CN112280795A - Use of glycosyltransferase genes - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Abstract
The invention discloses an application of a glycosyltransferase gene, namely the application of the glycosyltransferase gene in improving the synthesis of extracellular polysaccharide of lactobacillus plantarum; glycosyltransferase geneorf1595 or genescps4I, Geneorf1595 the nucleotide sequence is shown in SEQ ID NO:1, the genecpsThe nucleotide sequence of 4I is shown as SEQ ID NO. 2; will be provided withorf1595 orcps4I radicalConstructing a knockout vector by recombination with temperature sensitive plasmid, introducing the knockout vector into food-borne lactobacillus plantarum competent cells, and further constructing a gene knockout strain by homologous recombination; the extracellular polysaccharide producing capacity of the strain is measured by adopting a phenol-sulfuric acid method, and the extracellular polysaccharide producing capacity of the knock-out strain is lower than that of a wild strain,orf1595 orcpsThe 4I gene plays a key role in extracellular polysaccharide synthesis, and the invention has great potential in the fields of extracellular polysaccharide biosynthesis research and application.
Description
Technical Field
The invention belongs to the field of functions and applications of microbial genes, and particularly relates to a glycosyltransferase geneorf1595、cps4I in improving the biosynthesis of extracellular polysaccharide of Lactobacillus plantarum YM 4-3.
Background
Lactic Acid Bacteria (Lactic Acid Bacteria) are a general term for a class of microorganisms that are Generally Recognized As Safe (GRAS) to ferment glucose or produce Lactic Acid from lactose. Not only in inorganic environments, but also in environments such as human and animal intestines, which are traditionally related to fermented foods and have been closely linked to human culture and welfare, lactic acid bacteria have historically been widely known for their positive contribution to the organoleptic, quality and safety aspects of fermented foods.
Exopolysaccharides (EPS) are important secondary metabolites produced by lactic acid bacteria, and have attracted much attention and recognition in recent years. The microorganism EPS is a high molecular weight, long-chain, linear or branched biopolymer, often composed of repeating saccharide units or saccharide derivatives linked by alpha-and beta-glycosidic bonds, is a metabolite secreted extracellularly by microorganisms during growth, can be divided into two types, namely capsular polysaccharide adhering to the cell surface and mucopolysaccharide free in the culture medium, and has not only protective and adhesive effects on microorganisms but also other beneficial properties for humans, plants or animals. Exopolysaccharides produced by lactic acid bacteria, for example, play an important role in helping microorganisms to fight adverse conditions such as dehydration, nutrient deficiency, bacteriophages, osmotic pressure, antagonists and toxicants. Compared with plant-derived polysaccharides and animal-derived polysaccharides, the extracellular polysaccharide of the microorganism has the characteristics of easy culture, no influence of diseases and insect pests, less environmental and seasonal limiting factors, large-scale culture and fermentation, various physiological activities and the like, and has more excellent application potential. The existence of various culturable microorganisms provides great convenience for human production and life, and metabolites of the microorganisms can be applied to industries such as agriculture, pharmacy, cosmetics, food and the like, and enrich the life of people.
With the economic development, the demand of human health is continuously rising, and the source of the EPS safety of the lactic acid bacteria and the various physiological activities thereof make the lactic acid bacteria more competitive in the market. However, in general, the EPS synthetic amount is generally low, the strain stability is poor, and the large-scale application of the lactic acid bacteria EPS in industrial production is restricted by factors such as high extraction and purification costs, so that the EPS synthetic amount can be effectively increased and the cost can be reduced by combining the genetic engineering technology which is mature to the corresponding strain with the optimal culture condition, and the industrial production of the EPS can be possible.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of a glycosyltransferase gene, namely the glycosyltransferase geneorf1595 orcps4I in increasing lactobacillus plantarumLactobacillus plantarum) Use in exopolysaccharide synthesis, said genesorf1595 the nucleotide sequence is shown in SEQ ID NO:1, the genecpsThe nucleotide sequence of 4I is shown as SEQ ID NO. 2; the invention is toorf1595、cps4I gene and temperature sensitive plasmid pFED760 are recombined to construct knockout vector pFED 760-deltaorf1595、pFED760-Δcps4I, and introducing the strain into the Lactobacillus plantarum competence to construct the strain by homologous recombinationorf1595、cps4I Gene Single knockout Strain Deltaorf1595、Δcps4I; demonstration of physiological and biochemical differences and EPS production by comparing wild-type strains and knockout strainsorf1595 orcpsThe 4I gene plays a key role in EPS synthesis, and the invention has great potential in the field of research and application of EPS biosynthesis.
