CN110643561A

CN110643561A - Application of glms gene in lactobacillus biosafety screening marker

Info

Publication number: CN110643561A
Application number: CN201911042417.3A
Authority: CN
Inventors: 武国干; 唐雪明; 孙宇; 邓廷山; 曾海娟
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2020-01-03

Abstract

The invention provides the application of the glms gene in the biological safety screening marker of lactic acid bacteria, the glms gene is used as the biological safety screening marker of lactic acid bacteria, and has no antibiotic pollution and no drug resistance diffusion, thereby being beneficial to the environment, convenient to screen and low in cost; according to the invention, the glms gene of the lactic acid bacteria is knocked out through homologous recombination to obtain a defective strain, the strain cannot survive under the condition of not adding glucosamine, the strain can normally grow under the condition of adding glucosamine, and the growth condition of bacterial cells of the strain is close to that of the original strain without being knocked out; the glms gene is used as a biosafety screening marker of lactic acid bacteria, has extremely high biosafety, is different from the prior selection marker gene derived from the traditional metabolic pathway, has the advantages of convenient operation process, low cost, convenient screening, no limitation of culture medium components and the like, and provides a new important means for the genetic engineering of food microorganisms.

Description

Application of glms gene in lactobacillus biosafety screening marker

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an application of glms gene in a lactic acid bacteria biosafety screening marker.

Background

Glutamine, the 6-phosphofructosyl aminotransferase (L-Glutamine D-Fructose 6-phosphate Amidotransferase, GFAT, EC2.6.1.16), also known as glucosamine-6-phosphate synthase (Glms), is an intracellular enzyme that catalyzes the formation of glucosamine-6-phosphate (glucosamine-6-p) and glutamic acid (glutamate) from the substrates glutamyl (Glutamine) and Fructose-6-phosphate (Fructose-6-p).

GFAT catalyzes the first reaction step in the hexosamine anabolic pathway, the rate-limiting enzyme in this pathway, which is usually irreversible, and consists of two combined enzymatic reactions, the first reaction step being the hydrolysis of glutamine to produce a glutamic acid and a free amino group for transfer to fructose 6-phosphate, and the second reaction step being the conversion of the isoketofructose 6-phosphate to the aldofructose 6-phosphate.

In all eukaryotes and prokaryotes, glucosamine-6-phosphate, one of the products of GFAT metabolism, is catalyzed by a series of conserved enzymatic reactions to form uracil diphosphate-N-acetylglucosamine (UDP-GlcNAc), the final product of the hexosamine pathway. In fungi and arthropods, UDP-GlcNAc is polymerized by chitinase to form chitin.

Chitin is formed by connecting linear N-acetylglucosamine (GlcNAc) residues by β (1-4) glycosidic bonds, and is an important component of cell walls. Experimental studies have shown that chitin synthesis is mainly regulated by GFAT. Uracil diphosphate-N-acetylglucosamine is also a donor for O-glycosylation of intracellular lipids, proteins, and carbohydrates. O-glycosylation is a dynamic process, the transformation rate of which is fast, and the regulation of protein function and assembly between proteins is performed together with the protein phosphorylation process, and the activation of hexosamine pathway leads to the increase of glycosylation of these proteins, thereby regulating the function of cells.

The mutation of the gene coding for GFAT is generally lethal to organisms, the mutant strain of Saccharomyces cerevisiae cannot grow normally, glucosamine is added into a culture medium, and is transported into cells through a hexose transport system, and then forms 6-phosphoglucosamine to directly participate in a hexosamine pathway under the action of hexokinase, and the yeast mutant cells can grow normally without catalysis of GFAT enzyme. After addition of 0-23mM glucosamine to s.cerevisiae medium (YPD medium), the chitin content of the yeast cells rose from the normal 4-5nM GlcN to 14 nM.

The recombinant DNA technology formally established in 1973 has brought modern biotechnology into a completely new genetic engineering era, and from now on, people can design and construct organisms according to their own will to modify organisms and further modify the whole nature, but from now on, the safety problem of the recombinant DNA technology has caused a further violent debate, and from now on, the biological safety becomes an important problem which must be considered in the development of genetic engineering.

