CN113699087A - Lactobacillus plantarum engineering strain for converting lactose to generate lactulose and construction method and application thereof - Google Patents

Abstract

The invention relates to a lactobacillus plantarum engineering strain for converting lactose to generate lactulose, a construction method and application thereof. The invention mainly takes lactobacillus plantarum NZ0203 as a basis, and the cellobiose 2-epimerase is overexpressed in the lactobacillus plantarum NZ 0203. The constructed lactobacillus plantarum engineering strain does not consume lactose and lactulose, is beneficial to the progress of lactose conversion reaction and the accumulation of lactulose products, obviously improves the yield of the lactulose, and is suitable for the high-efficiency production of the lactulose. Different from the enzymatic method for producing lactulose, the method for producing lactulose provided by the invention utilizes whole cells for catalysis, has the advantages of reducing enzyme purification cost and multiple use, and is more acceptable by people by utilizing prebiotics produced by probiotics.

Description

Lactobacillus plantarum engineering strain for converting lactose to generate lactulose and construction method and application thereof

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to a lactobacillus plantarum engineering strain for converting lactose to generate lactulose, and a construction method and application thereof.

Background

Lactulose (4-O- β -D-galactopyranosyl-D-fructose) is a disaccharide made by linking a molecule of fructose with a molecule of galactose via a β -1, 4-glycosidic linkage. Lactulose can be produced by isomerization using lactose as a substrate. This reaction is of great value to the dairy industry, as lactose is widely present in whey, and about 30-50% of the world's cheese whey remains unutilized. Besides reducing the environmental pollution caused by whey, lactulose has high added value. Lactulose is used in the pharmaceutical industry because of its efficacy in treating hepatic encephalopathy and chronic constipation. It is used as prebiotic, and lactulose can be added to infant formula (for functional food) as a bifidogenic factor. Furthermore, according to the european food safety agency, lactulose can reduce potentially pathogenic gastrointestinal microorganisms and reduce intestinal transit time.

Isomerization of lactose to lactulose can be accomplished by chemical and enzymatic means. Both of these methods have their inherent limitations, but the synthesis of lactulose using enzymatic reactions is more attractive than chemical methods based on synthetic feasibility and related environmental considerations. Because the enzymatic method is safe, non-toxic, environmentally friendly and less costly. Generally, there are two suitable enzymes that catalyze the synthesis of lactulose, glycosyltransferases and glycosidases. Of these two classes of enzymes, glycosidases are more closely related to industrial applications (e.g., β -galactosidase) because they are commercially available, relatively inexpensive, and have been widely used in the food industry. The conversion yield of lactulose achieved with these two enzymes is 5% -15%. It can be seen that low reaction yields are a major challenge for the enzymatic synthesis of lactulose. In recent years, the use of cellobiose 2-epimerase in the production of lactulose has been gaining attention, which is capable of partially isomerizing glucose in lactose to fructose to produce lactulose. Cellobiose 2-epimerase from the saccharolytic cellulose bacterium (Caldicellulosriptor saccharolyticus) has been reported to give a yield of 58.3%.

In the process of converting lactose into lactulose by using enzymatic reaction, purified enzyme is generally used, but the catalytic reaction is performed by using the whole cells of the enzyme-producing strain, so that the enzyme can be repeatedly used and the purification of the enzyme is avoided, thereby improving the economic benefit and simplifying the production method. Chinese patent document CN104004699A (application No. 201410262301.1) discloses a method for producing lactulose by whole-cell catalysis, which uses recombinant Escherichia coli BL21(DE3) producing cellobiose epimerase as a production strain, uses lactose to replace isopropyl-beta-D-thiogalactoside (IPTG) as an inducer for fermentation culture, uses thalli obtained by centrifugation as a cell biocatalyst after ethanol permeabilization and vacuum freeze drying treatment, and directly converts lactose to produce lactulose. Chinese patent document CN105255805A (application No. 201510782905.3) discloses a method for producing lactulose using recombinant bacillus subtilis, which uses recombinant bacillus subtilis as a production strain, the recombinant bacillus subtilis contains cellobiose epimerase capable of catalyzing lactose to produce lactulose, the expression is induced by lactose or isopropyl-beta-D-thiogalactoside (IPTG), and the biocatalytic reaction is performed using lactose or whey as a substrate. Lactobacillus plantarum is an important lactic acid bacterium, not only having recognized safety, but also being a cell factory with potential application, but has little research in the production of lactulose.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a lactobacillus plantarum engineering strain for converting lactose to generate lactulose, and a construction method and application thereof.