In order to achieve the above object of the present invention, the technical solution of the present invention is as follows:
1. glycosyltransferase geneorf1595、cpsPreparation of a 4I knockout vector, said knockout vector being obtained by: using food-borne lactobacillus plantarum genome as a template and utilizing a primer pair (orf1595-up-F+ orf1595-up-R；orf1595-down-F+ orf1595-down-R；cps4I-up-F + cps4I-up-R；cps4I-down-F + cps4I-down-R) respectively amplifying upstream and downstream homologous arms, using PCR products as templates, and using a primer pair (orf1595-up-F + orf1595-down-R；cps4I-up-F + cps4I-down-R) to obtain a knockout fragment, purifying the fragment, and respectively using restriction enzyme to separate the fragment and the temperature-sensitive plasmid pED760EcoRⅠ、Hind III, synchronously carrying out enzyme digestion, carrying out gel cutting recovery on an enzyme digestion product, carrying out a ligation experiment, introducing the ligation product into escherichia coli DH5 alpha competent cells, and extracting plasmids to obtainorf1595、cps4I Gene knockout vector pFED 760-Deltaorf1595、pFED760-Δcps4I, wherein the primer sequences are as follows:
orf1595-up-F 5’- CCG GAATTC GATGGATCTAAAGCGGTGTT-3’，
orf1595-up-R 5’- CGCTGAATGAATTGCCTTTGTGCCGCATGTATTTGTAAGG-3’；
orf1595-down-F 5’- CCTTACAAATACATGCGGCACAAAGGCAATTCATTCAGCG-3’，
orf1595-down-R 5’- CCC AAGCTT GTGGCAAGCCGTCAACGA-3’；
cps4I-up-F 5’- CCG GAATTC GACAAGTGAAACGGTCTT-3’，
cps4I-up-R 5’- TCTGCTCCCGTCCGTTTACGCTCCCTCGGCAAATAGGTT-3’；
cps4I-down-F 5’- AACCTATTTGCCGAGGGAGCGTAAACGGACGGGAGCAGA-3’，
cps4I-down-R 5’- CCC AAGCTT AGGTCCATACCATTAGAAGC-3’；
in the inventionorf1595、cps4I gene and the sequences of the upstream and downstream homologous arms thereof are shown as SEQ ID NO 3 and SEQ ID NO 4, and are derived from food-borne lactobacillus plantarum (A)Lactobacillus plantarum) (ii) a 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 of the lactobacillus plantarum is also safe and harmless to human bodiesThe invention provides theoretical safety guarantee for application in the field of exopolysaccharide production in the future.
2. Glycosyltransferase geneorf1595、cps4I knockout strain construction and screening, including the glycosyltransferase geneorf1595、cpsConstructing a 4I knockout vector, transforming a late knockout vector and screening knockout strains, and comprises the following steps:
(1) knockout vector pFED 760-. DELTA.orf1595、pFED760-Δcps4I transformation: respectively adding 10 mu L knockout carrier pFED 760-delta into 90-100 mu L of lactobacillus plantarum receptive stateorf1595、pFED760-Δcps4I, gently mixing, carrying out ice bath for 5min, transferring into a precooled electric shock cup, carrying out electric shock according to parameters of 1.25kv/cm and 200 omega, quickly adding 900 mu L of fresh MRS culture solution into the electric shock cup after the electric shock is finished, gently blowing and uniformly mixing by using a gun head, transferring the mixed solution into a sterile 1.5mL centrifuge tube, and carrying out static culture at 28 ℃ for 2.5-3 h to resuscitate cells. Centrifuging the cultured bacterial liquid at 8000-10000 rpm for 3 min, discarding 900 mu L of supernatant, resuspending the thallus with the residual supernatant, coating the thallus on an MRS solid plate containing 50 mu g/mL of erythromycin, and standing and culturing at 28 ℃.