In genetic engineering, selection markers are important components of vectors, commonly used selection markers mainly comprise auxotroph markers and resistance selection markers, and the biological safety is firstly the safety of selection marker genes. The resistance screening marker has the problems of antibiotic residue, drug resistance diffusion and the like, and has great influence on the environment; the auxotrophy marker used in the prior art is mainly related genes in amino acid and nucleotide biosynthesis pathways, but the isolation of auxotrophy complementary strains is difficult, and the auxotrophy is used as a screening marker, so that the reversion is easy to generate, the requirements on a culture medium are strict, and the auxotrophy cannot contain trace components capable of complementing the auxotrophy, so that only a limit culture medium can be used frequently, and the growth of the strains and the industrial fermentation application are seriously influenced.

The biosafety marker genes discovered in recent years mainly comprise a green fluorescent protein Gene (GFP), a ribitol operon (rtl), a 6-phosphomannose isomerase gene (pmi), a xylose isomerase gene (xylA), a glutamic acid-1-semialdehyde transaminase gene (hemL) and the like, the screening efficiency of the marker genes is not very high, the application is not very wide, certain requirements on enzyme systems of hosts are met, and the development of biosafety screening markers for lactic acid bacteria is not reported.

Lactic Acid Bacteria (LAB) with a wide range of application values are one of the most extensively studied microorganisms of the safe as safe (GRAS) type. However, some plasmid vectors and corresponding molecular elements such as selectable markers applied to lactic acid bacteria carry antibiotic resistance genes such as chloramphenicol and erythromycin, which brings great biological safety hazards.

In order to avoid the transfer of antibiotic resistance genes into the environment or other organisms, the U.S. FDA and other authorities prohibit the use of antibiotic selection markers for GRAS microorganisms, and a selection marker system which does not meet the standard limits the deep development and utilization of lactic acid bacteria, so that a lactic acid bacteria non-antibiotic resistance selection marker system becomes a research frontier and a hotspot in the field.

Disclosure of Invention

The invention aims to provide application of glms gene in a lactic acid bacteria biosafety screening marker, wherein the glms gene of lactic acid bacteria is knocked out through homologous recombination to obtain a defective strain, the strain cannot survive under the condition of not adding glucosamine, the strain can normally grow under the condition of adding glucosamine, and the growth condition of the bacterial cells is close to that of the original strain which is not knocked out; the glms gene is used as a biosafety screening marker of the lactic acid bacteria, has no antibiotic pollution and no drug resistance diffusion, is beneficial to the environment, convenient to screen and low in cost, is used for a series of genetic engineering of gene knockout, gene expression and the like of the lactic acid bacteria, and lays a solid foundation for the application of the novel screening marker in the genetic engineering of the lactic acid bacteria.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides an application of glms gene in a lactic acid bacteria biosafety screening marker.

Preferably, the lactic acid bacteria are lactococcus lactis.

A construction method of a glms gene deficient lactobacillus strain comprises the following steps:

1) extracting lactobacillus genome DNA and dissolving in TE buffer solution;

2) activating lactobacillus strain to prepare competent cells;

3) taking the genomic DNA of the lactobacillus as a template, taking sequences of 200-300bp respectively at the upstream and downstream of the glms gene of the lactobacillus as homologous arms, and obtaining homologous integration plasmids through homologous recombination;

4) transforming the homologous integration plasmid into a lactic acid bacteria competent cell for culture, knocking out glms gene of lactic acid bacteria, screening glms gene deletion strains, and obtaining the glms gene deletion lactic acid bacteria strain through PCR and physiological and ecological double verification.

Further, in the step 3), in the homologous recombination process, the sequence of the homology arm is respectively cloned into a pUC19 vector, and a homologous integration plasmid pUTery-glms containing the upstream and downstream homology arms is constructed.

Further, in the step 4), 2-5 groups of primers are used for verification during PCR verification.

