CN114634938A

CN114634938A - Application of lactobacillus plantarum gene fol KE in folic acid biosynthesis

Info

Publication number: CN114634938A
Application number: CN202210212625.9A
Authority: CN
Inventors: 柳陈坚; 孙洪琪; 李晓然
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2022-03-06
Filing date: 2022-03-06
Publication date: 2022-06-17

Abstract

The invention discloses a lactobacillus plantarum (A)Lactobacillus plantarum) GenefolNew application of KE, namely application of KE in folic acid biosynthesis, and genesfolKE is shown as SEQ ID NO 1; the invention constructs a temperature-sensitive plasmid pFED760 through a homologous recombination technologyfolKnockout strain delta of KE genefolKE, observing the cell shape and the growth state of the strain, measuring the folate production capability of the strain, and displaying that compared with the wild strain, the DeltafolThe folic acid production amount of the KE bacterial strain is obviously reduced,folthe KE gene plays a key role in folic acid synthesis, and the invention is favorable for improving the position of the key gene in folic acid synthesisThe yield of folic acid lays the early foundation for the development of folic acid food.

Description

Application of lactobacillus plantarum gene fol KE in folic acid biosynthesis

Technical Field

The invention belongs to the field of folic acid biosynthesis application, and particularly relates to application of a lactobacillus plantarum gene folKE in lactobacillus plantarum folic acid biosynthesis.

Background

Folic acid (also called pteroylglutamic acid), vitamin B9, anti-anemia factor, vitamin M, vitamin Bc, etc., is a water-soluble vitamin. Folic acid is yellow crystal, slightly soluble in water, insoluble in ethanol, and easily decomposed by light. Folate deficiency causes a number of diseases, commonly seen as megaloblastic anemia, cleft lip and palate, and depression.

Natural folic acid, although widely available, is not satisfactory for human consumption on a daily basis, particularly in pregnant women, due to its own instability and high rate of loss during cooking. In addition, due to geographical location factors, more people living in remote areas are less able to ingest daily needs. Therefore, the development of folic acid-rich foods or related preparations is the best solution to the problem.

Folic acid can be synthesized by industrial production, but in order to protect the environment, a plurality of plants are shut down, and thus it is imperative to seek an environmentally friendly synthesis method. Biosynthetic folic acid has incomparable advantages, most notably natural folic acid exists mostly in the form of pteroylpolyglutamic acid, and is not easy to form dependency on folic acid compared with pteroylmonoglutamic acid of synthetic folic acid. Since the liver is bound to be burdened if dependency is formed, naturally synthesized folic acid is required to solve the problem of insufficient folic acid intake.

Folic acid synthesized by lactobacillus plantarum is more competitive in the market due to its safety, but also suffers from a problem of low yield of folic acid. Therefore, it is necessary to explore the action mechanism of the key enzyme gene for the biosynthesis of folic acid by lactobacillus plantarum, so as to lay the foundation for the development of folic acid food in the future.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of a Lactobacillus plantarum (Lactobacillus plantarum) gene folKE gene, namely the application of the gene folKE in Lactobacillus plantarum folic acid synthesis, wherein the nucleotide sequence of the gene folKE is shown as SEQ ID NO. 1.

The invention clones the upstream and downstream homologous arms (991 bp and 1000 bp respectively) of gene fol KE from Lactobacillus plantarum YM-4-3 by the following primers:

up-KEF: 5'-GCGTCGACACGCACTTTTTTCGGTTCC-3' and up-KER: 5'-CCTGTATCCACTCAATCACCTAACTCACTCACATTTCT-3';

down-KEF: 5'-AGAAATGTGAGTGAGTTAGGTGATTGAGTGGATACAGG-3' and down-KER: 5'-CGGAATTCGCCACTAGTGCTGCCATGC-3', respectively;

the upstream and downstream homologous arms of folKE gene and temperature sensitive plasmid pFED760 are respectively cut by SalI and EcoRI and then connected to obtain a knockout vector, the knockout vector is then electrically transferred into Lactobacillus plantarum YM-4-3 competent cells, a knockout strain delta folKE is obtained after bacterial liquid verification is selected, strain growth states and folic acid yields of wild type strain YM-4-3 and knockout strain delta folKE are compared through experiments, and therefore the folKE gene plays a key role in strain growth state and folic acid synthesis.