（2）orf1595、cps4I gene knockout strain screening: transferring the single colony grown in the step (1) into an MRS liquid culture medium containing 50 mu g/mL erythromycin, carrying out static culture at 28 ℃ until the OD600 of the bacterial liquid is 0.2-0.3, transferring the bacterial liquid to 37 ℃, continuing the static culture overnight, diluting the cultured bacterial liquid by 103~105Spreading the multiplied cells on an MRS solid plate containing 50 mug/mL erythromycin, performing static culture at 37 ℃ for 24 hours, selecting monoclonals, inoculating the monoclonals into 1mL of MRS liquid culture medium containing 50 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 an MRS solid plate without antibiotics after doubling, performing static culture at 37 ℃ until single colonies grow out, selecting small single colonies, scribing on the MRS solid plate containing erythromycin of 50 microgram/mL and without antibiotics in a one-to-one correspondence manner, performing static culture at 37 ℃ for 24 hours, selecting colonies which cannot grow on an MRS agar plate containing antibiotics and can grow on a plate without antibiotics, and performing bacteria culture on the colonies which can grow on the MRS agar plate without antibioticsPerforming PCR verification to obtain Lactobacillus plantarum YM 4-3-deltaorf1595 and Lactobacillus plantarum YM 4-3-. DELTA.cps4I single knockout strains; the screening method improves the screening efficiency of the knockout strain.
The bacterial liquid PCR verification of the invention uses the front primer located in the upstream genome of the upstream homology arm (orf1595-uu-F：5’- CTTTGAGGTGGTGGTAGAC-3’，cps4I-uu-F: 5'-GACGTGCAAGTTGTCAGAGTT-3'), a back primer located in the genome downstream of the downstream homology arm(s) ((iii)orf1595-dd-R：5’- AATATAGCGCGATACCAGA -3’，cps4I -dd-R: 5'-AGGCACCGTAAGCGACAC-3'); ratio of PCR fragments of wild type strainorf1595 the knockout strain is 250bp larger thancpsThe 4I knockout strain is 591bp larger.
3. Demonstration of food-borne Lactobacillus plantarum Using successfully constructed knock-out strainsorf1595、cpsThe 4I gene plays a key role in the synthesis of lactobacillus plantarum EPS.
One of the characteristics of the invention is that the research gene is from food-borne food lactobacillus, 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 third characteristic of the invention is that the research gene of the invention is provedorf1595、cpsThe key role of the 4I gene in the synthesis of extracellular polysaccharide provides a certain theoretical basis for the research and development of EPS synthetic functional food, and further discovers the role of the gene in cell morphology and bacterial strain growth.
Compared with the prior art, the invention has the following advantages: 1) the research gene is derived from food-grade microorganisms and has safety; 2) the present invention relates to a geneorf1595、cpsThe 4I gene plays a key role in EPS synthesis.
Drawings
FIG. 1 shows PCR verification of a knockout strain of the present invention, wherein FIG. A: YM 4-3-. DELTA.orf1595 Strain screening, M is 5000bp DNA molecule Marker, and lanes 1-5 are YM 4-3-. DELTA.orf1595 Strain, lane 6 blank control (sterile ddH)2O）PCR product control, and YM4-3 strain control in the remaining lanes; and B: YM 4-3-. DELTA.cps4I strain screening, M is 5000bp DNA molecule Marker, and lanes 1-2 are YM 4-3-deltacps4I strain, and YM4-3 strain control in the remaining lanes;
FIG. 2 is a comparison of exopolysaccharide production by wild-type and knockout strains according to the present invention, wherein 4-3 represents wild-type YM4-3 strain; qc1595 denotesorf1595 knockout strain YM 4-3-. DELTA.orf1595; qc4I denotescps4I knockout strain YM 4-3-deltacps4I。
Detailed Description
The present invention is further described in detail with reference to the following 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, unless otherwise specified, are conventional reagents and conventional methods; the temperature sensitive plasmid pPED760 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: glycosyltransferase gene (A)orf1595、cps4I) Knockout homology arm cloning
1. Upstream and downstream homology arms for PCR amplification
Extracting the genome of the food-borne lactobacillus plantarum YM4-3 by using a TAKARA bacterial genome DNA extraction kit (Takara Bio-engineering Co., Ltd.), and specifically performing the following operations according to the kit instructions:
targeting genesorf1595 using the extracted genome as template, respectivelyorf1595-up-F（5’- CCGGAATTCGATGGATCTAAAGCGGTGTT-3' underline the cleavage siteEcoRⅠ)、orf1595-up-R (5'-CGCTGAATGAATTGCCTTTGTGCCGCATGTATTTGTAAGG-3') andorf1595-down-F （5’- CCTTACAAATACATGCGGCACAAAGGCAATTCATTCAGCG-3’）、orf1595-down-R（5’- CCCAAGCTTGTGGCAAGCCGTCAACGA-3', the restriction sites are underlinedHind III) carrying out amplification to obtain upstream and downstream homologous armsorf1595-up（992bp）、orf1595-down（1156bp）；
Targeting genescps4I, using the extracted genome as a template, and dividingRespectively with a primer paircps4I-up-F (5’- CCGGAATTCGACAAGTGAAACGGTCTT-3', the restriction sites are underlinedEcoRⅠ )、cps4I-up-R (5'-TCTGCTCCCGTCCGTTTACGCTCCCTCGGCAAATAGGTT-3') andcps4I-down-F（5’- AACCTATTTGCCGAGGGAGCGTAAACGGACGGGAGCAGA-3’）、cps4I-down-R (5’- CCCAAGCTTAGGTCCATACCATTAGAAGC-3', the restriction sites are underlined HindIII) amplification of upstream and downstream homology armscps4I-up（1018bp）、cps4I-down (775 b), the PCR reaction system and amplification conditions were 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 55 ℃ for 15 s; extension at 72 ℃ for 50 s; circulating for 30 times; extending for 5min at 72 ℃, and storing at 12 ℃. After the reaction was completed, 5 μ L of the product was subjected to electrophoresis analysis in 1% agarose gel.
