacaccatca tttcgattaa acgccatatg ggaacggatt ataaggtaga aattgaaggg 1440
aaaaaatata caccacaaga aatttcggca attattctac aatacttaaa atcatatgca 1500
gaagactatt taggcgaacc agtgacaaga gcggtaatta ccgttccggc gtactttaac 1560
gatgcgcaac gccaagcgac aaaagacgca gggcgcatcg ctggattgga agtagaacgg 1620
attattaacg agccgacagc agcggcgctt gcgtacggtt tggataaaga agaagatcaa 1680
acgatcctcg tttacgactt aggcggcggt acgtttgacg tatcgatcct tgaacttggc 1740
gacggggtat ttgaagtaaa agcgactgcc ggcgacaacc atcttggcgg cgacgatttt 1800
gaccaagtca tcatcgatta cttagtagag caattcaaac aagaacacgg catcgattta 1860
tccaaagaca aaatggcgtt gcaacgcctt aaagatgctg cggaaaaagc gaaaaaagaa 1920
ctttccggcg taacgcaaac gcaaatttca ttgccgttca ttagcgcaaa tgaaacgggg 1980
ccattacact tagaaacgac attaacaaga gcgaaatttg aagagctatc cgcccatcta 2040
gttgaacgga caatgggacc ggtacgtcaa gcgttgcaag atgcgggatt gacgccttcg 2100
gatatcgata aagtgatcct tgtcggcggt tcgacgcgca ttccagcggt acaagaagcg 2160
attaaacgcg agcttggaaa agagccgcat aaaggcgtta acccagacga agtggtagcg 2220
atcggcgcgg cgattcaagg cggtgtcatt gccggtgaag taaaagacgt cgttcttctt 2280
gacgtaacgc cgctatcgct tggaattgaa acaatgggtg gtgtgtttac aaaattaatt 2340
gaacgcaaca caacgattcc aacaagtaaa tcacaaattt tcactacggc agccgataac 2400
caaacaacgg ttgacattca tgtgctgcaa ggcgaacgtc cgatggctgc tgacaacaaa 2460
acgctcggcc gtttccaatt aacagatatc ccacctgcac cacgcggcgt accgcaaatc 2520
gaagtaacgt ttgacatcga cgccaacggt attgtccatg ttcgcgcgaa agatttgggc 2580
acaaacaaag aacaatccat tacgattaaa tcttcatccg gtctttctga agaagagatt 2640
caacgaatga ttaaagaagc agaagaaaac gcggaagcgg acagaaaacg taaagaagcg 2700
gcagaacttc gcaacgaagc cgaccaatta gtattcacga ccgaaaaaac attaaaagag 2760
gtagaaggaa aagtagacga agcagaagtg aaaaaagcgc aagaagcaaa agacgcgtta 2820
aaagctgcgc ttgagaaaaa cgacatcgat gacattcgca aaaagaaaga agcgcttcaa 2880
gaaatcgtac agcagctttc catcaagcta tacgaacaag cggcaaaaca agcacaagct 2940
cagcagcagg caggagccgg cggcgctgcg aaaaaagacg aaaatgttgt cgatgcagaa 3000
tttgaagaag taaaagacga caaataattt acactttatg cttccggctc gtatgttgaa 3060
taattttgtt taactttaag aaggagatat accatgggca gcagcatgga aaaagagcag 3120
aaagcagcac aagaacaggc tacatacgaa caggaaccgt taaatacgga accgcaagag 3180
gaaaaagtag agcaacatga agtaaatgaa catcaagagg aaatcgaaat agaagggcaa 3240
gaaaaagcac aagaagagca aaacgatgaa ttggcggcgg caaacgcaaa aattgcggaa 3300
ctagaagcga aaataaaaga aatggagaac cgctatcttc gtttatacgc cgattttgaa 3360
aatttccgcc gtcgtacaaa aatggaaatg gaagcagctg aaaaatatcg cgcccaaagc 3420
ttggttagcg atcttttgcc tgctttggac aactttgagc gtgcgttaaa gatagaggct 3480
gataacgaac aagcaaaatc gattctgcaa ggaatggaaa tggtgtatcg ctccgtgttg 3540
gatgcgctga aaaaagaagg agtagaagcg atcgaagcgg ttggcaaacc gtttgatccg 3600
aacttgcatc aagccgtgat gcaagtagaa gacagcaatt atgagccgaa tacagttgtg 3660
gaagaatttc agaaaggtta taaactgaaa gatcgtgtca ttcgtccagc aatggtgaaa 3720
gtaagccaat aaaataattt tgtttaactt taagaaggag atataccatg ggcagcagca 3780