Compared with the prior art, the invention has the following advantages:

1. the gene is from food-borne lactobacillus plantarum, has high safety and can be used in the field of later-stage food fermentation;

2. the food-borne lactobacillus plantarum is used for generating the folic acid, so that the production cost is reduced, and the economic benefit can be improved;

3. compared with the industrial synthesis of folic acid, the biological synthesis of folic acid has better environmental friendliness;

4. the folKE gene plays a key role in folic acid synthesis, provides a certain theoretical basis for research and development of folic acid synthesis functional foods, is simple to operate, and is suitable for industrial production and market popularization and application.

Drawings

FIG. 1 shows PCR verification of bacterial liquid of folKE gene knockout strain of the present invention, wherein lane M: BM5000 DNA marker;

lanes

1, 2, 4 and 5: knock out the genomic DNA of the strain folKE as a PCR product of the template; lane 3: taking the genome DNA of the lactobacillus plantarum wild strain as a PCR product of a template; 6 is blank control;

FIG. 2 is a scanning and transmission electron microscope image of a wild strain and a knock-out strain of Lactobacillus plantarum of the present invention; a: scanning electron microscope images of wild strains; b: knocking out a strain fol KE scanning electron microscope picture; c: transmission electron microscope picture of wild type strain; d: knocking out a strain fol KE transmission electron microscope picture;

FIG. 3 shows the growth status of the bacterial liquid of the wild Lactobacillus plantarum strain (A) and the Δ folKE (B) knockout strain of the invention;

FIG. 4 shows the OD of the wild type strain and the fated strain of Lactobacillus plantarum of the present invention₆₀₀And folic acid content.

Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and conventional methods. The temperature sensitive plasmid pFED760 was gifted by Philippines J Federle, university of Illinois; the results in the following examples are all the average values of three replicates unless otherwise specified.

Example 1: cloning of upstream and downstream homology arms of gene folKE

1. Upstream and downstream homology arms for PCR amplification

Extracting total DNA of a food-borne lactobacillus plantarum YM-4-3 genome by using a CTAB/enzyme method, taking the extracted genome as a template, and respectively amplifying upstream and downstream homologous arms of a folKE gene by using a primer pair up-KEF (5'-GCGTCGACACGCACTTTTTTCGGTTCC-3', underlined is SalI enzyme cutting site) + up-KER (5'-CCTGTATCCACTCAATCACCTAACTCACTCACATTTCT-3') and down-KEF (5'-AGAAATGTGAGTGAGTTAGGTGATTGAGTGGATACAGG-3') + down-KER (5'-CGGAATTCGCCACTAGTGCTGCCATGC-3', underlined is EcoRI enzyme cutting site), wherein a PCR reaction system and amplification conditions are as follows:

(1) PCR reaction system

；

(2) PCR amplification conditions

Pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 s; annealing at 60-63 ℃ for 30 s; extending for 1min at 72 ℃; circulating for 30 times; extending at 72 ℃ for 5min, storing at 12 ℃ (upstream annealing temperature 60 ℃ and downstream annealing temperature 63 ℃), taking 5 mu L after the reaction is finished, and carrying out electrophoretic analysis in 1% agarose gel, wherein the nucleotide sequences of the upstream and downstream homologous arms of the folKE gene are shown as SEQ ID NO: 2 and SEQ ID NO: 3.

2. Cloning and sequencing of gene knockout fragment

1 mu L of each downstream homology arm PCR product is taken as a template, up-KEF and down-KER are taken as primers, and overlapping PCR is carried out according to the PCR reaction system and the amplification conditions (the annealing temperature is 61 ℃, and the extension time is changed to 2 min). The PCR product (i.e., the knockout fragment) of the expected size was recovered by cutting the gel and ligated into the pMD19-T vector according to the TA cloning kit instructions of Dalibao Bio (China). The ligation products were introduced into E.coli DH 5. alpha. competent cells by heat shock transformation and plated on Amp-LB plates. After overnight culture at 37 ℃, randomly selecting 10-15 single colonies, extracting plasmids in cells, carrying out enzyme digestion verification by using SalI and EcoRI, and sending positive plasmids to a sequencing company for sequencing.