2. Construction and sequencing of gene knockout fragment clone
1 mu L of each downstream homology arm PCR product is taken as a template,orf1595-up-F andorf1595-down-R andcps4I-up-F andcps4I-down-R is used as a primer, and overlapped PCR is carried out according to the PCR reaction system and the amplification condition (the extension time is changed to 2 min); cutting the gel and recovering a PCR product (namely a gene knockout fragment) with an expected size, and connecting the PCR product to a pMD19-T vector according to the instruction of a TA cloning kit of Dalibao bioengineering Co., Ltd; introducing the ligation product into escherichia coli DH5 alpha competent cells by a heat shock transformation method, and coating the cells on an Amp-LB plate; after overnight culture at 37 ℃, randomly selecting 10-15 single colonies, extracting plasmids from cells of the single colonies, and respectively using the plasmidsEcoRⅠ、Hind III andEcoRⅠ、Hind III, enzyme digestion verification is carried out, and the positive plasmid is sent to a sequencing company for sequencing.
Example 2:orf1595、cpsconstruction of 4I Gene Single knockout vector
Limit of usePreparation of enzyme for incisionEcoRⅠ、HindIII andEcoRⅠ、Hind III, synchronously enzyme-cutting the gene knockout fragment with correct sequencing and the temperature-sensitive plasmid pED760 respectively;orf1595 the enzyme system is:EcoRⅠ，2µL；HindIII, 2 muL; 1 XH buffer, 4 μ L; knocking out a fragment or pFAD 760, 10-16 muL; and adding sterilized deionized water to 20 muL.cpsThe 4I enzyme cutting system is as follows:EcoRⅠ，1 µL；Hind III, 1 muL; 1 XM buffer, 4 muL; a gene knock-out fragment or pFAD 760, 30 μ L; and adding sterilized deionized water to 40 muL. And (3) enzyme digestion is carried out for 4h at 37 ℃, and then enzyme digestion products are recovered, wherein the enzyme digestion products are obtained according to the target genes: adding a carrier = 4: 1-2: 1 (molar ratio), adding T4 DNA ligase, and connecting for 12-16 h at 16 ℃; introducing the ligation product into escherichia coli DH5 alpha competent cells by a heat shock transformation method, and then coating the cells on an erythromycin-LB solid plate; after overnight culture at 28 ℃, extracting plasmids from 10-15 single colony cells, and performing double enzyme digestion verification by using corresponding endonuclease to obtain positive plasmids, wherein the positive plasmids are named as pFED 760-deltaorf1595 and pFED760-Δcps4I。
Example 3:orf1595 orcpsConstruction of 4I Gene Single knockout Strain
1、orf1595 orcpsIntroduction of 4I gene knockout vector into Lactobacillus plantarum competent cells
Lactobacillus plantarum competent cells were prepared according to the method reported in Ferigo (2015, master academic paper of university of southern China); respectively adding 10 mu L of gene knockout carrier pFED 760-delta into 90-100 mu L of lactobacillus plantarum receptive stateorf1595 or pFED 760-. DELTA.cps4I, mixing the materials gently, carrying out ice bath for 5min, transferring the mixture into a precooling electric shock cup, and carrying out electric shock according to the parameters of 1.25kv/cm and 200 omega; and after the electric shock is finished, quickly adding 900 mu L of fresh MRS culture solution into the electric rotating cup, 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 the cells. After the culture, the bacterial liquid is centrifuged for 3 min at 8000 rpm, 900 mu L of supernatant is discarded, the residual supernatant is used for resuspending the thalli, the thalli are coated on an MRS solid plate containing 50 mu g/mL of erythromycin, and the culture is carried out at the temperature of 28 ℃ in a standing way.
