CN113699087B

CN113699087B - Lactobacillus plantarum engineering strain for converting lactose to generate lactulose, construction method and application thereof

Info

Publication number: CN113699087B
Application number: CN202110968031.6A
Authority: CN
Inventors: 徐振上; 刘新利; 王婷
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2023-08-15
Anticipated expiration: 2041-08-23
Also published as: CN113699087A

Abstract

The invention relates to a lactobacillus plantarum engineering strain for converting lactose to generate lactulose, and a construction method and application thereof. The invention is mainly based on lactobacillus plantarum NZ0203, and the cellobiose 2-epimerase is overexpressed in lactobacillus plantarum NZ0203. The constructed lactobacillus plantarum engineering strain does not consume lactose and lactulose, is favorable for lactose conversion reaction and accumulation of lactulose products, remarkably improves the lactulose yield, and is suitable for efficient production of the lactulose. Unlike the enzymatic method for producing lactulose, the lactulose production method provided by the invention is catalyzed by whole cells, has the advantages of reducing enzyme purification cost and repeated use, and can be accepted by people more by utilizing probiotics produced by probiotics.

Description

Lactobacillus plantarum engineering strain for converting lactose to generate lactulose, 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-beta-D-galactopyranosyl-D-fructose) is a disaccharide, which is formed by connecting one molecule of fructose and one molecule of galactose through beta-1, 4-glycosidic bond. Lactose is used as a substrate, and can generate lactulose under the isomerization effect. This reaction is of great value to the dairy industry, as lactose is widely present in whey, while about 30-50% of cheese whey in the world is still not utilized. Besides reducing the environmental pollution caused by whey, lactulose has high added value. Lactulose is useful in the pharmaceutical industry because it has the efficacy of treating hepatic encephalopathy and chronic constipation. As a prebiotic, lactulose can be added as a bifidobacterial factor to infant formula (for use in functional foods). Furthermore, lactulose can reduce potentially pathogenic gastrointestinal microorganisms and reduce intestinal transit time according to the European food Security agency.

Isomerization of lactose to lactulose can be achieved chemically and enzymatically. Both of these methods have their inherent limitations, but the synthesis of lactulose using enzymatic reactions is more attractive than chemical methods, based on the feasibility of the synthesis and related environmental considerations. Because the enzymatic method is safe and nontoxic, environment-friendly and has lower cost. In general, there are two suitable enzymes that catalyze the synthesis of lactulose, namely glycosyltransferases and glycosidases. Among these two classes of enzymes, glycosidases are more closely related to industrial applications (e.g., β -galactosidases) 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 was 5% -15%. It can be seen that low reaction yields are a major challenge for enzymatic synthesis of lactulose. In recent years, the use of cellobiose 2-epimerase in the production of lactulose has been emphasized, which is capable of isomerizing glucose in lactose to fructose in part to produce lactulose. The cellobiose 2-epimerase of the saccharide cellulolytic bacterium (Caldicellulosiruptor saccharolyticus) has been reported to obtain a yield of 58.3%.

In the process of converting lactose into lactulose by utilizing enzymatic reaction, the process is generally carried out by utilizing purified enzyme, but the whole cells of enzyme-producing strains are utilized for catalyzing reaction, so that the process can be repeatedly utilized 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 takes recombinant escherichia coli BL21 (DE 3) producing cellobiose epimerase as a production strain, takes lactose instead of isopropyl-beta-D-thiogalactoside (IPTG) as an inducer for fermentation culture, and takes the cells obtained by centrifugation as a cell biocatalyst through ethanol permeabilization and vacuum freeze drying treatment, so as to directly convert lactose to produce lactulose. Chinese patent document CN105255805A (application No. 201510782905.3) discloses a method for producing lactulose by using recombinant bacillus subtilis, wherein the recombinant bacillus subtilis is used as a production strain, the recombinant bacillus comprises cellobiose epimerase capable of catalyzing lactose to produce the lactulose, lactose or isopropyl-beta-D-thiogalactoside (IPTG) is used for carrying out induction expression, and lactose or whey is used as a substrate for carrying out biocatalytic reaction. Lactobacillus plantarum is an important lactic acid bacterium, which not only has recognized safety, but also is a cell factory with application potential, but is rarely studied 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 lactobacillus plantarum gene knockout strain which does not utilize lactose and lactulose, inserts optimized cellobiose 2-epimerase gene, overexpresses cellobiose 2-epimerase, and utilizes the obtained engineering strain to produce lactulose by adopting a whole-cell catalysis method or a synchronous growth fermentation method.

