CN111235169A - GTP cyclohydrolase I gene folE and application thereof - Google Patents

GTP cyclohydrolase I gene folE and application thereof Download PDF

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CN111235169A
CN111235169A CN202010078968.1A CN202010078968A CN111235169A CN 111235169 A CN111235169 A CN 111235169A CN 202010078968 A CN202010078968 A CN 202010078968A CN 111235169 A CN111235169 A CN 111235169A
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fole
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lactobacillus plantarum
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柳陈坚
李梦月
李晓然
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Kunming University of Science and Technology
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Abstract

The invention belongs to the field of microbial genetic engineering, and discloses a GTP cyclohydrolase I gene folE and application thereof, wherein the nucleotide sequence of the GTP cyclohydrolase I gene folE is shown as SEQ ID NO. 1. The gene is from food-borne lactobacillus plantarum, has safety, and can be used in the field of later-period food fermentation. The screening method of the gene knockout strain improves the screening efficiency of the knockout strain. The invention proves the key role of the gene folE in folic acid synthesis, and provides a certain theoretical basis for the research and development of folic acid synthesis functional foods. The invention analyzes that the gene is derived from food-grade microorganisms, and has safety; the invention analyzes that the gene folE plays an important role in folic acid synthesis and is unrelated to cell morphology and strain growth.

Description

GTP cyclohydrolase I gene folE and application thereof
Technical Field
The invention belongs to the field of microbial genetic engineering, and particularly relates to GTP cyclohydrolase I gene folE and application thereof. In particular to application of GTP cyclohydrolase I gene folE in improving the biosynthesis of the folic acid of Lactobacillus plantarum (Lactobacillus plantarum).
Background
Currently, the current state of the art commonly used in the industry is such that: folic acid, chemically known as pteroylglutamic acid (pteroylglutamic acid), is a water-soluble B-complex vitamin, vitamin B9, formed by the combination of pterin, p-aminobenzoic acid (pABA) and one or more glutamic acids. Folic acid is stable in alkaline or neutral solution, is very unstable under acidic conditions, and is easily damaged under light conditions, especially under ultraviolet irradiation.
Folic acid has important physiological functions. Folate has been reported to be involved in physiological metabolic processes such as DNA and RNA biosynthesis, DNA repair, amino acid metabolism, and hemoglobin synthesis. In addition, folic acid is an important chemical raw material substance for forming nerve endings and constituting transmission nerve impulses because a large amount of free carbon ions can be provided by folic acid, and further, the normal development of a nervous system is ensured, so that folic acid is particularly important in the process of pregnancy and infant development.
The human body cannot synthesize folic acid by itself, and folic acid has important physiological functions, so that different diseases can be caused by deficiency of folic acid, and the folic acid can only be supplemented or ingested from food to meet the needs of the human body. Since most folic acid preparations are synthetic folic acid, chemically synthesized folic acid differs in many ways compared to naturally synthesized folic acid. Ingestion of a large amount of chemically synthesized folic acid brings many side effects to human health, such as masking clinical manifestations of early anemia caused by vitamin B12 deficiency, changing the activity of dihydrofolate reductase in the liver, promoting carcinogenesis, inducing cognitive dysfunction, etc., whereby increasing the production of natural folic acid becomes of great importance. However, the instability of folic acid and the heat loss rate of folic acid in the cooking process are more than 50 percent, folic acid is synthesized by using microorganisms, and then functional food rich in folic acid is developed and is a good method for effectively solving the problems.
The folic acid is synthesized by utilizing food-grade microorganisms (such as lactic acid bacteria), and has the characteristics of higher economic benefit, less environmental pollution, high safety and the like. The microorganism has similar folic acid biosynthesis pathway to that of plant, including pterin metabolism branch and p-aminobenzoic acid metabolism branch. The folE gene encodes GTP cyclohydrolase I (GCHI), which catalyzes the first step of the metabolic pathway of pterin and is therefore crucial in controlling the overall folate biosynthesis pathway. In order to increase the yield of natural folate and thus meet human needs, considerable attention has been paid to the study of key genes for the biosynthesis of folate in the above pathways or to the modification of folate metabolism in plants and microorganisms by genetic engineering.
In summary, the problems of the prior art are as follows:
(1) at present, the research on the regulation mechanism of the microbial folate biosynthesis pathway only deeply researches the branch of aminobenzoic acid metabolism, but the research on the other pterin metabolism branch is less, and the effect of the relevant genes on the pathway on the regulation of folate biosynthesis and even on the metabolism of the whole strain is lack of systematic research.
(2) Lactic acid bacteria have quite different abilities to synthesize folic acid using microbial media. Several studies have shown that some lactobacillus plantarum in lactic acid bacteria can synthesize natural folic acid, unlike other vitamins which can be industrially produced by microbial fermentation, almost all artificially added folic acid is chemically synthesized at present.