In addition, in the case of physiological and ecological verification, glucosamine concentration test is performed on the deficient strain, and the glucosamine concentration is 0.5-1 mM.

Preferably, the lactic acid bacterium is lactococcus lactis NZ 9700.

The biosafety screening marker gene selected by the invention is a glms gene, which is a coding gene of GFAT, is a first-step rate-limiting enzyme in a hexosamine pathway, is generally present in various species, is a relatively conservative gene, is a housekeeping gene in many species, is lethal to deletion and mutation of the gene, and can normally grow by adding a small amount of cheap glucosamine in a culture medium.

The invention knocks out glms gene of lactococcus lactis through homologous recombination to obtain defective strain, the strain can not survive without adding glucosamine, glms gene from different strains of the same strain is cloned into lactococcus lactis expression vector pNZ8030 to obtain recombinant expression plasmid pNZ8030-glms, the recombinant plasmid is transformed into defective strain for expression, the defective strain can grow normally without adding glucosamine, and the glms plays a role in lactococcus lactis as a biosafety screening marker.

The conserved enzyme gene glms of the invention is used as a screening marker gene, has extremely high biological safety, is different from the prior selection marker gene derived from the traditional metabolic pathway, and is used as a biological safety screening marker of genetic engineering, has the advantages of convenient operation process, low cost, convenient screening, no limitation of culture medium components and the like, and provides a new important means for the genetic engineering of food microorganisms.

Compared with the prior art, the invention has the following beneficial effects:

when the upstream and downstream homologous arms of the glms gene are selected, the length of 200 bp and 300bp respectively at the upstream and downstream is selected, so that cloning is easy, and homologous recombination is realized.

The gene source selected by the invention is the glms gene from different strains of lactococcus lactis, so that the gene can be correctly expressed in defective strains to realize anaplerosis.

The glms gene deletion strain obtained in the invention can normally grow under the condition of adding 1mM glucosamine, and the growth condition of the bacterial cells is close to that of the original strain without deleting the glms gene.

Drawings

FIG. 1 shows the results of restriction enzyme identification of plasmid pUC19-up in the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, and 2 is pUC19-up EcoRI, NcoI cut fragment.

FIG. 2 shows the results of the restriction enzyme identification of plasmid pUC19-ud in the example of the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, and 2, 3, and 4 are fragments of pUC19-ud digested with EcoRI and NcoI.

FIG. 3 is a PCR verification chart of upper and lower glms upper and lower homology arm primers up-N, down-C in the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, 2, 3 are electrophoresis bands of upper and lower homology arm primers of glms of gene homology integration transformant, and 4 is original strain control.

FIG. 4 is a PCR verification chart of primers glms-N and glms-C of glms gene in the example of the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, 2 and 3 are amplification bands of glms gene of transformant with homologous integration of gene, and 4 is a control of original strain.

FIG. 5 is a PCR verification chart of primers ec-1 and ec-3 flanking sequences of homologous regions in the example of the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, 2 is an original strain control, and 3 and 4 are amplification bands of gene homologous integration transformants.

FIG. 6 is a PCR verification chart of primers ec-2 and ec-4 flanking sequences of homologous regions in the example of the present invention, wherein 1 is lambda-EcoT 14I DNA Marker, 2 is an original strain control, and 3 and 4 are amplification bands of gene homologous integration transformants.

FIG. 7 is a PCR verification chart of primers ec-1 and ec-2 flanking sequences of homologous regions in the example of the present invention, wherein 1 is a lambda-EcoT 14I DNA Marker, 2 and 3 are amplification bands of gene homologous integration transformants, and 4 is an original strain control.

FIG. 8 is a schematic diagram of the position of the PCR verification primer in the embodiment of the present invention.

FIG. 9 shows the growth of L.lactis NZ9700 Δ g in M17 medium at various glucosamine concentrations in examples of the present invention.

FIG. 10 is a cleavage map of the vector pNZ8030 in the present embodiment.

FIG. 11 shows the restriction identification of plasmid pNZ8030g in the present invention, wherein 1 and 2 are NcoI of pNZ8030g, Hind III is restriction verified, and 3 is a lambda-EcoT 14I DNA Marker.