The technical scheme of the invention is as follows:

an engineering strain of lactobacillus plantarum for converting lactose to generate lactulose is based on lactobacillus plantarum with beta-galactosidase coding genes lacA and lacLM knocked out, and the coding genes of cellobiose 2-epimerase are inserted.

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

Further preferably, the lactobacillus plantarum NZ0203 cannot utilize lactose and lactulose.

According to the invention, preferably, the cellobiose 2-epimerase is derived from cellulolytic bacteria (Caldicellulosiruptor saccharolyticus), the amino acid sequence of which 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 the 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 of which the beta-galactosidase encoding genes lacA and lacLM are knocked out;

(2) Will carry promoter P _lac The cellobiose 2-epimerase-encoding gene of (E) was ligated to the expression vector pNZ8148, and the inducible promoter P in the expression vector pNZ8148 was used _nis Substitution with lactose-inducible strong promoter P _lac Obtaining recombinant plasmid;

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

According to the present invention, the Lactobacillus plantarum NZ0203 in the step (1) is preferably prepared by the method described in patent document 201911223427.7.

According to the preferred embodiment of the present invention, the nucleotide sequence of the coding gene of cellobiose 2-epimerase in the step (2) is shown in SEQ ID NO. 2.

According to a preferred embodiment of the invention, the promoter P is provided in step (2) _lac The method for obtaining the coding gene of cellobiose 2-epimerase is as follows:

first, primer P was used _lac -F and P _lac R, PCR amplification of the promoter P with Lactobacillus plantarum WCFS1 genomic DNA as template _lac Coding gene, promoter P _lac The nucleotide sequence of the coding gene is shown as SEQ ID No. 3; simultaneously using primers CE-F and CE-R, taking the coding gene of the artificially synthesized cellobiose 2-epimerase as a template, and amplifying the coding gene of the cellobiose 2-epimerase by PCR; then splicing the PCR amplified products by using an overlap splice PCR method to obtain the PCR amplified product with the promoter P _lac A cellobiose 2-epimerase encoding gene of (a);

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 _lac The 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. Mu.L, lactobacillus plantarum WCFS1 genomic DNA 1. Mu.L, ex taq 1. Mu.L, double distilled water 13. Mu.L;

the amplification procedure was:

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

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

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

the amplification procedure was:

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

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

ex taq buffer 25. Mu.L, dNTP 4. Mu.L, primer P _lac F2. Mu.L, primer CE-R2. Mu.L, promoter P _lac 1 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 3min; melting at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extending at 72 ℃ for 2min for 30s, repeating for 30 cycles; maintaining at 72deg.C for 10min; preserving at 4 ℃.

According to a preferred embodiment of the invention, the ligation in step (2) is performed by ligation of a promoter P _lac The coding gene of cellobiose 2-epimerase and the expression vector pNZ8148 respectively adopt BglII and SacIRestriction endonuclease is used for cutting, and T is adopted for the target fragment after the restriction endonuclease is cut ₄ The DNA ligase performs ligation.

According to a preferred embodiment of the invention, the method of conversion in step (3) is an electroconversion method.

According to a preferred embodiment of the present invention, the screening method in step (3) comprises: the transformant obtained after transformation is cultivated on MRS solid medium added with 10 mug/mL chloramphenicol, and can grow normally, namely the 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 hydrogen phosphate, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0mL of tween 80, 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 that lactobacillus plantarum NZ0203 cannot utilize lactulose for the first time, and aims at the prior art problem of lactulose production, the lactobacillus plantarum NZ0203 which does not utilize lactose and lactulose is adopted as a base (lactobacillus plantarum NZ0203 does not have the capacity of degrading lactose and lactulose and is an ideal starting strain for generating the lactulose), cellobiose 2-epimerase is overexpressed, the obtained engineering strain is utilized to produce the lactulose by adopting a whole cell catalysis method or a synchronous growth fermentation method, and the constructed lactobacillus plantarum engineering strain does not consume lactose and lactulose, thereby being beneficial to the progress of lactose conversion reaction and accumulation of lactulose products, so that the lactulose yield is obviously improved, and the invention is suitable for high-efficiency production of the lactulose. Unlike the enzymatic method for producing lactulose, the lactulose production method provided by the invention is catalyzed by whole cells, has the advantages of reducing enzyme purification cost and repeated use, and can be accepted by people more by utilizing probiotics produced by probiotics.