The invention is based on a lactobacillus plantarum gene knockout strain which does not use lactose and lactulose, an optimized cellobiose 2-epimerase gene is inserted, cellobiose 2-epimerase is over-expressed, and lactulose is produced by using the obtained engineering strain by a whole-cell catalysis method or a synchronous growth fermentation method.

The technical scheme of the invention is as follows:

a lactobacillus plantarum engineering strain for transforming lactose to generate lactulose is based on lactobacillus plantarum in which beta-galactosidase coding genes lacA and lacLM are knocked out, and a coding gene of cellobiose 2-epimerase is inserted.

According to the present invention, the lactobacillus plantarum in which the β -galactosidase encoding genes lacA and lacLM are knocked out is lactobacillus plantarum NZ0203, which is prepared according to the method described in patent document 201911223427.7.

Further preferably, said lactobacillus plantarum NZ0203 is not able to utilize lactose and lactulose.

Preferably, the cellobiose 2-epimerase is derived from a saccharolytic cellulose bacterium (Caldcellulosri) whose amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the inserted cellobiose 2-epimerase encoding gene is shown in SEQ ID NO. 2.

In the invention, in the lactobacillus plantarum engineering strain, the expression of the inserted cellobiose 2-epimerase is promoted by knocking out beta-galactosidase coding genes lacA and lacLM.

The construction method of the lactobacillus plantarum engineering strain for converting lactose to generate lactulose comprises the following steps:

(1) constructing lactobacillus plantarum NZ0203 for knocking out beta-galactosidase coding genes lacA and lacLM;

(2) will carry a promoter P_lacThe coding gene of cellobiose 2-epimerase is connected to an expression vector pNZ8148, and an inducible promoter P in the expression vector pNZ8148_nisReplacement by lactose-inducible Strong promoter P_lacObtaining recombinant plasmids;

(3) and (3) transforming the recombinant plasmid in the step (2) into the lactobacillus plantarum NZ0203 in the step (1), and screening to obtain the lactobacillus plantarum engineering strain for transforming lactose to generate lactulose.

According to a preferred embodiment of the present invention, lactobacillus plantarum NZ0203 described in step (1) is prepared according to the method described in patent document 201911223427.7.

Preferably, the nucleotide sequence of the cellobiose 2-epimerase encoding gene of step (2) is shown in SEQ ID NO. 2.

According to the invention, preferably, the promoter P is present in step (2)_lacObtaining the encoding gene of cellobiose 2-epimeraseThe method comprises the following steps:

first, primer P was used_lac-F and P_lac-R, using Lactobacillus plantarum WCFS1 genome DNA as template, PCR amplifying promoter P_lacCoding gene, promoter P_lacThe nucleotide sequence of the coding gene is shown as SEQ ID No. 3; simultaneously, primers CE-F and CE-R are used, and the cellobiose 2-epimerase encoding gene is amplified by PCR by taking the artificially synthesized cellobiose 2-epimerase encoding gene as a template; then splicing the PCR amplification products by using an overlapping splicing PCR method to obtain a gene with a promoter P_lacThe gene encoding cellobiose 2-epimerase of (a);

wherein, the primer sequences used for PCR amplification are as follows:

P_lac-F：5’-GCGAGATCTGAAAAATCCCTCCGTAAATAG-3’，

P_lac-R：5’-GAGGAAGCTCCTTTCAAAG-3’，

CE-F：5’-CTTTGAAAGGAGCTTCCTCATGGATATCACTCGTTTC-3’，

CE-R：5’-CGCGAGCTCTTAATCAACACGCTTGATG-3’。

further preferably, the promoter P_lacThe PCR amplification system of the coding gene is as follows:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lac-F2. mu.L, primer P_lac-R2 μ L, lactobacillus plantarum WCFS1 genomic DNA 1 μ L, Ex taq 1 μ L, double distilled water 13 μ L;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extending at 72 ℃ for 30s, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 ℃.