2、orf1595、cps4I Gene knockout Strain ScreenSelection and verification
Randomly selecting 2-3 single colonies, transferring the single colonies into an MRS liquid culture medium containing 50 mu g/mL erythromycin, carrying out static culture at 28 ℃ until the OD600 of the bacterial liquid is 0.2-0.3, transferring the bacterial liquid to 37 ℃, continuing the static culture overnight, diluting the cultured bacterial liquid by 103~105Spreading the multiplied cells on an MRS solid plate containing 50 mug/mL erythromycin, performing static culture at 37 ℃ for 24 hours, selecting monoclonals, inoculating the monoclonals into 1mL of MRS liquid culture medium containing 50 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%3~105And coating the double single colonies on an MRS solid plate without antibiotics, performing static culture at 37 ℃ until single colonies grow out, selecting small single colonies, scribing the small single colonies on the MRS solid plate containing erythromycin of 50 microgram/mL 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 antibiotics, and performing bacterial liquid PCR (polymerase chain reaction) verification on the colonies which can grow on the plate without antibiotics.orf1595 PCR verification of bacterial liquid of knockout strain: front primer: (orf1595-uu-F: 5'-CTTTGAGGTGGTGGTAGAC-3' is located on the upstream genome of the upstream homology arm, and the rear primer: (orf1595-dd-R: 5'-AATATAGCGCGATACCAGA-3') is located on the genome downstream of the downstream homology arm; the PCR fragment of the wild strain is 250bp larger than that of the knockout strain, and the knockout strain YM 4-3-delta is obtainedorf1595。cps4I PCR verification of the bacterial liquid of the knockout strain: front primer: (cps4I-uu-F: 5'-GACGTGCAAGTTGTCAGAGTT-3' is located on the upstream genome of the upstream homology arm, and the rear primer: (cps4I-dd-R: 5'-AGGCACCGTAAGCGACAC-3') is located on the genome downstream of the downstream homology arm; the PCR fragment of the wild strain is knocked out by 591bp to obtain a single knocked-out strain YM 4-3-deltacps4I; the screening method improves the screening efficiency of the knockout strain, and the result is shown in figure 1.
Example 4: detection of extracellular polysaccharide yield of knockout strain
1. Extraction of exopolysaccharides
Activating wild type Lactobacillus plantarum YM4-3 and knock-out Lactobacillus plantarum YM 4-3. deltaorf1595、YM 4-3Δ cps4I，Respectively inoculating the strains in MRS liquid culture medium according to the inoculation amount of 2%, and culturing at 37 ℃ for 24 h; centrifuging at 12000rpm/min for 10min, collecting supernatant, adding 4% trichloroacetic acid (TCA) to remove protein, adding into rotor, and stirring on magnetic stirrer for 30 min; 12000rpm/min, centrifuging for 10min, taking the supernatant, adding 3 times of volume of absolute ethyl alcohol, and precipitating at-20 ℃ overnight; centrifuging at 12000rpm/min for 15min, removing supernatant, dissolving precipitate with appropriate amount of distilled water, placing into a pretreatment dialysis bag, dialyzing for 2 days with water change every 12 h; subpackaging the dialysate obtained in the dialysis bag, fully freezing at-80 ℃, and carrying out vacuum freeze-drying to obtain the EPS.
2. Extracellular polysaccharide yield assay
Referring to the phenol-sulfuric acid method of Kanmani Paulraj et al (Bioresource Technol, 2011), FIG. 2 shows that the extracellular polysaccharide content of wild type Lactobacillus plantarum YM4-3 is 122.642 + -0.781 mg/L, while that of single knockout type YM 4-3-. DELTA.orf1595 extracellular polysaccharide content of 53.7 + -2.76 mg/L, single knockout strain YM 4-3-ΔcpsThe 4I exopolysaccharide content is 23.8 +/-0.364 mg/L, which is lower than that of the wild strain, thereby proving that the glycosyltransferase geneorf1595、cps4I plays a key role in the synthesis process of extracellular polysaccharide in the lactobacillus plantarum YM4-3 strain. Therefore, the yield of extracellular polysaccharide can be improved in a mode of overexpression of glycosyltransferase subsequently, so that the purpose of production expansion is achieved, and a theoretical basis is provided for industrial application of the lactobacillus plantarum YM4-3 strain.