Example 2: construction of fol KE Gene knockout vector

Synchronously digesting the gene knockout fragment with correct sequencing and the temperature-sensitive plasmid pED 760 by using restriction enzymes SalI and EcoRI respectively, wherein the digestion system is as follows: SalI, 1 muL; EcoRI, 1 μ L; 10 XH buffer, 2 μ L; knocking out a fragment or pFAD 760, 10-16 muL; adding sterilized deionized water to 20 mu L, and carrying out enzyme digestion at 37 ℃ for 4 h; and (3) recovering the enzyme digestion product, adding the sample according to the molar ratio of the target gene to the vector = 4: 1-2: 1, adding T4 DNA ligase, and connecting for 12-16 h at 16 ℃. The ligation products were introduced into E.coli DH 5. alpha. competent cells using heat shock transformation and subsequently plated onto erythromycin-LB solid plates. After overnight culture at 28 ℃, plasmids in 10-15 single colony cells are extracted and subjected to enzyme digestion verification by SalI and EcoRI to obtain positive plasmids which are named as pFED 760-folKE.

Example 3: folKE Gene knock-out Strain construction

1. Introduction of folKE gene knockout vector into lactobacillus plantarum competent cells

Lactobacillus plantarum competent cells were prepared as follows: thawing Lactobacillus plantarum YM4-3 strain, inoculating to MRS broth culture medium at 4 ‰ inoculum size, standing at 37 deg.C for 12h, inoculating 1mL into 50mL MRS broth culture medium containing 2.5% glycine, culturing for 6h, and stopping culturing when OD600 value reaches 0.6. Centrifuging at 4 deg.C and 4000 rpm/min for 10min to collect bacterial liquid, washing with 25mL sterilized sterile water twice, centrifuging again, and performing low temperature operation. Discarding the supernatant, resuspending the cells in 0.05mol/L EDTA solution, ice-cooling for 5min, adding 25mL ice-cold sterilized water, centrifuging at 4 deg.C and 8000rpm/min for 5min, washing with 25mL ice-cold sterilized water again, centrifuging with 25mL shock buffer (0.5mol/L sucrose, 10% glycerol) at 4 deg.C and 8000rpm/min for 10min, repeating the above steps once, finally resuspending the cells in 0.8mL shock buffer, packaging the above mixture in 90 μ L volumes into sterilized 1.5mL centrifuge tubes (handling on ice when dispensing is noted), and storing at-80 deg.C.

Adding 10 mu L of gene knockout carrier pFED760-folKE into 90 mu L of lactobacillus plantarum YM-4-3 competent cells, gently mixing uniformly, carrying out ice bath for 5min, and then transferring into a precooling electric shock cup (with the distance of 2mm) for electric shock. Quickly adding 900 mu L of fresh MRS culture solution into an electric rotating cup after electric shock is finished, slightly blowing and beating the mixture by using a gun head, uniformly mixing the mixture, transferring the mixture into a sterile 1.5mL centrifuge tube, and statically culturing the mixture at the temperature of 28 ℃ for 2.5-3 h to resuscitate cells; after the culture, the bacterial liquid is centrifuged for 3min at 8000rpm, 900 mu L of supernatant is discarded, the residual supernatant is used for resuspending the thalli, the thalli is coated on an MRS solid plate containing 5 mu g/mL of erythromycin, and the culture is carried out at the temperature of 28 ℃ in a standing way.