Further preferably, the PCR amplification system of the cellobiose 2-epimerase encoding gene is:

25 muL of Ex taq buffer, 4 muL of dNTP, 2 muL of primer CE-F, 2 muL of primer CE-R, 1 muL of coding gene of artificially synthesized cellobiose 2-epimerase, 1 muL of Ex taq and 13 muL of double distilled water;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 deg.C for 30s, annealing at 50 deg.C for 30s, extending at 72 deg.C for 2min, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 ℃.

Further preferably, the amplification system of the overlap-and-splice PCR is:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lacF2. mu.L, primer CE-R2. mu.L, promoter P _lac1 mu L of coding gene, 1 mu L of cellobiose 2-epimerase coding gene, 1 mu L of Ex taq and 12 mu L of double distilled water;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min for 30s, repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 ℃.

Preferably, according to the invention, the linkage in step (2) is to carry the promoter P_lacThe coding gene of the cellobiose 2-epimerase and an expression vector pNZ8148 are respectively cut by BglII and SacI restriction endonucleases, and the cut target fragment is cut by T₄DNA ligase is used for ligation.

Preferably, according to the present invention, the method for transformation in step (3) is an electrical transformation method.

Preferably, the screening method in step (3) comprises the following steps: and culturing the transformant obtained after conversion on an MRS solid culture medium added with 10 mu g/mL chloramphenicol, wherein the transformant which can normally grow is a positive transformant.

Further preferably, the MRS solid medium comprises the following components per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween, 20.0g of agar powder and the balance of water.

The Lactobacillus plantarum engineering strain is applied to the production of lactulose by taking lactose or whey as a substrate.

The invention has the beneficial effects that:

the invention discovers for the first time that lactobacillus plantarum NZ0203 can not utilize lactulose, and based on lactobacillus plantarum NZ0203 which does not utilize lactose and lactulose (lactobacillus plantarum NZ0203 does not have the capacity of degrading lactose and lactulose and is an ideal starting strain for generating lactulose), the invention overexpresses cellobiose 2-epimerase, and utilizes the obtained engineering strain to produce lactulose by a whole-cell catalysis method or a synchronous growth fermentation method. Different from the enzymatic method for producing lactulose, the method for producing lactulose provided by the invention utilizes whole cells for catalysis, has the advantages of reducing enzyme purification cost and multiple use, and is more acceptable by people by utilizing prebiotics produced by probiotics.

In addition, the inventors surprisingly found that the expression of cellobiose 2-epimerase can be significantly promoted by knocking out beta-galactosidase encoding genes lacA and lacLM by inserting the optimized cellobiose 2-epimerase encoding gene into Lactobacillus plantarum NZ0203, and the activity of the inserted cellobiose 2-epimerase in Lactobacillus plantarum WCFS1 is determined to be 85.3% of that in Lactobacillus plantarum NZ0203, thereby further increasing the conversion efficiency of lactulose.

Drawings

FIG. 1 shows the growth of Lactobacillus plantarum NZ0203 in MRS-lactose medium and MRS-lactulose medium.

FIG. 2 is a schematic diagram of a recombinant plasmid constructed in example 3; wherein CE is cellobiose 2-epimerase encoding gene, P_lacAnd Terminator are promoter and Terminator, respectively, cm is chloramphenicol resistance gene, repA and repC are replication elements.