The difficulty and significance for solving the technical problems are as follows: at present, a great deal of research is limited to improving the yield of folic acid by over-expressing key genes for folic acid biosynthesis, and the function of the genes is researched by using a gene knockout technology. By fully understanding plant folate synthesis genes and enzymes, the gene folE for guanosine triphosphate cyclohydrolase type I (GTPCHI) which has made folate metabolism engineering the most widely possible. Therefore, the folE gene is a key gene for high folate regulation throughout the synthesis of folate, but the function of the folE gene in the folate synthesis pathway in Lactobacillus plantarum is yet to be determined. Not all species contain the folE gene. However, some experiments have shown the possibility of lactobacillus plantarum developing folate-rich functional foods. The research knocks out the folE gene of Lactobacillus plantarum YM4-3 strain by homologous recombination gene knockout technology, so as to research the function of the strain on the biosynthesis and growth of Lactobacillus plantarum folic acid. China is one of the main countries for industrial production of synthetic folic acid, so that it is important to find a folic acid synthetic method which is both environment-friendly and economical. Natural folates present in food products or produced metabolically by microorganisms are selected to be safer than chemically synthesized drugs.
The invention deeply analyzes the folate synthesis gene of the pterin metabolic branch, can systematically explain the regulation and control mechanism of the folate synthesis pathway of the microorganism, and completes the industrial development and utilization of the microorganism and the metabolite thereof.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a GTP cyclohydrolase I gene folE and application thereof. Namely the application of GTP cyclohydrolase I gene folE in improving the folic acid biosynthesis of microbial strains or the application in improving the biomass of the microbial strains.
The invention is realized by the GTP cyclohydrolase I gene folE, and the nucleotide sequence of the GTP cyclohydrolase I gene folE is shown as SEQ ID NO. 1.
Another object of the present invention is to provide a protein encoded by the GTP cyclohydrolase I gene folE. This protein is described in the Crystal structure of the simulation complex of GTPcysteine I and its feedback regulation protein GFRP.
The invention also aims to provide a knockout vector pFED760-folE constructed by utilizing the GTP cyclohydrolase I gene folE, wherein the knockout vector pFED760-folE is introduced into the lactobacillus plantarum competent cell to construct a folE gene knockout strain delta folE through homologous recombination; by comparing the physiological and biochemical differences of the wild-type strain and the delta folE strain, the folE gene is analyzed to be independent of cell morphology and strain growth state, but plays a key role in folic acid synthesis.
Another object of the present invention is to provide a method for constructing the knock-out vector pFED760-folE, which comprises:
the method comprises the steps of taking a food-borne lactobacillus plantarum genome as a template, utilizing a primer pair up-EF + up-ER and a primer pair down-EF + down-ER to respectively amplify upstream and downstream homologous arms, using a PCR product as a template, utilizing the primer pair up-EF + down-ER to amplify to obtain a homologous arm knockout fragment, purifying the fragment, synchronously carrying out enzyme digestion on the fragment and a temperature sensitive plasmid pED760 by using restriction enzymes HindIII and Spe I respectively, carrying out a ligation experiment after the enzyme digestion product is subjected to gel cutting recovery, introducing the ligation product into escherichia coli DH5 α competent cells, and extracting a plasmid to obtain a folE gene knockout vector pFED 760-folE.
Further, the primer sequences are as follows:
up-EF：5’-CCCAAGCTTGAACGGCGTAATGGTCAAC-3’；
up-ER：5’-TTGAATTGACCACTGTAATTAATTATCAAATGGTGCGG-3’；
down-EF：5’-CCGCACCATTTGATAATTAATTACAGTGGTCAATTCAA-3’；
down-ER：5’-GCACTAGTACACGGTCAACCGGCCTGG-3’。
furthermore, the GTP cyclohydrolase I gene folE and the sequence of the upstream and downstream homologous arms are shown in SEQ ID NO. 2 and are derived from food-borne lactobacillus plantarum.
The invention also aims to provide a screening method of the knockout vector pED 760-folE, which comprises the following steps:
(1) knock-out vector pED 760-folE transformation: adding 10 mu L of knockout carrier pFED760-folE into 90-100 mu L of lactobacillus plantarum receptive state, mixing gently, carrying out ice bath for 5min, transferring into a precooling electric shock cup, and carrying out electric shock according to parameters of 12.5kv/cm and 200 omega; 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 1.5mL sterile centrifuge tube, and statically culturing the mixture at 28 ℃ for 2.5-3 h to resuscitate cells; centrifuging the cultured bacterial liquid at 8000-10000 rpm for 3min, discarding 900 mu L of supernatant, resuspending the thallus with the rest supernatant, coating the thallus on an MRS solid plate containing 5 mu g/mL of erythromycin, and standing and culturing at 28 ℃;
(2) and (3) screening of folE gene knockout strains: transferring the single colony grown in the step (1) 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 percent3～105Coating the double suspension on an MRS solid plate containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ for 24h, selecting a monoclonal antibody, inoculating the monoclonal antibody into 1mL of MRS liquid culture medium containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ overnight, inoculating 1% of the culture bacterial liquid into the MRS liquid culture medium without antibiotics, performing static culture at 28 ℃ overnight, and diluting the bacterial liquid by 10%3～105Coating the double single colonies on an MRS solid plate without antibiotics, performing static culture at 37 ℃ until single colonies grow out, selecting smaller single colonies, scribing the smaller single colonies on the MRS solid plate containing 5 mug/mL of erythromycin and without antibiotics one by one, performing static culture at 37 ℃ for 24 hours, selecting colonies which cannot grow on an MRS agar plate containing the antibiotics and can grow on the MRS agar plate without the antibiotics, and performing bacterial liquid PCR (polymerase chain reaction) verification;
in the PCR verification of the bacterial liquid, a front primer folE-FF: 5'-CAGACGCAAATGACGCAGT-3' is located on the upstream genome of the upstream homology arm, and the rear primer folE-RR: 5'-CGGATAACGCTCGTTCTGGAT-3' are located on the downstream genome of the downstream homology arm; the PCR fragment of the wild strain is 335bp larger than that of the knockout strain;
(3) the folE gene of the food-borne lactobacillus plantarum is analyzed by using the successfully constructed knockout strain, is irrelevant to the cell morphology of the lactobacillus plantarum and the growth state of the strain, and plays a key role in folic acid synthesis of the lactobacillus plantarum.