In addition, the inventor unexpectedly found that the optimized cellobiose 2-epimerase coding gene is inserted into lactobacillus plantarum NZ0203, the expression of cellobiose 2-epimerase can be obviously promoted after the beta-galactosidase coding genes lacA and lacLM are knocked out, and the activity of the inserted cellobiose 2-epimerase in lactobacillus plantarum WCFS1 is determined to be 85.3 percent in lactobacillus plantarum NZ0203, so that the conversion efficiency of lactulose is further improved.

Drawings

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

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

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

Detailed Description

The present invention will be further described with reference to specific examples, but the scope of the present invention is not limited thereto. The materials and reagents involved in the examples, unless specified, are all commonly commercially available products; the methods and steps involved in the examples are, unless specifically stated, conventional in the art.

The material sources are as follows:

lactobacillus plantarum WCFS1: commercially available from Guangdong Cryptographic microorganisms, inc., a common known strain;

coli XL-Blue1 competent cells: commercially available strains from Beijing full gold biotechnology Co., ltd;

plasmid pNZ8148: the cell protein antibody gene collection of the strain cell of the plasmid vector from Biovector NTCC, a common commercial product;

bacterial genome extraction kit: purchased from beijing tiangen biotechnology limited;

column type plasmid DNA small extraction kit: purchased from south genio Wei Zan biotechnology limited;

erythromycin, chloramphenicol, agarose, nucleic acid dyes, etc. are purchased from Shanghai Biotechnology Inc.;

polymerase such as Ex taq and rtaq, restriction endonuclease (PstI, xhoI, bglII, sacI), T ₄ DNA ligase, DNA Marker, DNA gel recovery kit were purchased from Beijing Bao Ri doctor materials technology Co., ltd;

the MRS-lactose medium described in the examples was composed per liter as follows:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of lactose, 2.0g of dipotassium hydrogen phosphate, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0mL of tween 80 and the balance of water. The solid medium was further supplemented with 20.0g of agar powder.

The MRS-lactulose medium described in the examples was composed per liter as follows:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of lactulose, 2.0g of dipotassium hydrogen phosphate, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0mL of tween 80 and the balance of water.

The MRS solid medium described in the examples, per liter of composition, is as follows:

The MRS liquid medium described in the examples was composed per liter as follows:

10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1.0mL of tween 80 and the balance of water.

Example 1: construction of Lactobacillus plantarum NZ0203

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

(1) Extracting genome DNA of lactobacillus plantarum WCFS1 (Lactobacillus plantarum WCFS 1);

(2) Amplifying an upstream homology arm of the gene lacA and a downstream homology arm of the lacA by using the genomic DNA of the step (1) as a template and utilizing a PCR amplification technology; then, connecting an upstream homologous arm of lacA with a downstream homologous arm of lacA by using an overlap splice PCR method to prepare a lacA gene knockout connecting arm;

(3) Performing double enzyme digestion on plasmid pGhost9 by using PstI and XhoI, then connecting the lacA gene knockout connecting arm prepared in the step (2) to pGhost9 by using homologous recombinase, transforming the connecting product into competent E.coli XL-Blue1, picking up 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, culturing the transformant to generate first homologous exchange, screening by using erythromycin, then performing continuous passage to generate second homologous exchange, screening the strain with the erythromycin marker lost, and obtaining the lacA gene knockout strain after PCR amplification verification;

(5) Amplifying an upstream homology arm of a gene lacLM and a downstream homology arm of the lacLM by using the genome DNA obtained in the step (1) as a template and using a PCR amplification technology, and then connecting the upstream homology arm of the lacLM and the downstream homology arm of the lacLM by using an overlapping splicing PCR method to obtain a lacLM gene knockout connecting arm;

(6) Performing double digestion on plasmid pGhost9 by using PstI and XhoI, then connecting the lacLM gene knockout connecting arm prepared in the step (5) to pGhost9 by using homologous recombinase, transforming the connecting product into competent E.coli XL-Blue1, picking up a transformant which is verified to be correct, and extracting recombinant plasmids;

(7) And (3) transforming the recombinant plasmid obtained in the step (6) into lactobacillus plantarum WCFS1 with lacA gene knocked out in the step (4), culturing thalli subjected to first homologous exchange through a transformant, screening by using erythromycin, then carrying out continuous passage to generate second homologous exchange, screening strains with erythromycin marker lost, and obtaining lactobacillus plantarum with lacA and lacLM knocked out simultaneously after PCR amplification verification, wherein the lactobacillus plantarum is named lactobacillus plantarum NZ0203.