FIG. 3 is a graph showing the results of measuring the conversion rate of Lactobacillus plantarum NZ0203/CE to lactose to lactulose and the recycling thereof in example 4.

Detailed Description

The invention is further illustrated with reference to the following specific examples, without limiting the scope of the invention thereto. Materials and reagents mentioned in the examples are all common commercial products unless otherwise specified; the methods and steps described in the examples are conventional in the art unless otherwise specified.

Material sources are as follows:

lactobacillus plantarum WCFS 1: a commonly commercially available known strain purchased from Kyork, Guangdong, Microbiol technologies, Inc.;

coli XL-Blue1 competent cells: known strains purchased from Beijing Quanjin Biotechnology Ltd, and commonly commercially available;

plasmid pNZ 8148: purchased from Biovector NTCC plasmid vector strain cell protein antibody gene collection center, a common commercial product;

bacterial genome extraction kit: purchased from Beijing Tiangen Biotech Ltd;

column type plasmid DNA small extraction kit: purchased from Nanjing Novowedam Biotech, Inc.;

erythromycin, chloramphenicol, agarose, nucleic acid dyes, and the like were purchased from Shanghai Biotechnology, Inc.;

polymerases such as Ex taq and rtaq, restriction enzymes (PstI, XhoI, BglII, SacI), and T₄DNA ligase, DNA Marker and DNA gel recovery kit are purchased from Beijing Baoriri doctor technology GmbH;

the MRS-lactose medium described in the examples had the following composition per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of lactose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween and the balance of water. Agar powder (20.0 g) was added to the solid medium.

The MRS-lactulose medium described in the examples had the following composition per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of lactulose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween and the balance of water.

The MRS solid culture medium described in the examples comprises the following components per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween, 20.0g of agar powder and the balance of water.

The MRS liquid culture medium described in the examples comprises the following components per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween and the balance of water.

Example 1: construction of Lactobacillus plantarum NZ0203

The method for constructing lactobacillus plantarum NZ0203 is described in detail in patent ZL201911223427.7, example 1, and is briefly as follows:

(1) extracting the genome DNA of the Lactobacillus plantarum WCFS1(Lactobacillus plantarum WCFS 1);

(2) amplifying an upstream homology arm of a gene lacA and a downstream homology arm of the lacA by using the genome DNA of the step (1) as a template and utilizing a PCR amplification technology; then, connecting an upstream homologous arm of lacA and a downstream homologous arm of lacA by using an overlapped splicing PCR method to prepare a lacA gene knockout connecting arm;

(3) carrying out double enzyme digestion on plasmid pGhost9 by using PstI and XhoI, connecting the lacA gene knockout connecting arm prepared in the step (2) to pGhost9 by using a homologous recombinase, converting the connecting product into competent escherichia coli XL-Blue1, selecting a transformant which is verified to be correct, and extracting a recombinant plasmid;

(4) transforming the recombinant plasmid obtained in the step (3) into lactobacillus plantarum WCFS1, performing first homologous exchange through transformant culture, screening by using erythromycin, performing continuous passage to perform second homologous exchange, screening a strain with an erythromycin marker lost, and performing PCR amplification verification to obtain a lacA gene knockout strain;

(5) taking the genome DNA obtained in the step (1) as a template, amplifying an upstream homologous arm of the lacLM and a downstream homologous arm of the lacLM by utilizing a PCR amplification technology, and then connecting the upstream homologous arm of the lacLM and the downstream homologous arm of the lacLM by utilizing an overlapped splicing PCR method to prepare a lacLM gene knockout connecting arm;

(6) carrying out double enzyme digestion on plasmid pGhost9 by using PstI and XhoI, connecting the lacLM gene knockout connecting arm prepared in the step (5) to pGhost9 by using a homologous recombinase, converting the connecting product into competent escherichia coli XL-Blue1, selecting a transformant which is verified to be correct, and extracting a recombinant plasmid;

(7) and (3) transforming the recombinant plasmid obtained in the step (6) into the lactobacillus plantarum WCFS1 with the lacA gene knocked out in the step (4), culturing thalli with primary homologous exchange through a transformant, screening by using erythromycin, then carrying out continuous passage to generate secondary homologous exchange, screening a strain with lost erythromycin markers, and obtaining the lactobacillus plantarum with the lacA and the lacLM knocked out simultaneously after PCR amplification verification, wherein the lactobacillus plantarum is named as lactobacillus plantarum NZ 0203.