2. Screening and verification of folKE gene knockout strain

Randomly selecting 2-3 single colonies, transferring the single colonies into an MRS liquid culture medium containing 5 mu g/mL erythromycin, and standing and culturing at 28 ℃ until bacterial liquid OD₆₀₀When the concentration is 0.2-0.3 ℃, transferring the bacterial liquid to 37 ℃, continuing to perform standing culture overnight, and diluting the cultured bacterial liquid by 10 percent³~10⁵Spreading the multiplied cells on an MRS solid plate containing 5 mug/mL erythromycin, performing static culture at 37 ℃ for 24 hours, selecting monoclonals, inoculating the monoclonals into 1mL of MRS liquid culture medium containing 5 mug/mL erythromycin, performing static culture at 37 ℃ overnight, inoculating 1% of culture liquid into the MRS liquid culture medium without antibiotics, performing static culture at 28 ℃ overnight, and then diluting the culture liquid by 10%³~10⁵Coating on MRS solid plate without antibiotic, static culturing at 37 deg.C until single colony grows out, picking out smaller single colonyAnd scribing on MRS solid plates containing 5 mug/mL of erythromycin and containing no antibiotics in a one-to-one correspondence manner, carrying out static culture at 37 ℃ for 24h, and selecting colonies which cannot grow on MRS agar plates containing antibiotics and can grow on plates containing no antibiotics for carrying out bacteria liquid PCR verification. PCR verification of bacterial liquid: pFED760 FF: 5'-CTAAAAATCAGTTTCATCAAGCAAT-3', respectively; folKE-RR: 5'-CATCGCAGCCGTGGTCATA-3', respectively; the PCR fragment of the wild strain was 570bp larger than that of the knockout strain, and the result is shown in FIG. 1.

Example 4: detection of cell morphology and growth state of delta folKE strain

1. Cell morphology observation

1.5mL of cultured Lactobacillus plantarum wild strain and gene knockout strain delta folKE are respectively taken, centrifuged for 5min at 5000rpm, after supernatant is discarded, thalli are firstly fixed by 3.5% glutaraldehyde stationary liquid, and then an electron microscope observation sample is processed according to the following steps.

(1) Preparation of scanning electron microscope sample

Glutaraldehyde pre-fixation followed → phosphate buffer washing → 1% osmic acid fixation → phosphate buffer washing → different gradient ethanol dehydration → tert-butanol replacement → critical point freeze drying → ion sputtering gold → scanning electron microscopy.

(2) Preparation of sample for projection electron microscope

Glutaraldehyde pre-fixation and post-fixation → phosphate buffer washing → 1% osmic acid fixation → phosphate buffer washing → ethanol, acetone stepwise dehydration → epoxy resin 618 permeation → embedding → semithin section → optical lens localization, block modification → Leica-R microtome section → lead citrate-uranium acetate double staining → transmission electron microscopy observation.

As shown in FIG. 2A, the wild-type strain cells were rod-shaped, smooth in surface, intact in shape, and without separation of the cytoplasmic wall. Compared with lactobacillus plantarum wild strains, scanning electron microscopy shows that cytoplasm of the gene knockout strain delta folKE is shriveled, plasmolysis occurs, the adhesion among strains is enhanced, and some thalli are irregular rod-shaped and even sunken (figure 2B); in addition, the transmission electron microscope result shows that the cell wall of partial cells of the gene knockout strain delta folKE is thinned, the cell contents are condensed into particles, and even some cell walls are cracked, the contents leak (fig. 2C and 2D); these results indicate that the folKE gene knockout alters the cellular structural morphology, and that the folKE gene is closely associated with the lactobacillus plantarum cellular morphology maintenance.

2. Observation of macroscopic bacteria liquid state

Inoculating the activated lactobacillus plantarum wild type strain and the gene knockout strain delta folKE into 5mL of fresh MRS liquid culture medium according to the same inoculation amount, and performing static culture at 37 ℃ for 18 h; the growth condition of the strain is shown in figure 3, compared with the wild strain, the growth speed of the knocked-out strain delta folKE is obviously reduced, and the thallus is obviously settled at the bottom of the liquid, namely the thallus is precipitated to grow, so that the growth state of the strain is changed after the folKE gene is knocked out.

3. Gene knockout strain delta folKE dynamic growth monitoring

After the strains were activated and counted, the number of the strains was 1.0X 10⁶Inoculating the CFU/mL inoculum size into 300mL fresh MRS liquid culture medium, standing at 37 deg.C for 120h, sampling every 2h, and measuring OD with spectrophotometer₆₀₀The results are shown in FIG. 4 by comparison of OD₆₀₀It was found that the gene knockout strain Δ folKE grew slowly and the OD of the wild-type YM-4-3 strain₆₀₀Rises sharply during 8h-12h, remains relatively stable after 24h, OD₆₀₀Is 7.709. OD of knockout strain delta fol KE₆₀₀The strain rises steadily until 36h, the rising amplitude is relatively small, and then the strain fluctuates slightly up and down, is basically maintained at about 2.5 and has a large difference with the wild strain.