Example 2: utilization of lactose and lactulose by Lactobacillus plantarum NZ0203

Preparing MRS-lactose culture medium and MRS-lactulose culture medium, transferring Lactobacillus plantarum NZ0203 activated in MRS liquid culture medium into MRS-lactose culture medium and MRS-lactulose culture medium, respectively, culturing at 37deg.C for 24 hr, and measuring OD by spectrophotometer ₆₀₀ Values. As shown in FIG. 1, lactobacillus plantarum NZ0203 did not grow in both MRS-lactose and MRS-lactulose media, whereas the control wild-type strain Lactobacillus plantarum WCFS1 grew vigorously, indicating that Lactobacillus plantarum NZ0203 is no longer able to utilize lactose and lactulose.

EXAMPLE 3 construction of Lactobacillus plantarum engineering Strain

Based on cellobiose 2-epimerase from saccharolyticum pyrolyzing cellulose bacteria (Caldicellulosiruptor saccharolyticus), the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is optimized according to the preference of lactobacillus plantarum codon usage, the nucleotide sequence of the optimized cellobiose 2-epimerase coding gene is shown as SEQ ID NO.2, and the gene synthesis is carried out by Shanghai Paeno Biotechnology Co Ltd.

Using coding genes of the cellobiose 2-epimerase synthesized artificially as templates, and using primers CE-F and CE-R, amplifying the coding genes of the cellobiose 2-epimerase by PCR;

wherein, the nucleotide sequences of the 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:

the reaction procedure for PCR amplification was as follows:

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

meanwhile, the lactobacillus plantarum WCFS1 genome DNA is used as a template, and a primer P is used _lac -F and P _lac -R, PCR amplified promoter P _lac Coding gene, promoter P _lac The nucleotide sequence of the coding gene is shown as SEQ ID No. 3;

wherein, primer P for PCR amplification _lac -F and P _lac The 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:

the reaction procedure for PCR amplification was as follows:

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

then the PCR amplified products are connected by using an overlap splice PCR method to obtain the PCR amplified product with the promoter P _lac A cellobiose 2-epimerase encoding gene of (a);

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

the reaction procedure for PCR amplification was as follows:

The expression vector pNZ8148 is digested with BglII and SacI restriction enzymes, and has a promoter P _lac The coding gene of cellobiose 2-epimerase of (2) is also subjected to enzyme digestion by adopting BglII and SacI restriction enzymes, and the enzyme digestion is finishedT is adopted as a vector and a target gene ₄ The DNA ligase is connected to obtain recombinant plasmid, the schematic diagram of the finally obtained recombinant plasmid is shown in figure 2, the coding gene of cellobiose 2-epimerase is inserted into the recombinant plasmid, and the inducible promoter P in the expression vector pNZ8148 _nis Substitution with lactose-inducible strong promoter P _lac 。

Then, the constructed recombinant plasmid is electrically transformed into lactobacillus plantarum NZ0203, the transformant obtained after transformation is cultured on MRS solid culture medium added with 10 mug/mL chloramphenicol, positive transformant which grows normally is selected, namely lactobacillus plantarum engineering strain, named lactobacillus plantarum NZ0203/CE.

Example 4: lactose conversion to lactulose Using Lactobacillus plantarum NZ0203/CE

Inoculating lactobacillus plantarum NZ0203/CE to MRS liquid culture medium added with 2% lactose, wherein lactose is an inducer of cellobiose 2-epimerase, and culturing at 37 ℃ for 24 hours, and collecting thalli; the cells were added to 100g/L lactose solution, the amount of the added cells was 8g/L, and the reaction was carried out at 60℃for 4 hours, followed by detection of the sugar component in the reaction solution by liquid chromatography. Wherein the detection conditions of the liquid chromatography are as follows: the column was Utime XB-NH 2-3.6X1250 mm (5 μm), the detector was a differential detector, the mobile phase was acetonitrile-phosphate buffer (1.15 g sodium dihydrogen phosphate in 1000mL water) =84: 16 (v/v) at 40℃and a flow rate of 1.5mL/min.

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

The method is characterized in that the method comprises the steps of collecting bacterial cells again, adding the bacterial cells into 100g/L lactose solution, reacting for 4 hours at 60 ℃ with the addition of 8g/L bacterial cells, repeating the step, and after 5 rounds of reactions, the conversion rate of lactulose can still reach 41.4%, which means that the invention adopts a whole cell catalysis method to produce lactulose, thereby realizing the repeated use of lactobacillus plantarum engineering strain NZ0203/CE and reducing the production cost.