The invention also aims to provide application of the GTP cyclohydrolase I gene folE in food-borne lactobacillus plantarum in food fermentation.
The invention also aims to provide application of the GTP cyclohydrolase I gene folE in folic acid synthesis functional food.
The invention also aims to provide an application of the GTP cyclohydrolase I gene folE in improving the biomass of microbial strains.
In summary, the advantages and positive effects of the invention are: one of the characteristics of the invention is that the research gene is from food-borne lactobacillus plantarum, has safety and can be used in the field of later-stage food fermentation.
The invention is characterized in that the screening method of the gene knockout strain improves the screening efficiency of the knockout strain.
The invention is further characterized in that the key role of the gene folE gene in folic acid synthesis is proved, and a certain theoretical basis is provided for research and development of folic acid synthesis functional food.
Compared with the prior art, the invention has the following advantages: the gene is analyzed to be derived from food-grade microorganisms, and the safety is realized; the invention analyzes that the gene folE plays an important role in folic acid synthesis and is unrelated to cell morphology and strain growth. The folKE gene is related to cell morphology, and further proves that folE and folK gene separately encode proteins.
Drawings
Fig. 1 is a bacterial liquid PCR verification of the folE knockout strain provided in the embodiment of the present invention, wherein lane M: DNA marker; lanes 1 and 4: taking the genome DNA of the lactobacillus plantarum wild strain as a template PCR product; lane 2: knocking out the delta folE genome DNA of the strain as a template PCR product; lane 3: water was the template PCR product.
FIG. 2 is a scanning and transmission electron microscope image of a wild Lactobacillus plantarum strain, a knockout strain Δ folE and a knockout strain Δ folKE provided in an embodiment of the present invention; a: scanning electron microscope images of wild strains; b: knocking out a strain delta folE scanning electron microscope picture; c: knocking out a scanning electron microscope image of the strain delta folKE; d: transmission electron microscope picture of wild type strain; e: knocking out a strain delta folE transmission electron microscope picture; f: knockout strain delta folKE transmission electron microscopy.
FIG. 3 shows the bacterial liquid growth status of Lactobacillus plantarum wild type strain (A) and knockout strain Δ folE (B) provided in the examples of the present invention.
FIG. 4 shows the growth curves, OD, of wild type strain and Δ folE knockout strain of Lactobacillus plantarum provided in the examples of the present invention600And pH dynamic curves.
FIG. 5 shows the total folate content of the fermentation broth of the wild Lactobacillus plantarum strain and the knockout strain Δ folE provided by the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The reagents and methods used in the examples of the present invention are, unless otherwise specified, those which are conventional and those which are conventional in the art. The temperature sensitive plasmid pPED760 was gifted by Philippi Federle, university of Illinois; the results in the following examples are all the average values of three replicates unless otherwise specified.
Aiming at the problems in the prior art, the invention provides GTP cyclohydrolase I gene folE and application thereof, and the invention is described in detail with reference to the accompanying drawings.
The nucleotide sequence of the GTP cyclohydrolase I gene folE provided by the embodiment of the invention is shown in SEQ ID NO 1; according to the invention, a folE gene and a temperature-sensitive plasmid pED760 are recombined to construct a knockout vector pED 760-folE, and the knockout vector pED 760-folE is introduced into lactobacillus plantarum competence to construct a folE gene knockout strain delta folE through homologous recombination; by comparing the physiological and biochemical differences of the wild strain and the delta folE strain, the folE gene is proved to be irrelevant to the cell morphology and the growth state of the strain, but plays a key role in folic acid synthesis, and the folKE gene is also relevant to the cell morphology, so that the folE gene and the folK gene are proved to independently encode proteins. The invention has great potential in the research and application fields of folic acid biosynthesis.