Example 5: determination of folate content of gene knockout strain delta folKE

Activating wild strain and gene knockout strain delta folKE of Lactobacillus plantarum at a ratio of 1.0 × 10⁷Inoculating CFU/mL into 30mL of FACM liquid culture medium, standing at 37 ℃ for 72h, taking out 5mL of bacterial liquid every 12h, carrying out light-shielding ultrasonic crushing treatment for 20min, centrifuging at 12000rpm for 10min, and then taking 1mL of supernatant for freeze drying; then 1mL of 1% ammonia water was added for dissolution, sonicated for 5min, centrifuged at 12000rpm for 10min, and the supernatant was used for HPLC analysis of the folate content.

(1) Chromatographic conditions are as follows: chromatography column, Waters ACQUITY UPLC BEH Amide column (2.1 mm × 100mm, 1.7 μm); the mobile phase is methanol (containing 5mmol/L ammonium formate) and water (containing 5mmol/L ammonium formate); gradient elution: 0-5 min, 98% -95% methanol; 5-10 min, 95% -55% methanol; 10-12 min, 55% methanol; 12-14 min, 55% -98% methanol; 14-20 min, 98% methanol. The flow rate is 0.2 mL/min, the column temperature is 35 ℃, and the sample injection amount is 5 muL.

(2) Mass spectrum conditions: 4500QTrap mass spectral parameters were set as follows: the method comprises the following steps of detecting by using a positive ion mode, wherein the ion source is an ESI ionization source, and the detection is carried out by using a gas curtain gas (CUR) 25, a collision gas (CAD) and the like, wherein the ion source gas is 1 (GS 1) 45, the ion source gas is 2 (GS 2) 50, the electrospray voltage is 5500V, the heater temperature is 350 ℃.

As can be seen from FIG. 4, the folic acid content in the fermentation broth of the Lactobacillus plantarum wild type strain and the gene knockout strain delta folKE is increased or decreased in a fluctuating manner; the subsequent fluctuation of the folate content of the gene knockout strain delta folKE is always lower than that of the wild strain, and the result shows that the folKE gene knockout causes slow growth, and the OD is verified before₆₀₀The change of (3) seriously affects the folic acid yield of the strain, and proves that the folKE gene plays a key role in the synthesis of the lactobacillus plantarum folic acid, so that the folic acid yield can be improved by changing the folKE gene subsequently, and a theoretical basis is provided for the industrial application of the lactobacillus plantarum YM-4-3 strain and metabolites thereof.

Sequence listing

<110> university of Kunming science

<120> application of Lactobacillus plantarum gene fol KE in folic acid biosynthesis

<160> 9

<170> SIPOSequenceListing 1.0

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<211> 570

<212> DNA

<213> Lactobacillus plantarum YM4-3 (Lactobacillus plantarum YM 4-3)

<400> 1

atgattgatg agaagaacca agcaaagatt gagcatgcag tacgagaaat tttaagtgca 60

gttggtgaag atcctgaccg accgggatta gtagaaacac cagcacgggt tgcacgaatg 120

tatgcggaag ttttcgctac taagaccgcc gcaccatttg ataattataa actgttcaag 180

gttgagcatc cgactgaaat ggtattactt aaggatattc cattctattc gatgtgtgag 240

catcaccttt tgccgttttt tggcacggtt caagttgctt atgtgccgca gcatgaacaa 300

gtgattggct tgagtaagat tcctcgcttg attgactatt gcagtcaaca gccgaacgtt 360

caggagcggt tgacagtttc cattgcaaca gaattacaac gaattcttga cccggctggg 420

atcgcggtct caatcacggc gcggcacatg tgcatggaga tgcggggtgt tagcaaaccg 480

ggtgtgcata cggaaagtag ctattacagt ggtcaattca agacggattt agacttgaaa 540

cgagaattct tacagcgaat cgcaaagtag 570

<210> 2

<211> 991

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tatcatattg agcgtccttc atgctatgat tataagtaaa aatgagcaag aaagaggctg 120