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

Lactobacillus plantarum NZ0203/CE was inoculated into MRS liquid medium supplemented with 10% lactose, and after incubation at 37℃for 24 hours, the sugar content of the fermentation broth was detected by liquid chromatography under the same conditions as in example 4. The results show that the conversion rate of lactulose can reach 45.6%.

Comparative example 1: determination of cellobiose 2-epimerase Activity in different strains

The recombinant plasmids constructed in example 3 were electrotransformed into Lactobacillus plantarum WCFS1 and Lactobacillus plantarum NZ0203, respectively, to obtain engineering strains named Lactobacillus plantarum WCFS1/CE and Lactobacillus plantarum NZ0203/CE.

Lactobacillus plantarum WCFS1/CE and Lactobacillus plantarum NZ0203/CE are respectively inoculated into MRS liquid culture medium added with 2% lactose, wherein lactose is an inducer of cellobiose 2-epimerase, after the culture is carried out for 24 hours at 37 ℃, thalli are collected, and intracellular cellobiose 2-epimerase activity is measured, and the result shows that the activity of the cellobiose 2-epimerase in the Lactobacillus plantarum WCFS1/CE is 85.3% in the Lactobacillus plantarum NZ0203/CE, and the result 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 obviously promoted after beta-galactosidase coding genes lacA and lacLM are knocked out, so that lactose is promoted to be converted into lactulose.

SEQUENCE LISTING

<110> Qilu university of industry

<120> engineering strain of lactobacillus plantarum 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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Val Tyr Val Gln Ala Phe Gly Ile Tyr Gly Leu Ser Glu Tyr Tyr Glu

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Ala Ser Gly Asp Glu Glu Ala Leu His Met Ala Lys Arg Leu Phe Glu

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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

1. An engineering strain of lactobacillus plantarum for converting lactose to generate lactulose is characterized in that the engineering strain is based on lactobacillus plantarum with beta-galactosidase coding genes lacA and lacLM knocked out, and the coding genes of cellobiose 2-epimerase are inserted;

the lactobacillus plantarum from which the beta-galactosidase encoding genes lacA and lacLM are knocked out is lactobacillus plantarum NZ0203, and the lactobacillus plantarum is prepared according to the following method;

(7) Transforming the recombinant plasmid obtained in the step (6) into lactobacillus plantarum WCFS1 with lacA gene knocked out in the step (4), culturing thalli subjected to first homologous exchange through a transformant, screening by using erythromycin, then carrying out continuous passage to generate second homologous exchange, screening strains with erythromycin markers lost, and obtaining lactobacillus plantarum with lacA and lacLM knocked out simultaneously after PCR amplification verification, wherein the lactobacillus plantarum is named lactobacillus plantarum NZ0203;

the lactobacillus plantarum NZ0203 cannot utilize lactose and lactulose;

the cellobiose 2-epimerase is derived from cellulolytic bacteria (Caldicellulosiruptor saccharolyticus), the amino acid sequence of the cellobiose 2-epimerase is shown as SEQ ID NO.1, and the nucleotide sequence of the inserted cellobiose 2-epimerase coding gene is shown as SEQ ID NO. 2.

2. The method for constructing the lactobacillus plantarum engineering strain for converting lactose into lactulose as claimed in claim 1, which is characterized by comprising the following steps:

3. The method of claim 2, wherein in step (2) the promoter P is present _lac The method for obtaining the coding gene of cellobiose 2-epimerase is as follows:

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’。

4. a method as claimed in claim 3The construction method of (1) is characterized in that the promoter P _lac The PCR amplification system of the coding gene is as follows:

the amplification procedure was:

5. The construction method according to claim 3, wherein the PCR amplification system of the cellobiose 2-epimerase encoding gene is:

the amplification procedure was:

6. The method of claim 3, wherein the overlap splice PCR amplification system is:

the amplification procedure was:

7. The method of claim 2, wherein the ligation in step (2) is performed using a promoter P _lac Coding gene of cellobiose 2-epimerase and expression vector pNZ8148 adopts BglII and SacI restriction enzymes to cut respectively, and the target fragment after enzyme cutting adopts T ₄ The DNA ligase performs ligation.

8. The method of construction according to claim 2, wherein the method of transformation in step (3) is an electrotransformation method.

9. The method of construction according to claim 2, wherein the screening in step (3) is performed by: the transformant obtained after transformation is cultivated on MRS solid medium added with 10 mug/mL chloramphenicol, and can grow normally, namely the positive transformant.

10. The method of construction of claim 9, wherein the MRS solid medium comprises the following per liter of components:

11. Use of the lactobacillus plantarum engineering strain according to claim 1 for producing lactulose by using lactose or whey as a substrate.