FIG. 12 shows the transformation results of plasmid pNZ8030g on L.lactis NZ 9700. delta.g in the example of the present invention, wherein A is the growth of the medium without glucosamine added thereto, and B is the growth of the medium with glucosamine added thereto.

Detailed Description

The present invention is further illustrated by the following specific examples.

Expression vector pUCERY sources: pUC19 is a laboratory preservation plasmid, and pUCERY is obtained through cloning and transformation.

Example construction of a glms Gene deficient Strain of lactococcus lactis NZ9700

1. Extraction and detection of lactococcus lactis genomic DNA

Lactococcus lactis genomic DNA was extracted according to literature (Ausubel f.m., Brent r.and Kingston R.E, et al short procedures In Molecular biology.3rd ed.john Wiley & Sons, inc., 1995) In the following specific procedures:

culturing 5ml of lactococcus lactis bacterial liquid to a saturated state, and centrifuging 1.5ml of bacterial liquid for 2 min; adding 567 μ l TE buffer solution into the precipitate, repeatedly pipetting with a pipette to resuspend, adding 30 μ l 10% SDS and 3 μ l 20mg/ml proteinase K, mixing, and incubating at 37 deg.C for 1 h; adding 100 μ l of 5M NaCl, mixing, adding 80 μ l of CTAB/NaCl solution, mixing, and incubating at 65 deg.C for 10 min; equal volume of chloroform was added: isoamyl alcohol (24: 1), mixing uniformly, and centrifuging for 4-5 min. Transferring the supernatant into a new tube, and removing the interface substances with toothpick if the supernatant is difficult to remove; equal volume of phenol was added: chloroform: isoamyl alcohol (25: 24: 1), mixing, centrifuging for 5min, and transferring the supernatant into a new tube; adding 0.6 volume of isopropanol, mixing gently until DNA precipitates, and washing with 70% ethanol; centrifuge for 5min, discard the supernatant, air dry, redissolve in 100. mu.l TE buffer.

2. Preparation and electrotransformation of lactis competent cells

Activating lactococcus lactis strain NZ9700, culturing in small amount, inoculating to prepare competent L.lactis cell, and converting:

inoculating single colony of L.lactis NZ9700 strain of lactococcus lactis into 10ml of M17 culture solution, and culturing at 30 ℃ overnight; adding 5ml of culture solution into a 2L triangular flask containing 500ml of LB culture solution, and culturing at 30 ℃ until OD600 is 0.3-0.4; cooling the bacteria culture solution in ice water bath for 10-15 min, transferring to a precooled 1L centrifugal bottle, centrifuging at 2 ℃ for 10min at 5000g, and dissolving the precipitate with 5ml precooled water; adding 300ml of ice-cold water, mixing uniformly, repeatedly centrifuging for 1 time according to the step 3, immediately pouring out supernatant, and washing with ice-cold water again; adding 200ml of ice-cold 10% (v/v) glycerol, mixing uniformly, then centrifuging for 10min at 2 ℃ and 5000g, estimating the volume of the precipitate, adding the same volume of ice-cold 10% (v/v) glycerol, resuspending the cells, subpackaging 50-100 μ l of the suspension in a precooled microcentrifuge tube, and storing at-80 ℃ to obtain competent cells.

And (3) electric conversion: adjusting the electric converter to 2.5kV and 25 muF, adjusting the pulse controller to 200-400 omega, adding the DNA to be converted into the competent cells frozen and thawed on ice, and mixing uniformly; transferring the mixture to be converted into a pre-cooled electric conversion pool, sucking the outer surface of the pool, and then putting the pool into a sample groove; pulse electrotransformation is carried out, 1ml of culture solution is immediately added, and the selective plate is coated after static culture for 2 hours at 30 ℃.