In the embodiment of the invention, the construction method of the GTP cyclohydrolase I gene folE knockout vector comprises the following steps:
the method comprises the steps of using a food-borne Lactobacillus plantarum (Lactobacillus plantarum) genome as a template, respectively amplifying upstream and downstream homologous arms by using a primer pair (up-EF + up-ER; down-EF + down-ER), using a PCR product as a template, amplifying by using the primer pair (up-EF + down-ER) to obtain a homologous arm knockout fragment, purifying the fragment, synchronously carrying out enzyme digestion with a temperature sensitive plasmid pFED760 by using restriction enzymes Hind III and Spe I respectively, carrying out a ligation experiment after the enzyme digestion product is subjected to gel cutting recovery, introducing the ligation product into Escherichia coli DH5 α competent cells, and extracting plasmids to obtain a folE gene knockout vector pFED 760-folE.
Wherein the primer sequences are as follows:
up-EF：5’-CCCAAGCTTGAACGGCGTAATGGTCAAC-3’；SEQ ID NO:3.
up-ER：5’-TTGAATTGACCACTGTAATTAATTATCAAATGGTGCGG-3’；SEQ ID NO:4。
down-EF：5’-CCGCACCATTTGATAATTAATTACAGTGGTCAATTCAA-3’；SEQ ID NO:5。
down-ER：5’-GCACTAGTACACGGTCAACCGGCCTGG-3’；SEQ ID NO:6。
the GTP cyclohydrolase I gene folE and the sequence of the upstream and downstream homologous arms thereof are shown as SEQ ID NO. 2 and are derived from food-borne Lactobacillus plantarum (Lactobacillus plantarum); the lactobacillus plantarum is widely applied to the fields of food, medicines and the like due to the characteristics of safety, harmlessness and the like, so that the gene is safe and harmless to human bodies, and theoretical safety guarantee is provided for the future application of the lactobacillus plantarum in the field of folic acid production.
In the embodiment of the invention, the screening method of the GTP cyclohydrolase I gene folE knockout strain comprises the steps of constructing a GTP cyclohydrolase I gene folE knockout vector, converting the later knockout vector and screening knockout strains; the method comprises the following steps:
(1) knock-out vector pED 760-folE transformation: adding 10 mu L of knockout carrier pFED760-folE into 90-100 mu L of lactobacillus plantarum receptive state, mixing gently, carrying out ice bath for 5min, transferring into a precooling electric shock cup, and carrying out electric shock according to parameters of 12.5kv/cm and 200 omega; 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 1.5mL sterile centrifuge tube, and statically culturing the mixture at 28 ℃ for 2.5-3 h to resuscitate cells; centrifuging the cultured bacterial liquid at 8000-10000 rpm for 3min, discarding 900 mu L of supernatant, resuspending the thallus with the residual supernatant, coating the thallus on an MRS solid plate containing 5 mu g/mL of erythromycin, and standing and culturing at 28 ℃.
(2) And (3) screening of folE gene knockout strains: transferring the single colony grown in the step (1) toIn MRS liquid culture medium containing 5 mu g/mL erythromycin, 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 percent3～105Coating the double suspension on an MRS solid plate containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ for 24h, selecting a monoclonal antibody, inoculating the monoclonal antibody into 1mL of MRS liquid culture medium containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ overnight, inoculating 1% of the culture bacterial liquid into the MRS liquid culture medium without antibiotics, performing static culture at 28 ℃ overnight, and diluting the bacterial liquid by 10%3～105Coating the double single colonies on an MRS solid plate without antibiotics, performing static culture at 37 ℃ until single colonies grow out, selecting smaller single colonies, scribing the smaller single colonies on the MRS solid plate containing 5 mug/mL of erythromycin and without antibiotics one by one, performing static culture at 37 ℃ for 24 hours, selecting colonies which cannot grow on an MRS agar plate containing the antibiotics and can grow on the MRS agar plate without the antibiotics, and performing bacterial liquid PCR (polymerase chain reaction) verification;
in the PCR verification of the bacterial liquid, a front primer (folE-FF: 5'-CAGACGCAAATGACGCAGT-3' SEQ ID NO:7) is positioned on an upstream genome of an upstream homology arm, and a rear primer (folE-RR: 5'-CGGATAACGCTCGTTCTGGAT-3' SEQ ID NO:8) is positioned on a downstream genome of a downstream homology arm; the PCR fragment of the wild strain is 335bp larger than that of the knockout strain.
(3) The successfully constructed knockout strain is utilized to prove that the folE gene of the food-borne lactobacillus plantarum is irrelevant to the cell morphology and the strain growth state of the lactobacillus plantarum, but plays a key role in folic acid synthesis of the lactobacillus plantarum.
The invention is further described with reference to specific examples.