Sequence listing
<110> university of Kunming science
<120> use of glycosyltransferase gene
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 717
<212> DNA
<213> Lactobacillus plantarum YM4-3(Lactobacillus plantarum YM4-3)
<400> 1
gtgaagattg tttacatcat tactcaagcg acttggggtg gggcccaggc gcatctatat 60
agtttgatca aagcgcaagt gatgcgtggc aatgccgttg ccttagtata cggcgttgaa 120
ggacgcctga gtgcaagcgt cgcgaaagaa tttcaagacg tgcaagttgt cagagttgcc 180
agcctggtac atccgattgc accgctgagt gatttgaaag caatctacac gttaaggaaa 240
ttagtaaaaa attggcagcc agatattatt catttgcatt cttcgaaggc tggtatgatt 300
gggattgtcg gcgatggtag cttgagcgca cacatcgcta aacagattgc agccaccccg 360
caaatacggt ggttagggtt taaaagtaac ccttacaaat acatgcggca tgcaaaagtt 420
gtgctgtcga cgtccaaatc agacgcgttt ggattgacca tggtcgaggc agccttattg 480
ggtgcgattc cgtttgcccc ccgcattggg ggcatctctc aaacggccgc aagggtaaat 540
ggcctggttt atgcgagtga tgctgaatta ttggcagtac tgacacgtct cttttcggat 600
gggaaatttt atcaggaaac caaggctaaa atagcagcgg tcgattttag tgactatgaa 660
caaaggcaat tcattcagcg gatccagcaa gtttataaag gagtaatgga acgatga 717
<210> 2
<211> 969
<212> DNA
<213> Lactobacillus plantarum YM4-3(Lactobacillus plantarum YM4-3)
<400> 2
atggcaatca gcgtaattat gagtgtatat aatgaacgac ctgagcaagt ccagcaagcg 60
gttgactcga ttttgaagca aacctatttg ccgagggagt ttgtgatcgt actggataat 120
ccagaacgga gtgatttaaa agacttgtta atggattacg attgccgtgt ggaaatgatc 180
aaactagtgt gcaatccaga aaacctcggc ctggccgcta gtctcaataa ggcgattgag 240
ctagcaagta atgagttgat tgcgcggatg gatgcagatg atatttctgt cactaatcgg 300
ctcgaagtgg aattagaggc gctaaaaacg cgcgatttgg atttaatttc cggaaatatt 360
gcttatcttg atgagcagga tgaggttgtc ggtgaaaaat cggccattcc cgaggctgaa 420
ccactaattc aaaaaatcct gccctatggt tcgacaatta ttcatcccac cgttttaatg 480
cggaaaacgg ccgtccaaca agtcggtggt taccgtttat taccaacggc tgaggactat 540
gatctctggc tgcggctgct tgctaatgga ttcaaaatcg ggtcaattaa ccgccgagtg 600
ttgaactatc ggttacgcgg taatagtatg accagcgacg cgtggaaaac gtatctggta 660
tcgcgctata ttcaacaatt gtatgcggaa cgtaaacgga cgggagcaga cagtttcggc 720
cgcgggatgt cggcgttaac agacaaaata aatgacgtcc gtgcccaaag tacgtttaat 780
cgtgggcaaa aatattttac gatggcgatg gccagtatgc gcaaacgaaa acttggtcag 840
gccctggctg cgttagtccg cgcaatggtg acatccccag ataattgtta ctatgtgatc 900
aactatttac gtttaagaac gatttggtcg gtaaatgggc gccggtatcg acaacaacca 960
agtgattga 969
<210> 3
<211> 2126
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
agatggatct aaagcggtgt ttcgatctag cagtcggctt attgggcaca ctgctcagcg 60
ttcccatcgt attgtgtttg ccatacttat agttgacttc tgacggtccc gttttctaca 120
agcaggagcg agtcggttgg atggggacga cctttgacgt gattaagctg cgatcaatgt 180
atcaggacgc tgaggcgcag acgggtgcga tctgggcaca aaaaaatgat cctcgggtga 240
cgccggttgg acgctggatg cgcaaaactc ggatcgatga gttaccacaa ttttggaatg 300
ttttgaaggg cgatatgagt ttggtcggac ctcgcccgga acggccagaa ctaaccgaac 360
aattcagtga acgctatcct gattttccga aacggctacg aatcattcct ggcattaccg 420
gctatgcgca gatcaatggt ggctatgata ttacaccggg tgccaagtgt cagtacgata 480
actattacat tgagcacttt tcgatttggt ttgatatcaa gatgctgatg ggaacggtta 540
gggtgattat ttccggggat ggtgcgcggt agtgaagatt gtttacatca ttactcaagc 600