tggcaaaaga aattaaattc agcgaagaag ctcgtcgtgc gatgttacgt ggtgtggaca 3840
aattagctga tgcggtaaaa gtaacgttag gtccaaaagg ccgtaacgtc gtattagaga 3900
aaaaattcgg ttctccatta attacgaatg acggtgtaac gatcgcgaaa gaaatcgaat 3960
tagaagatcc atttgaaaac atgggtgcga agcttgttgc tgaagttgca agcaaaacaa 4020
acgatgttgc tggggacggt acaacaacgg caacagtttt agcgcaagca atgatccgcg 4080
aaggattgaa aaacgttaca gctggcgcta acccaatggg catccgtaaa ggtattgaaa 4140
aagcggtcgc tgtggcagta gaagaattaa aagcaatctc caaaccaatc aaaggaaaag 4200
aatcgattgc tcaagttgcg gctatctctg cagctgacga agaagttggc caattaatcg 4260
cagaagcaat ggaacgcgtt ggtaacgacg gtgttatcac attagaagaa tcaaaaggct 4320
tcacaacaga attagatgtt gtggaaggta tgcaatttga ccgtggttat gtatctccat 4380
acatgatcac agatacagaa aaaatggaag cagtgcttga aaatccatat atcttaatca 4440
cagacaaaaa aatctctaac atccaagaca tcttgcctat cttagaacaa gtagttcaac 4500
aaggcaaacc attattaatc atcgcagaag atgtcgaagg cgaagcactt gcgacattag 4560
tagtgaacaa acttcgcggt acattcactg ccgtagcagt taaagctcct ggcttcggtg 4620
atcgtcgtaa agcaatgctt gaagacatcg caatcttaac tggcggtgaa gttatctccg 4680
aagaactagg acgtgactta aaatctacaa caatcgcatc acttggccgc gcttcgaaag 4740
tagtcgtaac aaaagaaaat acaacaattg tagatggcgc tggcgactct gaacgcatta 4800
aagctcgcat caaccaaatt cgtgcgcaat tagaagaaac aacttctgaa ttcgatcgtg 4860
aaaaattgca agaacgtcta gcaaaattag ctggcggcgt agcggtaatc aaagttggtg 4920
cagctacaga aacagaattg aaagaacgca aattacgcat cgaagacgcg ctcaactcta 4980
ctcgtgccgc tgtggaagaa ggtatcgtag ccggcggtgg tacagcatta atgaacgtat 5040
acaataaagt tgctgcgatt gaagcagaag gcgatgaagc aactggtgtg aaaatcgttc 5100
ttcgcgcaat tgaagagcca gttcgccaaa tcgcgcaaaa cgctggtttg gaaggctctg 5160
tcattgttga acgcttaaaa acagaaaaac ctggcatcgg cttcaacgcg gctactggtg 5220
aatgggtaga catgattgaa gctggtatcg tagacccaac gaaagtaact cgttccgcac 5280
ttcaaaacgc agcttctgtt gccgctatgt tcttaacaac agaagcagtt gtcgctgaca 5340
aaccagaaga aaacaaaggc ggcaacccag gcatgcctga catgggcgga atgatgtaag 5400
gtacc 5405
Claims (4)
1. An engineering bacterium for efficiently expressing D-psicose 3-epimerase is characterized in that a chassis strain of the engineering bacterium comprises a recombinant expression vector and a co-expression vector;
the recombinant expression vector comprises a gene for expressing D-psicose 3-epimerase and a constitutive promoter HCE gene; the D-psicose 3-epimerase is a polypeptide having the amino acid sequence of SEQ NO:1, a protein having an amino acid sequence shown in the specification; the constitutive promoter HCE is a promoter having the sequence of SEQ NO:2, a DNA fragment of the nucleotide sequence shown in fig. 2;
the co-expression vector comprises a gene for chaperonin including dnaJ, dnaK, groEL and grpE;
the nucleotide sequence of the dnaJ gene is shown in a sequence table SEQ ID NO. 3;
the nucleotide sequence of the dnaK gene is shown in a sequence table SEQ ID NO. 4;
the nucleotide sequence of the GroEL gene is shown in a sequence table SEQ ID NO. 5;