Example 1: cloning of homologous arm for knocking out GTP cyclohydrolase I gene folE
1. Upstream and downstream homology arms for PCR amplification
A bacterial genome DNA extraction kit (Baitach Biotechnology Co., Ltd., China) is used for extracting the genome of the food-borne lactobacillus plantarum YM4-3, and the specific operation is carried out according to the kit instruction. The upstream 970bp sequence of the folE gene coding region is used as an upstream homologous arm, and the downstream 1001bp sequence is used as a downstream homologous arm; using extracted genome as template and primer pair
up-EF(5’-CCCAAGCTTGAACGGCGTAATGGTCAAC-3 ', the Spe I cleavage site is underlined) + up-ER (5'-TTGAATTGACCACTGTAATTAATTATCAAATGGTGCGG-3') and
down-EF(5’-CCGCACCATTTGATAATTAATTACAGTGGTCAATTCAA-3’)+down-ER(5’-GCACTAGTACACGGTCAACCGGCCTGG-3', underlined Hind III restriction enzyme sites) were amplified separately from the upstream and downstream homology arms, and the PCR reaction system and amplification conditions were as follows:
(1) PCR reaction system
Figure BDA0002379589960000091
(2) PCR amplification conditions
Pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 s; annealing at 56 ℃ for 30 s; extending for 1min at 72 ℃; circulating for 30 times; extending for 5min at 72 ℃, and storing at 12 ℃. After completion of the reaction, 5. mu.L of the reaction mixture was electrophoresed on a 1% agarose gel.
2. Cloning and sequencing of gene knockout fragment
1 mu L of each downstream homology arm PCR product is used as a template, up-EF and down-ER are used as primers, overlapping PCR is carried out according to the PCR reaction system and the amplification condition (the extension time is changed to 2min), gel cutting is carried out to recover a PCR product (a gene knockout fragment) with an expected size, the PCR product is connected to a pMD19-T vector according to the instruction of a TA cloning kit of Dalibao biology company (China), the connection product is introduced into escherichia coli DH5 α competent cells through a heat shock transformation method, the cells are coated on an Amp-LB plate, after overnight culture at 37 ℃, 10-15 single colonies are randomly selected, plasmids in the cells are extracted, SpeI and Hind III are used for enzyme digestion verification, and the positive plasmids are sent to a sequencing company for sequencing.
Example 2: folE gene knockout vector construction
Synchronously digesting a correctly sequenced gene knockout fragment and a temperature-sensitive plasmid pFED760 respectively by using restriction enzymes SpeI and Hind III in a SpeI, 1 muL, Hind III, 1 muL, 1 XHbuffer, 2 muL and 10-16 muL respectively, adding sterilized deionized water to 20 muL, digesting for 4H at 37 ℃, recovering a digested product, adding T4 DNA ligase to connect for 12-16H at 16 ℃ after adding a target gene, namely a vector 4: 1-2: 1 (molar ratio), introducing the ligated product into escherichia coli DH5 α competent cells by a heat shock transformation method, then coating the cells on an erythromycin-LB solid plate, culturing overnight at 28 ℃, extracting plasmids in 10-15 single colony cells, and digesting and verifying by using SpeI and Hind III to obtain a positive plasmid which is named as pFED 760-lE.
Example 3: folE Gene knockout Strain construction
1. Introduction of folE gene knockout vector into lactobacillus plantarum competent cells
Lactobacillus plantarum competent cells were prepared according to the method reported in Ferigo (2015, master's academic paper, university of southern China). Adding 10 mu L of gene knockout vector pFED760-folE into 90-100 mu L of lactobacillus plantarum receptive state, mixing gently, carrying out ice bath for 5min, transferring into a precooling electric shock cup, and carrying out electric shock according to the parameters of 12.5kv/cm and 200 omega. 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 1.5mL sterile centrifuge tube, and statically culturing the mixture at 28 ℃ for 2.5-3 h to resuscitate cells; centrifuging the cultured bacterial liquid at 8000-10000 rpm for 3min, discarding 900 mu L of supernatant, resuspending the thallus with the residual supernatant, coating the thallus on an MRS solid plate containing 5 mu g/mL of erythromycin, and standing and culturing at 28 ℃.
2. Screening and verification of folE 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 percent3～105Coating the double suspension on an MRS solid plate containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ for 24h, selecting a monoclonal antibody, inoculating the monoclonal antibody into 1mL of MRS liquid culture medium containing 5 mu g/mL erythromycin, performing static culture at 37 ℃ overnight, inoculating 1% of the culture bacterial liquid into the MRS liquid culture medium without antibiotics, performing static culture at 28 ℃ overnight, and diluting the bacterial liquid by 10%3～105Post-doubling coated with antibiotic-free MAnd (3) performing static culture on the RS solid plate at 37 ℃ until a single clone grows out, selecting small single colonies, scribing on the MRS solid plate containing 5 mu g/mL erythromycin and no antibiotics one by one, performing static culture at 37 ℃ for 24h, selecting colonies which cannot grow on the MRS agar plate containing the antibiotics and can grow on the plate without the antibiotics, and performing bacterial liquid PCR verification. PCR verification of bacterial liquid: the front primer (folE-FF: 5'-CAGACGCAAATGACGCAGT-3') is positioned on the upstream genome of the upstream homology arm, and the rear primer (folE-RR: 5'-CGGATAACGCTCGTTCTGGAT-3') is positioned on the downstream genome of the downstream homology arm; the PCR fragment of the wild strain is 335bp larger than that of the knockout strain. The screening method improves the screening efficiency of the knockout strain, and the result is shown in figure 1.
Example 4: detection of cell morphology and growth state of delta folE strain
1. Cell morphology observation
1.5mL of cultured Lactobacillus plantarum wild strain, gene knockout strain delta folE and gene knockout strain delta folKE are respectively taken, centrifuged for 5min at 5000rpm, after supernatant is discarded, the 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 transmission electron microscope sample
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.