3. Construction of homologous integration plasmid pUTery-glms

Taking the provided L.lactis NZ9700 genome DNA as a template, obtaining a glms gene sequence and upstream and downstream flanking sequences thereof according to a genome sequence (NC-013656) of L.lactis MG1363 in Genbank, designing primers up-N and up-C, amplifying the upstream flanking sequence, wherein EcoRI and NcoI enzyme cutting sites are underlined, and PCR amplification parameters are as follows: denaturing at 95 deg.c for 5min and adding Pyrobest DNA polymerase; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. After the PCR amplification product is purified, cutting enzyme by EcoRI and NcoI, tapping and recovering, precipitating by ethanol, and concentrating to obtain a purified target fragment, wherein the primer sequences are as follows:

up-N：5’-CCCGAATTCTTTTGTAGGGGTCCATTATCCG-3’；

up-C：5’-CCCCCATGGAATAAAGCTCCACTTAACTCACC-3’。

the downstream flanking sequence was amplified using the proposed L.lactis NZ9700 genomic DNA as template and primers down-N, down-C, the underlined parts being NcoI, XbaI restriction sites. PCR amplification parameters: denaturing at 95 deg.c for 5min and adding Pyrobest DNA polymerase; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extending for 10min at 72 ℃; preserving heat at 4 ℃, purifying a PCR amplification product, performing enzyme digestion and purification to obtain a target fragment, wherein the primer sequence is as follows:

down-N：5’-CCCCCATGGAAATTTACTGACAGAAAGGTC-3’；

down-C：5’-CCCTCTAGAAAAATTATCCAATGTCCCAAAA-3’。

the upstream and downstream flanking sequences were cloned into the vector pUC19 digested with the same enzymes, respectively, to obtain a plasmid pUC-glms.

The expression vector pUCERY was digested with BamHI and XbaI to obtain a fragment containing the erythromycin resistance gene (ery), the cohesive ends were filled in with Klenow fragment of E.coli DNA polymerase I, and the fragment was cloned into BamHI-digested plasmid pUC-glms to obtain a homologous integration plasmid pUCEry-glms for homologous recombination.

The gene of glms for coding glutamine-6-phosphofructosyl aminotransferase in lactococcus lactis NZ9700 is 1818bp, in this example, 300bp outside the open reading frame of the gene are selected as the sequences of upstream and downstream homology arms in order to completely knock out the glms gene from the genome.

The upstream fragment was inserted between EcoRI and NcoI cleavage sites of plasmid pUC19 to obtain plasmid pUC19-up, and the constructed plasmid pUC19-up was verified by cleavage, and when cleaved with both EcoRI and NcoI, two fragments of about 2.6kb and 0.3kb were cleaved (FIG. 1).

The downstream fragment was inserted between NcoI and XbaI in plasmid pUC19-up using this plasmid as a vector, to obtain plasmid pUC 19-ud. The constructed plasmid pUC19-ud was verified by digestion, and when digested simultaneously with NcoI and XbaI, two fragments of about 2.9kb and 0.3kb were excised (FIG. 2). Thus, the upstream and downstream homology arms were cloned into the pUC19 vector, respectively.

And carrying out enzyme digestion on pUCERY with BamHI and XbaI to obtain a fragment of a 1.6kb erythromycin resistance gene (ery), cloning the fragment into a plasmid pUC19-ud subjected to enzyme digestion with BamHI to obtain a plasmid pUCEry-glms for homologous recombination, and carrying out enzyme digestion verification on the constructed plasmid pUCEry-glms to obtain the plasmid pUCEry-glms.

Knock-out of glms Gene

Electrically transforming the homologous integration plasmid pUTery-glms into L.lactis NZ9700 competent cells in a medium M17 (with 1% glucose) and 2mM glucosamine, screening erythromycin-resistant colonies on the plates, coating the plates without glucosamine after transformation, setting a control group without sample DNA, and standing at 30 ℃.