gacttggggt ggggcccagg cgcatctata tagtttgatc aaagcgcaag tgatgcgtgg 660
caatgccgtt gccttagtat acggcgttga aggacgcctg agtgcaagcg tcgcgaaaga 720
atttcaagac gtgcaagttg tcagagttgc cagcctggta catccgattg caccgctgag 780
tgatttgaaa gcaatctaca cgttaaggaa attagtaaaa aattggcagc cagatattat 840
tcatttgcat tcttcgaagg ctggtatgat tgggattgtc ggcgatggta gcttgagcgc 900
acacatcgct aaacagattg cagccacccc gcaaatacgg tggttagggt ttaaaagtaa 960
cccttacaaa tacatgcggc acaaaggcaa ttcattcagc ggatccagca agtttataaa 1020
ggagtaatgg aacgatgact gcagtaaatc gtcagattgg tgggccattc ttaagactag 1080
gactattgat tgccggattt tacctgattt accaacccaa cttttggagc agtagttatg 1140
tgcccctgat tgttacactg gggattactg gtggttgcgt cttgctattg tcgccaaagg 1200
acttatcgat tgctattaat cggctgcaca tttttgtgat tggtgtgatg ctatcagtga 1260
tttactttgc gttcagagcg aagctggcag gaactgatcc ccgggtgttt caaaatgttg 1320
tgattatttt gaatgtcctt gccttagtgt tatggctaga agttcttcgt aagtatttta 1380
acgtgacaag tgaaacggtc ttgacctgga tgctatggat ggtggttgtt caatgcttgt 1440
ttgctttggt catgctggca agttcgggta ttcgcgctgc gattttagca aaaacagcca 1500
ccggtattat tcagaatcag tttatttatg gtgaacgttt gtatggaatc agcagtgact 1560
acacattttt tacaccaatc taccatagtc tgatgggact agttgcgata tatatgggaa 1620
tgaaccttgg caaaaagtat tattggttcc tgccattttg tgtctttatt atcgtattaa 1680
acggccgaac tggcttgatc acgctggctt ttggttttgc cttgatgctg gtcaaacgaa 1740
tgctgtccag tgttcgaggg ctgtttcagg ttgcactgat tatcgtgttg agtgtgacct 1800
ttatcatact gggtttggct tttctgcaat ccatccagcc tgatacgtat acctggatcg 1860
tcagtggctt gaagatacgt ttgcactcgt ctttgaaggt agtaagcagg gcaattacga 1920
acaactgacg ggctcgttct tgatgtttcc gagtcagctt ttagattggg tgtttggtta 1980
tggcgttcgc gtatatggtg gcaatgccaa caatatttgg ttcggaacgt ccgacattgg 2040
ctttattaat gacttattta tggggggcat catttacatg gcgctactgt acggtacgat 2100
tatagcctcg ttgacggctt gccaca 2126
<210> 4
<211> 1774
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
tgacaagtga aacggtcttg acctggatgc tatggatggt ggttgttcaa tgcttgtttg 60
ctttggtcat gctggcaagt tcgggtattc gcgctgcgat tttagcaaaa acagccaccg 120
gtattattca gaatcagttt atttatggtg aacgtttgta tggaatcagc agtgactaca 180
cattttttac accaatctac catagtctga tgggactagt tgcgatatat atgggaatga 240
accttggcaa aaagtattat tggttcctgc cattttgtgt ctttattatc gtattaaacg 300
gccgaactgg cttgatcacg ctggcttttg gttttgcctt gatgctggtc aaacgaatgc 360
tgtccagtgt tcgagggctg tttcaggttg cactgattat cgtgttgagt gtgaccttta 420
tcatactggg tttggctttt ctgcaatcca tccagcctga tacgtatacc tggatcgtca 480
gtggctttga agatacgttt gcactcgtct ttgaaggtag taagcagggc aattacgaac 540
aactgacggg ctcgttcttg atgtttccga gtcagctttt agattgggtg tttggttatg 600
gcgttcgcgt atatggtggc aatgccaaca atatttggtt cggaacgtcc gacattggct 660
ttattaatga cttatttatg gggggcatca tttacatggc gctactgtac ggtacgatta 720
tagcctcgtt gacggcttgc cacaaaaagt ctggaaaggt tgttcgtgat agtaaaatct 780
ttctagcctt tggcgtagtt ctactaatcg caaattataa aggtgaggtt gcgcgcagcg 840
gcatggtact gacggcaata atattcttgg ggtatttact ttcaactgaa ttgaagattg 900
aggagaaaaa atggcaatca gcgtaattat gagtgtatat aatgaacgac ctgagcaagt 960
ccagcaagcg gttgactcga ttttgaagca aacctatttg ccgagggagc gtaaacggac 1020