The lactobacillus plantarum wild strain cells are rod-shaped, the surface is smooth, the shape is complete, and the texture and the wall are not separated. Compared with a lactobacillus plantarum wild strain, the scanning electron microscope shows that the cell morphology of the folE gene knockout strain is similar to that of the wild strain and is hardly changed. The cytoplasm of delta folKE is shriveled, plasmolysis occurs, the adhesion among strains is enhanced, and the shapes of some thalli are irregular rods and even pits appear (figure 2A, figure 2B and figure 2C); in addition, transmission electron microscopy results show that Δ folE cells are intact and have no change in interior compared to wild type strain cells. Cell walls of partial cells of the delta folKE strain are thinned, cell contents are condensed into particles, and even some cell walls of the cells are broken, and the contents leak. (FIG. 2D, FIG. 2E and FIG. 2F); these results indicate that the folE gene has no major influence on the cell morphology, and that the Δ folKE strain knocks out only 7 bases of the folK gene, but undergoes a major change relative to the cell morphology of the Δ folE strain, indicating that the folK and folE genes do not encode one protein.
2. Observation of macroscopic bacteria liquid state
Activating wild strain and gene knockout strain delta folE of Lactobacillus plantarum according to the ratio of 1.0 multiplied by 106Inoculating the CFU/mL inoculum size into 5mL fresh MRS liquid culture medium, and performing static culture at 37 ℃ for 18-24 h; as shown in FIG. 3, it was found by observing the bacterial liquid state that the growth state of the Δ folE strain was uniform in the liquid as the wild-type strain, but the growth rate was slightly slow, so that the growth state of the strain was not changed by knocking out the folE gene.
3. Gene knockout strain delta folE 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, performing static culture at 37 ℃ for 86h, sampling every 2h, and measuring the pH value by using a pH meter; determination of OD Using Spectrophotometer600(ii) a Meanwhile, carrying out 10-fold gradient dilution on the bacterial liquid, coating 100 mu L of diluted bacterial liquid on an MRS solid plate, then carrying out static culture in an incubator at 37 ℃ for 16-24 h, and selecting a plate with the colony number within the range of 30-300 in the plate for counting; the result shows that compared with the lactobacillus plantarum wild strain, the gene knockout strain delta folE has slow growth, prolonged growth period and reduced acid production capability; the concrete expression is as follows: the growth cycle of the gene knockout strain delta folE is prolonged by nearly 35h, and then the folE gene knockout seriously reduces the biomass of the strain to ensure that OD is ensured600Maximum ofThe value is 4.5 less than that of the wild strain; in addition, the pH of the knockout strain Δ folE decreased more slowly than that of the wild strain, and the final pH was also about 0.3 higher than that of the knockout strain (FIG. 4).
Example 5: determination of content of gene knockout strain delta folE folic acid
Activating wild strain and gene knockout strain delta folE of Lactobacillus plantarum according to the ratio of 1.0 multiplied by 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. times.100 mm, 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 was 0.2mL/min, the column temperature was 35 ℃ and the amount of sample was 5. mu.L.
(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(GS1)45, the ion source gas is 2(GS2)50, the electrospray voltage is 5500V, the heater temperature is 350 ℃.
As can be seen from FIG. 5, the folate content of the fermentation broth of the Lactobacillus plantarum wild type strain and the gene knockout strain Δ folE increased or decreased in a fluctuating manner. The folic acid content of the fermentation liquid of the delta folE strain is lower than that of the wild strain in 24h, 36h and 60h culture, but the folic acid content of the fermentation liquid of the delta folE strain suddenly rises and is larger than that of the wild strain at 72 h. This may be to meet self-demand and improve folic acid synthesis efficiency, but another possibility is that anaplerotic pathways occur when folic acid content is low to a certain limit. The result is enough to indicate that the folic acid yield of the strain is seriously influenced by the knockout of the folE gene, and the folE gene is proved to play a key role in the synthesis of the folic acid of the lactobacillus plantarum; therefore, the folic acid yield can be improved by over-expressing GTP cyclohydrolase I subsequently, so that the aim of expanding production is fulfilled, and a theoretical basis is provided for the industrial application of the Lactobacillus plantarum YM4-3 strain and metabolites thereof.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> university of Kunming science