In the case of the plates coated with the sample groups, more than one hundred single colonies were grown on the erythromycin-and glucosamine-containing M17 plates, and a few single colonies were grown on the erythromycin-only M17 plates, and theoretically, cells transformed into the fragment and integrated at the correct gene position could not grow on the erythromycin-only M17 plates without added glucosamine, but this most ideal case was almost impossible, and the colonies grown on the erythromycin-containing M17 plates should mostly be non-specifically integrated to obtain resistant cells, and some unstable homologous recombination cells, and a few transformants were likely to produce spontaneous resistant mutations. In the control group, the presence of sterile colonies on both erythromycin and glucosamine-containing M17 plates and erythromycin-only M17 plates was the most desirable, as was the control group, which had no transferred fragment, and the original cells did not acquire resistance, nor did there be any spontaneous resistance mutations.

5. Screening for glms Gene-deleted Strain

1) PCR validation

A single colony of l.lactis on an M17 plate containing erythromycin and 2mM glucosamine in the sample group was picked up in a liquid M17 medium to which 150g/ml of erythromycin and 2mM glucosamine were added, followed by further transfer to a liquid M17 medium to which only erythromycin-resistant and glucosamine was not added.

Extracting genome DNA respectively, measuring concentration, performing PCR verification by using the genome DNA as a template after running gel detection, using five pairs of primers in amplification, and using the primers and PCR conditions in verification as shown in the following table 1.

TABLE 1 primers for validation of glms Gene knockout

In the control of the sample group, 8 single colonies picked from the erythromycin-only M17 plate were not grown in the liquid medium of M17 supplemented with erythromycin and glucosamine, indicating that the colonies grown on the control erythromycin plate of the sample group are not stable resistant transformants, and six single colonies picked from the Ery-and glucosamine-containing M17 plate were grown and verified by PCR using the genomic DNA.

PCR verification with the first set of primers, up-N and down-C, shows that the length of the glms gene is 1818bp, the expression cassette of the Ery resistance gene is 1.6kb, if the glms gene is replaced by Ery, the upstream and downstream primers are used for amplification, the length of the homology arm is calculated, the correct knockout sample is 2.2kb, and the length when the glms gene is not replaced is about 3.0kb, and the result after amplification with the set of primers is shown in FIG. 3.

PCR verification using the second set of primers glms-N and glms-C, which should not be amplified if the target gene is knocked out, is shown in FIG. 4, wherein none of these samples amplified the corresponding band of the target gene. The original strain without gene knockout can be amplified to the size of Glms-encoding gene in the lactococcus lactis strain under the same PCR condition, and the band is 2.3 kb.

PCR verification is carried out by using a third group of primers ec-1 and ec-3, the primer ec-1 is positioned at 1.5kb upstream of an open reading frame of the glms gene, ec-3 is positioned at about 600bp away from an N-terminal promoter in an Ery resistance gene expression cassette, the correct length is 2.1kb by using the group of primers for amplification, the band cannot be amplified when the band is not correctly knocked out, and the result after amplification by using the group of primers is shown in figure 5.

PCR verification is carried out by using a fourth group of primers ec-2 and ec-4, wherein the primer ec-2 is positioned at 1.2kb downstream of an open reading frame of a glms gene, the ec-4 is positioned at about 600bp from the N end of a promoter in an Ery resistance gene expression cassette, 1.0kb from the C end of a terminator is positioned at the same position as the primer ec-3, only the sequence direction is opposite, the sequence is reversely complementary, the amplification is carried out by using the group of primers, the correct length is 2.2kb, the band cannot be amplified when the band is not correctly knocked out, and the result after the amplification is carried out by using the group of primers is shown in figure 6.

PCR verification was performed using the fifth set of primers ec-1 and ec-2, the correct length of the full-length knocked-out amplified with this set of primers should be 4.3kb, the length of the band not knocked-out correctly should be 5.1kb, the results after amplification with this set of primers are shown in FIG. 7, and the schematic position of the primers is shown in FIG. 8.

2) Morphological physiological verification

After single colonies of lactococcus lactis on M17 plates containing erythromycin and 2mM glucosamine in the sample groups were cultured in a liquid medium containing glucosamine, 10. mu.l of the bacterial solution was diluted and applied to M17 plates containing erythromycin and glucosamine and M17 plates containing erythromycin alone. After the single fungus grows out, the single fungus is picked into a liquid M17 culture medium, erythromycin and glucosamine are added into one tube of the culture medium, and erythromycin is added into the other tube.