ggatacttat gggcatgatt cgaattaata atttacgctt tcacacgttt aacggggtac 180

ttccggaaga acggcgtaat ggtcaacaac tagggctaga tattgccatt aaatatccta 240

tcgaaaccaa ggttcaacac gatgacgttc acgagaccat caattacgcg gcggtccgta 300

acgtggtcga tgaatttgta acgacccatt catacaagtt gattgaatcg ctagctaacc 360

acttattgca gacgttattg acaagttttc ccgcggcgga tgcaatcaat attaaaattc 420

gtaaatatag cgtaccaatg cctggaatct ttgatgatgt ggaaattgag gtggagggga 480

cgccgaatgg caagtaggga agaacgggtt tatttgagtg ttggttccaa tattcatccg 540

cgcgtccaaa atattcagca agcccttagc cgattacgag ccgtcaatgg ggtaaacgtg 600

attgacgaat ctcattggta tgagactcaa ccgtggggaa agcgtgatca ggccaatttt 660

tacaatgttt cggtatcctt aacgactaat ttgacaccag aagaactatt ggatgaatta 720

catacaattg agcaggcggg ccaccgccaa cgcttggttc actggggacc acgtacgatt 780

gatttggaca ttattttttg gggcgaccgg caaatcaaca cagcgacgct gacgattccg 840

catgcgcagg cagctaagcg caactttgtg ctactgccaa ctgctgaaat cgccaaaact 900

gatgtgttag ttggaccaca agtggcccaa ttgattgcgg ctaatcagga tcagagttgg 960

attaaaaaag taagaaatgt gagtgagtta g 991

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<213> Lactobacillus plantarum YM4-3 (Lactobacillus plantarum YM 4-3)

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gtgattgagt ggatacaggg acgattgaac aacgttatca ggacttatta gcccaactaa 60

atcaagccat gttagccaat aatcatcagc gtgttccgtt attacggcgc atcatggcac 120

atcttggcca ccctgaccat tattaccatg tgatccatat cgctggaact aatggcaaag 180

gctctacggg agccatgtta gccagtatcc tgcgggcgca agggtatcaa gttggccgct 240

tcagcagtcc cgcgattaat gatgcgcgtg aacaactgca atgcaatggg acgtggatca 300

gtccagcgga atttatcgat acgtatcgtg aaattctacc ggttctgcaa aatatgggac 360

tgacggctag cgatgtctcc atctttgaat ggttctttct cattagtgtc gtttggtttc 420

ggaatcaaaa cgtgcaatgg gcggtcattg aagccggttt gggtggcttg tatgatgcga 480

ctaacgcctt agccagtccc caactcacgg tatttactaa gattgccctc gatcatacca 540

ccattcttgg gcccacaatc accgcgattg cgcaaaataa gtcaaagatc attaagcccc 600

atacaacagc agtgacattg gctgaccaac acccagaagc cctggctgtt ttgcagaccg 660

aagcgctcaa tcaaggtgtt cggctagtga ccgctaagca cgcgcaactg acggtgactg 720

ggcaaacact cacgcaaacc gtcgttgatg cgcacagtca gctttttgat tggacgcaat 780

taacggtcgg actgagtggc acctatcagc tacaaaatct gcggttggtt ttaactgtgg 840

ttgcagtact acaacagcaa caagttaact tgacgaacag tgcggttcgc cgaggcttgc 900

aacaagtttc cttgccaggc cggttgaccg tgttacagga gcaacctgtc attattgccg 960

atggggcgca taatccggat ggcatggcag cactagtggc 1000

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

gcgtcgacac gcactttttt cggttcc 27

<210> 5

<211> 38

<212> DNA

<213> Artificial sequence (Artificial)

<400> 5

cctgtatcca ctcaatcacc taactcactc acatttct 38

<210> 5

<211> 38

<212> DNA

<213> Artificial sequence (Artificial)

<400> 5

agaaatgtga gtgagttagg tgattgagtg gatacagg 38

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence (Artificial)

<400> 6

cggaattcgc cactagtgct gccatgc 27

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial)

<400> 7

ctaaaaatca gtttcatcaa gcaat 25

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<211> 19

<212> DNA

<213> Artificial sequence (Artificial)

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catcgcagcc gtggtcata 19

Claims

1. A Lactobacillus plantarum (A)Lactobacillus plantarum) Genefol Application of KE in folic acid biosynthesis and genefol KE is shown in SEQ ID NO 1.