<120> GTP cyclohydrolase I gene folE and application
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>570
<212>DNA
<213> Lactobacillus plantarum YM4-3(Lactobacillus plantarum YM4-3)
<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>2342
<212>DNA
<213> Artificial sequence (Artificial)
<400>2
ttacgctttc acacgtttaa cggggtactt ccggaagaac ggcgtaatgg tcaacaacta 60
gggctagata ttgccattaa atatcctatc gaaaccaagg ttcaacacga tgacgttcac 120
gagaccatca attacgcggc ggtccgtaac gtggtcgatg aatttgtaac gacccattca 180
tacaagttga ttgaatcgct agctaaccac ttattgcaga cgttattgac aagttttccc 240
gcggcggatg caatcaatat taaaattcgt aaatatagcg taccaatgcc tggaatcttt 300
gatgatgtgg aaattgaggt ggaggggacg ccgaatggca agtagggaag aacgggttta 360
tttgagtgtt ggttccaata ttcatccgcg cgtccaaaat attcagcaag cccttagccg 420
attacgagcc gtcaatgggg taaacgtgat tgacgaatct cattggtatg agactcaacc 480
gtggggaaag cgtgatcagg ccaattttta caatgtttcg gtatccttaa cgactaattt 540
gacaccagaa gaactattgg atgaattaca tacaattgag caggcgggcc accgccaacg 600
cttggttcac tggggaccac gtacgattga tttggacatt attttttggg gcgaccggca 660
aatcaacaca gcgacgctga cgattccgca tgcgcaggca gctaagcgca actttgtgct 720
actgccaact gctgaaatcg ccaaaactga tgtgttagtt ggaccacaag tggcccaatt 780
gattgcggct aatcaggatc agagttggat taaaaaagta agaaatgtga gtgagttaga 840
tgattgatga gaagaaccaa gcaaagattg agcatgcagt acgagaaatt ttaagtgcag 900
ttggtgaaga tcctgaccga ccgggattag tagaaacacc agcacgggtt gcacgaatgt 960
atgcggaagt tttcgctact aagaccgccg caccatttga taattataaa ctgttcaagg 1020
ttgagcatcc gactgaaatg gtattactta aggatattcc attctattcg atgtgtgagc 1080
atcacctttt gccgtttttt ggcacggttc aagttgctta tgtgccgcag catgaacaag 1140
tgattggctt gagtaagatt cctcgcttga ttgactattg cagtcaacag ccgaacgttc 1200
aggagcggtt gacagtttcc attgcaacag aattacaacg aattcttgac ccggctggga 1260
tcgcggtctc aatcacggcg cggcacatgt gcatggagat gcggggtgtt agcaaaccgg 1320
gtgtgcatac ggaaagtagc tattacagtg gtcaattcaa gacggattta gacttgaaac 1380
gagaattctt acagcgaatc gcaaagtagg tgattgagtg gatacaggga cgattgaaca 1440
acgttatcag gacttattag cccaactaaa tcaagccatg ttagccaata atcatcagcg 1500
tgttccgtta ttacggcgca tcatggcaca tcttggccac cctgaccatt attaccatgt 1560
gatccatatc gctggaacta atggcaaagg ctctacggga gccatgttag ccagtatcct 1620
gcgggcgcaa gggtatcaag ttggccgctt cagcagtccc gcgattaatg atgcgcgtga 1680
acaactgcaa tgcaatggga cgtggatcag tccagcggaa tttatcgata cgtatcgtga 1740
aattctaccg gttctgcaaa atatgggact gacggctagc gatgtctcca tctttgaatg 1800
gttctttctc attagtgtcg tttggtttcg gaatcaaaac gtgcaatggg cggtcattga 1860
agccggtttg ggtggcttgt atgatgcgac taacgcctta gccagtcccc aactcacggt 1920
atttactaag attgccctcg atcataccac cattcttggg cccacaatca ccgcgattgc 1980
gcaaaataag tcaaagatca ttaagcccca tacaacagca gtgacattgg ctgaccaaca 2040
cccagaagcc ctggctgttt tgcagaccga agcgctcaat caaggtgttc ggctagtgac 2100
cgctaagcac gcgcaactga cggtgactgg gcaaacactc acgcaaaccg tcgttgatgc 2160
gcacagtcag ctttttgatt ggacgcaatt aacggtcgga ctgagtggca cctatcagct 2220
acaaaatctg cggttggttt taactgtggt tgcagtacta caacagcaac aagttaactt 2280
gacgaacagt gcggttcgcc gaggcttgca acaagtttcc ttgccaggcc ggttgaccgt 2340
gt 2342
<210>3
<211>28
<212>DNA
<213> Artificial sequence (Artificial)
<400>3
cccaagcttg aacggcgtaa tggtcaac 28
<210>4
<211>38
<212>DNA
<213> Artificial sequence (Artificial)
<400>4
ttgaattgac cactgtaatt aattatcaaa tggtgcgg 38
<210>5
<211>38
<212>DNA
<213> Artificial sequence (Artificial)
<400>5
ccgcaccatt tgataattaa ttacagtggt caattcaa 38
<210>6
<211>27
<212>DNA
<213> Artificial sequence (Artificial)
<400>6
gcactagtac acggtcaacc ggcctgg 27
<210>7
<211>19
<212>DNA
<213> Artificial sequence (Artificial)
<400>7
cagacgcaaa tgacgcagt 19
<210>8
<211>21
<212>DNA
<213> Artificial sequence (Artificial)
<400>8
cggataacgc tcgttctgga t 21

Claims (10)

1. A GTP cyclohydrolase I gene folE is characterized in that the nucleotide sequence of the GTP cyclohydrolase I gene folE is shown as SEQ ID NO. 1.