After single colonies of lactococcus lactis on M17 plates containing erythromycin and 1mM of gluconamine in the sample group were cultured in liquid M17 medium to which the same concentration of glucosamine was added, 10. mu.l of diluted lactococcus lactis was applied to M17 plates containing erythromycin and gluconamine and M17 plates containing erythromycin alone. Colonies could not grow on M17 plates containing only erythromycin, but could grow on a medium containing erythromycin and glucosamine, and when colonies on the plates were cultured in M17 liquid medium containing erythromycin and glucosamine, cells grew normally, whereas cells could not grow in the liquid medium containing only erythromycin.

3) Glucosamine concentration test

Glucosamine was added to the liquid M17 medium at various concentrations to give final concentrations of 0mM, 0.125mM, 0.25mM, 0.5mM and 1mM, and a single colony was picked from a plate containing erythromycin and glucosamine in the liquid M17 medium and inoculated in the same inoculum size in the liquid M17 medium containing glucosamine at various concentrations, as controlled by the L.lactis NZ9700 strain.

In the liquid M17 medium in which the final glucosamine concentration was 0mM, 0.125mM, 0.25mM, 0.5mM, and 1mM, respectively, the strain failed to grow normally without the addition of glucosamine, the growth of the lactococcus lactis strain was better as the glucosamine concentration increased, and the growth of L.lactis NZ 9700. delta.g was close to that of the cell of the original strain in which the gene was not knocked out in the medium in which the final glucosamine concentration was 1mM, and therefore, the optimum glucosamine concentration was 1mM for the glms gene-deficient strain L.lactis NZ 9700. delta.g (FIG. 9).

4) Glms gene deletion type lactococcus lactis strain

After the L.lactis NZ9700 strain with the complete glms gene knockout is purified by three rounds of streaking, the strain is preserved with 15% of glycerol and frozen at-70 ℃, and the strain is L.lactis NZ9700 delta g.

Expression of L.lactis MG 1363-derived glms Gene in L.lactis NZ 9700. delta.g

1) Construction of plasmid pNZ8030-glms

Using a vector pNZ8030 as a template, designing primers pNZ8030c1 and pNZ8030c2 to perform reverse PCR, removing a chloramphenicol resistance gene Cm on the vector, performing self-cyclization, performing ligation, and verifying to obtain a plasmid pNZ8030c, wherein the primer sequence is as follows:

pNZ8030c1：5’-ATGTGTGGGATTTTTGCTTACTG-3’；

pNZ8030c2：5’-TTACTCCGTAGTGACAGATTTAGC-3’。

using genome DNA derived from strain L.lactis MG1363 as a template, designing primers glms-N and human glms-C, amplifying glms gene, wherein the underlined parts of the primers are NcoI and Hind III enzyme cutting sites, and connecting the primers with pNZ8030C cut by the same enzyme to obtain a recombinant expression plasmid pNZ8030-glms, wherein the size of the vector pNZ8030 is 3.8kb as shown in a plasmid map in FIG. 10, and the plasmid is a lactobacillus-escherichia coli shuttle vector and comprises a replicon capable of replicating in escherichia coli, a chloramphenicol screening marker Cm, an inducible promoter nisA, a transcription terminator and the like.

Wherein the primer sequences are as follows:

glms-N：5’-CCCCCATGGTTTTGTAGGGGTCCATTATCCG-3’；

glms-C：5’-CCCAAGCTTAATAAAGCTCCACTTAACTCACC-3’。

in this example, glms was used as a selection marker for yeast, and a chloramphenicol selection marker Cm on the pNZ8030 vector was not required, so it was desired to remove the Cm marker from the vector, and a reverse PCR and self-circularization method were selected for smaller vectors to obtain the pNZ8030c vector, which was verified to have the correct size by single restriction with NcoI, and the glms gene derived from l.lactis MG1363 was cloned into this plasmid to obtain pNZ8030-glms, NcoI, and hindiii which were verified to have the correct size by restriction with enzyme (fig. 11).