gggagcagac agtttcggcc gcgggatgtc ggcgttaaca gacaaaataa atgacgtccg 1080
tgcccaaagt acgtttaatc gtgggcaaaa atattttacg atggcgatgg ccagtatgcg 1140
caaacgaaaa cttggtcagg ccctggctgc gttagtccgc gcaatggtga catccccaga 1200
taattgttac tatgtgatca actatttacg tttaagaacg atttggtcgg taaatgggcg 1260
ccggtatcga caacaaccaa gtgattgaac gtgtgcggtg cttatgagat ttgttattag 1320
taagcacggg atgtgatgct cagttcagat ctatgagaaa ggaatttatg ttagtcagta 1380
ttagaaataa atatcggcat ttgccaaagc aagttaaggc ctcactttgg ttcttaatct 1440
gtgctttttt tgaaaagagc atttcaataa tcgcaacgcc catttttacc agaataatgt 1500
ctacttccga gtatggtcaa tttaatgtgc tgtactcctg gctgactatc gtcacgatca 1560
tcgtctcact aaatctgtgc tacggcgttt atactcaagg tctcatcaaa ttcagccatg 1620
atcgacgtcg atattcggct gcattacaag gattaacggt tgtgttggta ttagcatgga 1680
cactagttta tcttggcttt cgtgattttt ggaatagtgt cttttcttta acgacgacgc 1740
agatgttggc gatgcttcta atggtatgga cctc 1774
<210> 5
<211> 29
<212> DNA
<213> Artificial sequence (Artificial)
<400> 5
ccggaattcg atggatctaa agcggtgtt 29
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 6
cgctgaatga attgcctttg tgccgcatgt atttgtaagg 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 7
ccttacaaat acatgcggca caaaggcaat tcattcagcg 40
<210> 8
<211> 27
<212> DNA
<213> Artificial sequence (Artificial)
<400> 8
cccaagcttg tggcaagccg tcaacga 27
<210> 9
<211> 27
<212> DNA
<213> Artificial sequence (Artificial)
<400> 9
ccggaattcg acaagtgaaa cggtctt 27
<210> 10
<211> 39
<212> DNA
<213> Artificial sequence (Artificial)
<400> 10
tctgctcccg tccgtttacg ctccctcggc aaataggtt 39
<210> 11
<211> 39
<212> DNA
<213> Artificial sequence (Artificial)
<400> 11
aacctatttg ccgagggagc gtaaacggac gggagcaga 39
<210> 12
<211> 29
<212> DNA
<213> Artificial sequence (Artificial)
<400> 12
cccaagctta ggtccatacc attagaagc 29
<210> 13
<211> 19
<212> DNA
<213> Artificial sequence (Artificial)
<400> 13
ctttgaggtg gtggtagac 19
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (Artificial)
<400> 14
aatatagcgc gataccaga 19
<210> 15
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 15
gacgtgcaag ttgtcagagt t 21
<210> 16
<211> 18
<212> DNA
<213> Artificial sequence (Artificial)
<400> 16
aggcaccgta agcgacac 18
Claims (1)
1. Glycosyltransferase gene in increasing lactobacillus plantarum (A)Lactobacillus plantarum) Use of the glycosyltransferase gene in the synthesis of exopolysaccharidesorf1595 or genescps4I, Geneorf1595 the nucleotide sequence is shown in SEQ ID NO:1, the genecpsThe nucleotide sequence of 4I is shown as SEQ ID NO. 2.
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| CN112852844A (en) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | Application of hydroxymethyl dihydropterin pyrophosphokinase gene folK |
| CN112961878A (en) * | 2021-03-08 | 2021-06-15 | 昆明理工大学 | Application of gene of lactobacillus plantarum in folic acid biological generation |
| CN113832167A (en) * | 2021-11-01 | 2021-12-24 | 昆明理工大学 | Gene and application thereof in improving yield of phenethyl alcohol and tryptophol |
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