Example 2: utilization condition of lactobacillus plantarum NZ0203 to lactose and lactulose

Preparing MRS-lactose culture medium and MRS-lactosucrose culture medium, respectively transferring activated Lactobacillus plantarum NZ0203 in MRS liquid culture medium to MRS-lactosucrose culture medium and MRS-lactosucrose culture medium, culturing at 37 deg.C for 24 hr, and measuring OD with spectrophotometer₆₀₀The value is obtained. The results are shown in fig. 1, wherein lactobacillus plantarum NZ0203 does not grow in MRS-lactose medium and MRS-lactulose medium, while the control wild strain lactobacillus plantarum WCFS1 grows vigorously, which indicates that lactobacillus plantarum NZ0203 is no longer able to utilize lactose and lactulose.

EXAMPLE 3 construction of engineering strains of Lactobacillus plantarum

Based on cellobiose 2-epimerase from saccharolytic cellulose bacteria (Caldcellulosis saccharolyticus), the amino acid sequence of which is shown in SEQ ID NO.1, the nucleotide sequence of which is optimized according to the codon usage preference of Lactobacillus plantarum, the nucleotide sequence of the optimized cellobiose 2-epimerase encoding gene is shown in SEQ ID NO.2, and the gene synthesis is carried out by Shanghai Senno Biotech GmbH.

Carrying out PCR amplification on the cellobiose 2-epimerase encoding gene by using an artificially synthesized cellobiose 2-epimerase encoding gene as a template and primers CE-F and CE-R;

wherein the nucleotide sequences of primers CE-F and CE-R used for PCR amplification are as follows:

CE-F：5’-CTTTGAAAGGAGCTTCCTCATGGATATCACTCGTTTC-3’，

CE-R：5’-CGCGAGCTCTTAATCAACACGCTTGATG-3’

the reaction system for PCR amplification is as follows:

25 muL of Ex taq buffer, 4 muL of dNTP, 2 muL of primer CE-F, 2 muL of primer CE-R, 1 muL of coding gene of artificially synthesized cellobiose 2-epimerase, 1 muL of Ex taq and 13 muL of double distilled water;

the reaction procedure for PCR amplification was as follows:

pre-denaturation at 95 ℃ for 3 min; melting at 95 deg.C for 30s, annealing at 50 deg.C for 30s, extending at 72 deg.C for 2min, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 deg.C;

meanwhile, the genome DNA of the lactobacillus plantarum WCFS1 is used as a template, and a primer P is used_lac-F and P_lac-R, PCR amplification promoter P_lacCoding gene, promoter P_lacThe nucleotide sequence of the coding gene is shown as SEQ ID No. 3;

wherein, the primer P used for PCR amplification_lac-F and P_lacThe nucleotide sequence of R is as follows:

P_lac-F：5’-GCGAGATCTGAAAAATCCCTCCGTAAATAG-3’，

P_lac-R：5’-GAGGAAGCTCCTTTCAAAG-3’，

the reaction system for PCR amplification is as follows:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lac-F2. mu.L, primer P_lac-R2 μ L, lactobacillus plantarum WCFS1 genomic DNA 1 μ L, Ex taq 1 μ L, double distilled water 13 μ L;

the reaction procedure for PCR amplification was as follows:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extending at 72 ℃ for 30s, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 deg.C;

then, the PCR amplification products are connected by using an overlapping splicing PCR method to obtain a gene with a promoter P_lacCellobiose 2-A gene encoding an epimerase;

wherein, the amplification reaction system of the overlapping splicing PCR is as follows:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lacF2. mu.L, primer CE-R2. mu.L, promoter P _lac1 mu L of coding gene, 1 mu L of cellobiose 2-epimerase coding gene, 1 mu L of Ex taq and 12 mu L of double distilled water;

the reaction procedure for PCR amplification was as follows:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min for 30s, repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 ℃.