2. A protein encoded by the GTP cyclohydrolase I gene folE of claim 1.
3. A knock-out vector pFED760-folE constructed by using the GTP cyclohydrolase I gene folE of claim 1, wherein the knock-out vector pFED760-folE is introduced into lactobacillus plantarum competent cells to construct a folE gene knock-out strain delta folE by homologous recombination; by comparing the physiological and biochemical differences of the wild strain and the delta folE strain, the folE gene is analyzed to be irrelevant to the cell morphology and the growth state of the strain and play a role in folic acid synthesis.
4. The method for constructing the knockout vector pED 760-folE according to claim 3, wherein the method for constructing the knockout vector pED 760-folE comprises:
the method comprises the steps of taking a food-borne lactobacillus plantarum genome as a template, utilizing a primer pair up-EF + up-ER and a primer pair down-EF + down-ER to respectively amplify upstream and downstream homologous arms, using a PCR product as a template, utilizing the primer pair up-EF + down-ER to amplify to obtain a homologous arm knockout fragment, purifying the fragment, synchronously carrying out enzyme digestion on the fragment and a temperature sensitive plasmid pFED760 by using restriction enzymes Hind III and Spe I respectively, carrying out a ligation experiment after the enzyme digestion product is subjected to gel cutting recovery, introducing the ligation product into escherichia coli DH5 α competent cells, and extracting a plasmid to obtain a folE gene knockout vector pFED 760-folE.
5. The method of claim 4 for constructing the knockout vector pED 760-folE, wherein the primer sequence is as follows:
up-EF：5’-CCCAAGCTTGAACGGCGTAATGGTCAAC-3’；
up-ER：5’-TTGAATTGACCACTGTAATTAATTATCAAATGGTGCGG-3’；
down-EF：5’-CCGCACCATTTGATAATTAATTACAGTGGTCAATTCAA-3’；
down-ER：5’-GCACTAGTACACGGTCAACCGGCCTGG-3’。
6. the method for constructing the knockout vector pED 760-folE according to claim 4, wherein the GTP cyclohydrolase I gene folE and the upstream and downstream homology arm sequences are shown in SEQ ID NO. 2 and are derived from Lactobacillus plantarum.
7. The method for screening for a knockout vector pED 760-folE according to claim 3, wherein the method for screening for a knockout vector pED 760-folE comprises the steps of:
(1) knock-out vector pED 760-folE transformation: adding 10 mu L of knockout carrier pFED760-folE into 90-100 mu L of lactobacillus plantarum receptive state, mixing gently, carrying out ice bath for 5min, transferring into a precooling electric shock cup, and carrying out electric shock according to parameters of 12.5kv/cm and 200 omega; 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 1.5mL sterile centrifuge tube, and statically culturing the mixture at 28 ℃ for 2.5-3 h to resuscitate cells; centrifuging the cultured bacterial liquid at 8000-10000 rpm for 3min, discarding 900 mu L of supernatant, resuspending the thallus with the rest supernatant, coating the thallus on an MRS solid plate containing 5 mu g/mL of erythromycin, and standing and culturing at 28 ℃;
(2) and (3) screening of folE gene knockout strains: transferring the single colony grown in the step (1) 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 on MRS solid plate containing 5 μ g/mL erythromycin, standing at 37 deg.C for 24 hr, picking out single clone, inoculating to 1mL solid plate containing 5 μ g/mL erythromycinThe MRS liquid culture medium is statically cultured at 37 ℃ for overnight, the culture bacterial liquid is inoculated into the MRS liquid culture medium without antibiotics according to 1 percent, the MRS liquid culture medium is statically cultured at 28 ℃ for overnight, and then the bacterial liquid is diluted by 10 percent3～105Coating the double single colonies on an MRS solid plate without antibiotics, performing static culture at 37 ℃ until single colonies grow out, selecting smaller single colonies, scribing the smaller single colonies on the MRS solid plate containing 5 mug/mL of erythromycin and without antibiotics one by one, performing static culture at 37 ℃ for 24 hours, selecting colonies which cannot grow on an MRS agar plate containing the antibiotics and can grow on the MRS agar plate without the antibiotics, and performing bacterial liquid PCR (polymerase chain reaction) verification;
in the PCR verification of the bacterial liquid, a front primer folE-FF: 5'-CAGACGCAAATGACGCAGT-3' is located on the upstream genome of the upstream homology arm, and the rear primer folE-RR: 5'-CGGATAACGCTCGTTCTGGAT-3' are located on the downstream genome of the downstream homology arm; the PCR fragment of the wild strain is 335bp larger than that of the knockout strain;
(3) the folE gene of the food-borne lactobacillus plantarum is analyzed by using the successfully constructed knockout strain, is irrelevant to the cell morphology of the lactobacillus plantarum and the growth state of the strain, and plays a key role in folic acid synthesis of the lactobacillus plantarum.
8. Use of the GTP cyclohydrolase I gene folE of claim 1 from Lactobacillus plantarum for food fermentation.
9. Use of the GTP cyclohydrolase I gene folE as defined in claim 1 in the synthesis of functional foods from folic acid.
10. Use of the GTP cyclohydrolase I gene folE as defined in claim 1 for increasing the biomass of a microbial strain.
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