the nucleotide sequence of the grpE gene is shown in a sequence table SEQ ID NO. 6;
the gene sequences of dnaJ, dnaK, groEL and grpE are fully synthesized according to the sequence of dnaJ-dnaK-GroEL-grpE or dnaJ-dnaK-grpE-GroEL, and the fully synthesized sequences are inserted into the multiple cloning sites of the vector to construct the co-expression vector.
2. The method for constructing engineering bacteria for efficiently expressing D-psicose 3-epimerase according to claim 1, wherein the recombinant expression vector and the co-expression vector according to claim 1 are transferred into a chassis strain to obtain the engineering bacteria.
3. The use of an engineering bacterium for the efficient expression of D-psicose 3-epimerase according to claim 1, for intracellular production of D-psicose 3-epimerase, catalyzing production of D-psicose from D-fructose.
4. A process for preparing D-psicose, comprising the steps of:
1) Inoculating the engineering bacteria of claim 1 into a fermentation medium for fermentation culture;
2) Adding an inducer into a fermentation medium for induced expression to obtain fermentation liquor;
3) Separating the fermentation liquor to obtain thalli, and adding a buffer solution for re-suspension to obtain a bacterial solution;
4) And adding bacterial liquid into the D-fructose solution to carry out bioconversion, so as to obtain the D-psicose.
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| CN202210768159.2A CN114934062B (en) | 2022-07-01 | 2022-07-01 | Engineering bacterium for efficiently expressing D-psicose 3-epimerase and application |
| PCT/CN2023/103067 WO2024002131A1 (en) | 2022-07-01 | 2023-06-28 | Engineered microorganism for efficiently expressing d-psicose 3-epimerase and use thereof |
| CN202380011780.7A CN118103513A (en) | 2022-07-01 | 2023-06-28 | Engineering bacterium for efficiently expressing D-psicose 3-epimerase and application |
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| CN105602879B (en) * | 2016-01-26 | 2019-09-03 | 中国科学院天津工业生物技术研究所 | Engineering strain, construction method and its application of one plant of efficient secretion D-Psicose 3- epimerase |
| WO2018116266A1 (en) * | 2016-12-23 | 2018-06-28 | Petiva Private Ltd. | D-psicose 3-epimerase mutant and uses thereof |
| CN107723307A (en) * | 2017-10-09 | 2018-02-23 | 中国科学院天津工业生物技术研究所 | A kind of method and its application for efficiently preparing the epimerase of D psicoses 3 |
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Denomination of invention: An engineered bacterium with efficient expression of D-alodone-3-epimerase and its application Granted publication date: 20230901 Pledgee: Industrial Bank Limited by Share Ltd. Chengdu branch Pledgor: SICHUAN INGIA BIOSYNTHETIC Co.,Ltd. Registration number: Y2024980008649 |
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