The expression vector pNZ8148 is cut by BglII and SacI restriction endonuclease and is provided with a promoter P_lacThe coding gene of the cellobiose 2-epimerase is also cut by BglII and SacI restriction enzyme, and the cut vector and the target gene are cut by T₄DNA ligase is ligated to obtain a recombinant plasmid, the finally obtained recombinant plasmid is shown in figure 2, the encoding gene of cellobiose 2-epimerase is inserted into the recombinant plasmid, and the inducible promoter P in the expression vector pNZ8148_nisReplacement by lactose-inducible Strong promoter P_lac。

And then the constructed recombinant plasmid is electrically transformed into lactobacillus plantarum NZ0203, a transformant obtained after transformation is cultured on an MRS solid culture medium added with 10 mu g/mL chloramphenicol, and a positive transformant which grows normally is screened, namely the lactobacillus plantarum engineering strain is named as lactobacillus plantarum NZ 0203/CE.

Example 4: method for converting lactose into lactulose by using lactobacillus plantarum NZ0203/CE

Inoculating lactobacillus plantarum NZ0203/CE in MRS liquid culture medium added with 2% lactose, wherein the lactose is an inducer of cellobiose 2-epimerase, culturing at 37 ℃ for 24 hours, and collecting thalli; adding thallus into 100g/L lactose solution, adding thallus at 8g/L, reacting at 60 deg.C for 4 hr, and detecting sugar component in the reaction solution by liquid chromatography. Wherein the detection conditions of the liquid chromatogram are as follows: the chromatographic column is Utimate XB-NH 2-34.6 × 250mm (5 μm), the detector is a differential detector, the mobile phase is acetonitrile-phosphate buffer (taking 1.15g of sodium dihydrogen phosphate, dissolving in 1000mL of water), 84: 16(v/v), temperature 40 ℃ and flow rate 1.5 mL/min.

As a result, as shown in FIG. 3, the conversion of lactulose reached 59.7%.

And (2) collecting bacterial cells again, adding the bacterial cells into 100g/L lactose solution, reacting for 4 hours at 60 ℃, repeating the step, and after 5 times of reaction, still enabling the conversion rate of lactulose to reach 41.4 percent, which shows that the invention adopts a whole cell catalysis method to produce lactulose to realize the repeated use of the lactobacillus plantarum engineering strain NZ0203/CE and reduce the production cost.

Example 5: synchronous growth and fermentation by using lactobacillus plantarum NZ0203/CE

Lactobacillus plantarum NZ0203/CE is inoculated in MRS liquid medium added with 10% lactose, and after 24 hours of culture at 37 ℃, sugar components in the fermentation broth are detected by liquid chromatography, wherein the detection conditions of the liquid chromatography are the same as those of example 4. The results show that the conversion rate of lactulose can reach 45.6%.

Comparative example 1: measurement of Cellobiose 2-epimerase Activity in different strains

The recombinant plasmids constructed in example 3 were respectively transformed into Lactobacillus plantarum WCFS1 and Lactobacillus plantarum NZ0203 to obtain engineered strains named Lactobacillus plantarum WCFS1/CE and Lactobacillus plantarum NZ 0203/CE.