2) Expression of recombinant plasmid pNZ8030-glms

Electrically transforming the recombinant expression plasmid pNZ8030-glms into L.lactis NZ9700 delta g competent cells, adding Nisin with the final concentration of 10 mu g/l into M17 culture medium without adding glucosamine for induced expression, culturing at 30 ℃, setting the control of unloaded plasmid, and simultaneously coating M17 culture medium with or without adding glucosamine. This transformation experiment was repeated three times, and a single colony of lactococcus lactis on a plate without glucosamine added to the sample group was picked up in a liquid medium in which glucosamine was also not added.

Electrically transforming a recombinant expression plasmid pNZ8030-Glms into L.lactis NZ9700 delta g competent cells for expression, adding Nisin with a final concentration of 10 mu g/l in M17 medium without adding glucosamine for induction expression, and then culturing at 30 ℃, wherein if a gene from L.lactis MG1363 encoding Glms can be expressed in L.lactis NZ9700 delta g, the Glms gene-deficient lactococcus lactis cells can be grown on the medium without adding glucosamine. After 2 days, colonies were grown on M17 medium without glucosamine, the control group was transferred to a plate without insert and empty plasmid, colonies were grown without glucosamine, and colonies with lactic acid bacteria were grown on M17 medium with glucosamine, as shown in FIG. 12.

Figure 12 shows that glms is an essential gene in lactococcus lactis and that the glms gene from l.lactis MG1363 can be correctly expressed in l.lactis NZ9700 Δ g, complementing the glms gene deficiency of l.lactis NZ 9700.

Under the condition of adding 1mM of glucosamine, the glms gene deletion strain L.lactis NZ9700 delta g can normally grow, and the growth condition of bacterial cells is close to that of the L.lactis NZ9700 of the original strain without knockout; cloning glms gene from L.lactisMG1363 into lactococcus lactis expression vector pNZ8030 to obtain recombinant expression plasmid pNZ8030-glms, transforming the recombinant plasmid into L.lactis NZ9700 delta g for expression, and enabling the defective strain to grow normally without adding glucosamine.

Claims

The application of glms gene in lactobacillus biological safety screening marker.
2. The use of the glms gene of claim 1 in a biosafety screening marker for lactic acid bacteria, wherein said lactic acid bacteria is lactococcus lactis.
3. A construction method of a glms gene deficient lactobacillus strain comprises the following steps:

1) extracting lactobacillus genome DNA, and dissolving in TE buffer solution;

2) activating lactobacillus strain to prepare competent cells;

3) taking the genomic DNA of the lactobacillus as a template, taking sequences of 200-300bp respectively at the upstream and downstream of the glms gene of the lactobacillus as homologous arms, and obtaining homologous integration plasmids through homologous recombination;

4) transforming the homologous integration plasmid into a lactic acid bacteria competent cell for culture, knocking out glms gene of lactic acid bacteria, screening glms gene deletion strains, and obtaining the glms gene deletion lactic acid bacteria strain through PCR and physiological and ecological double verification.
4. The method for constructing a glms gene-deficient lactic acid bacterial strain according to claim 3, wherein in step 3), the homology arm sequences are cloned into the pUC19 vector during the homologous recombination process, so as to construct a homologous integration plasmid pUTery-glms containing upstream and downstream homology arms.
5. The method for constructing a strain of lactic acid bacteria deficient in the glms gene as claimed in claim 3, wherein in step 4), the PCR is verified by using 2-5 sets of primers.
6. The method for constructing a strain of lactic acid bacteria deficient in the glms gene as claimed in claim 3, wherein the strain deficient in the physiological ecology test is subjected to glucosamine concentration test, wherein the glucosamine concentration is 0.5-1 mM.
7. The method of constructing a strain of lactic acid bacteria deficient in the glms gene according to any one of claims 3 to 6 wherein said lactic acid bacteria is lactococcus lactis.
8. The method of claim 7, wherein said L.lactis strain is L.lactis NZ 9700.