Lactobacillus plantarum WCFS1/CE and Lactobacillus plantarum NZ0203/CE are respectively inoculated in MRS liquid culture medium added with 2% of lactose, wherein the lactose is an inducer of cellobiose 2-epimerase, after the lactobacillus plantarum WCFS1/CE and the Lactobacillus plantarum NZ0203/CE are cultured for 24 hours at 37 ℃, thallus is collected, the activity of the cellobiose 2-epimerase in cells is measured, and the result shows that the activity of the cellobiose 2-epimerase in the Lactobacillus plantarum WCFS1/CE is 85.3% of that in the Lactobacillus plantarum NZ0203/CE, which shows that the expression level of the cellobiose 2-epimerase in the Lactobacillus plantarum NZ0203/CE is higher, and the expression of the cellobiose 2-epimerase can be remarkably promoted after the beta-galactosidase coding genes lacA and lacLM are knocked out, and the lactose conversion to lactulose is promoted.

SEQUENCE LISTING

<110> university of Qilu Industrial science

<120> lactobacillus plantarum engineering strain for converting lactose to generate lactulose, construction method and application thereof

<160> 3

<170> PatentIn version 3.5

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<213> Caldicellulosiruptor saccharolyticus

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aaggctgttt catacggtca cgatatcgaa gcttcatggt tattagatca agctgctaag 780

tacttaaagg atgaaaagtt aaaggaagaa gttgaaaagt tagctttaga agttgctcaa 840

atcactttaa aggaagcttt cgatggtcaa tcattaatca acgaaatgat cgaagatcgt 900

atcgatcgtt caaagatatg gtgggttgaa gctgaaactg ttgttggttt cttcaacgct 960

taccaaaaga ctaaggaaga aaagtactta gatgctgcta tcaagacttg ggaattcatc 1020

aaggaacact tagttgatcg tcgtaagaac tcagaatggt tatggaaggt taacgaagat 1080

ttagaagctg ttaacatgcc aatcgttgaa caatggaagt gtccatacca caacggtcgt 1140

atgtgtttag aaatcatcaa gcgtgttgat taa 1173

<210> 3

<211> 283

<212> DNA

<213> Lactobacillus plantarum promoter Plac

<400> 3

gaaaaatccc tccgtaaata gttttactaa aattttatta gtttgactat aatgcttttt 60

ttgattgttg tcaacacccc ataagacctt tagaacaaga tttggtaacg ctttacaaat 120

ttatcacgtt atcgattcaa attcttctta tcgcccgttg tcgttgtcag tagttgttgt 180

catttagtta aagtttaact aaaacgacat atacaaattt aatattttgt tttatgataa 240

ttgtaagcgt ttttatttat gtaactttga aaggagcttc ctc 283

Claims (10)

1. A lactobacillus plantarum engineering strain for transforming lactose to generate lactulose is characterized in that a coding gene of cellobiose 2-epimerase is inserted based on lactobacillus plantarum for knocking out coding genes lacA and lacLM of beta-galactosidase.

2. The engineered Lactobacillus plantarum strain according to claim 1, wherein the Lactobacillus plantarum from which the β -galactosidase coding genes lacA and lacLM have been knocked out is Lactobacillus plantarum NZ0203 prepared according to the method described in patent document 201911223427.7;

further preferably, said lactobacillus plantarum NZ0203 is not able to utilize lactose and lactulose.

3. The engineered strain of lactobacillus plantarum according to claim 1, wherein the cellobiose 2-epimerase is derived from saccharolytic cellulose (calidicellulosriptor saccharolyticus) and has the amino acid sequence shown in SEQ ID No.1, and the nucleotide sequence of the inserted cellobiose 2-epimerase encoding gene is shown in SEQ ID No. 2.

4. The method for constructing engineering strain of lactobacillus plantarum for converting lactose to lactulose in claim 1, characterized by comprising the following steps:

(1) constructing lactobacillus plantarum NZ0203 for knocking out beta-galactosidase coding genes lacA and lacLM;

(2) will carry a promoter P_lacThe coding gene of cellobiose 2-epimerase is connected to an expression vector pNZ8148, and an inducible promoter P in the expression vector pNZ8148_nisReplacement by lactose-inducible Strong promoter P_lacObtaining recombinant plasmids;

(3) and (3) transforming the recombinant plasmid in the step (2) into the lactobacillus plantarum NZ0203 in the step (1), and screening to obtain the lactobacillus plantarum engineering strain for transforming lactose to generate lactulose.

5. The method according to claim 4, wherein in step (1), Lactobacillus plantarum NZ0203 is prepared according to the method described in patent document 201911223427.7;

preferably, the nucleotide sequence of the cellobiose 2-epimerase encoding gene of step (2) is shown in SEQ ID NO. 2.

6. The method of claim 4, wherein the vector of step (2) contains a promoter P_lacThe method for obtaining the encoding gene of cellobiose 2-epimerase comprises the following steps:

first, primer P was used_lac-F and P_lac-R, using Lactobacillus plantarum WCFS1 genome DNA as template, PCR amplifying promoter P_lacCoding gene, promoter P_lacThe nucleotide sequence of the coding gene is shown as SEQ ID No. 3; simultaneously, primers CE-F and CE-R are used, and the cellobiose 2-epimerase encoding gene is amplified by PCR by taking the artificially synthesized cellobiose 2-epimerase encoding gene as a template; then splicing the PCR amplification products by using an overlapping splicing PCR method to obtain a gene with a promoter P_lacThe gene encoding cellobiose 2-epimerase of (a);

wherein, the primer sequences used for PCR amplification are as follows:

P_lac-F：5’-GCGAGATCTGAAAAATCCCTCCGTAAATAG-3’，

P_lac-R：5’-GAGGAAGCTCCTTTCAAAG-3’，

CE-F：5’-CTTTGAAAGGAGCTTCCTCATGGATATCACTCGTTTC-3’，

CE-R：5’-CGCGAGCTCTTAATCAACACGCTTGATG-3’；

further preferably, the promoter P_lacThe PCR amplification system of the coding gene is as follows:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lac-F2. mu.L, primer P_lac-R2 μ L, lactobacillus plantarum WCFS1 genomic DNA 1 μ L, Ex taq 1 μ L, double distilled water 13 μ L;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extending at 72 ℃ for 30s, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 deg.C;

further preferably, the PCR amplification system of the cellobiose 2-epimerase encoding gene is:

25 muL of Ex taq buffer, 4 muL of dNTP, 2 muL of primer CE-F, 2 muL of primer CE-R, 1 muL of coding gene of artificially synthesized cellobiose 2-epimerase, 1 muL of Ex taq and 13 muL of double distilled water;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 deg.C for 30s, annealing at 50 deg.C for 30s, extending at 72 deg.C for 2min, and repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 deg.C;

further preferably, the amplification system of the overlap-and-splice PCR is:

ex taq buffer 25. mu.L, dNTP 4. mu.L, primer P_lacF2. mu.L, primer CE-R2. mu.L, promoter P_lac1 mu L of coding gene, 1 mu L of cellobiose 2-epimerase coding gene, 1 mu L of Ex taq and 12 mu L of double distilled water;

the amplification procedure was:

pre-denaturation at 95 ℃ for 3 min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min for 30s, repeating for 30 cycles; maintaining at 72 deg.C for 10 min; storing at 4 ℃.

7. The method of claim 4, wherein the ligation in step (2) is performed by ligating the vector with a promoter P_lacThe coding gene of the cellobiose 2-epimerase and an expression vector pNZ8148 are respectively cut by BglII and SacI restriction endonucleases, and the cut target fragment is cut by T₄DNA ligase is used for ligation.

8. The method of claim 4, wherein the transformation in step (3) is an electrical transformation method.

9. The method of claim 4, wherein the screening in step (3) is performed by: culturing the transformant obtained after conversion on an MRS solid culture medium added with 10 mu g/mL chloramphenicol, wherein the transformant which can normally grow is a positive transformant;

further preferably, the MRS solid medium comprises the following components per liter:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate tribasic, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801.0 mL of tween, 20.0g of agar powder and the balance of water.

10. Use of the engineered strain of lactobacillus plantarum as claimed in claim 1 for the production of lactulose using lactose or whey as substrate.

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