CN117778391A - Novel lithocanol biosynthesis gene promoter of deep sea fungus phomopsis FS508 - Google Patents
Novel lithocanol biosynthesis gene promoter of deep sea fungus phomopsis FS508 Download PDFInfo
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Abstract
The invention discloses a novel lithocanol biosynthesis gene promoter of a deep sea fungus phomopsis FS508. The nucleotide sequence of the promoter litIP is shown as SEQ ID NO. 2. According to the invention, a promoter litIP of a Baeyer-Villiger oxidase coding gene litI is obtained by analyzing a lithocanol biosynthesis gene cluster and a key gene litI thereof. The promoter litIP has activity of promoting luciferase in Escherichia coli close to that of pgpdA, and can promote ampicillin expression in Escherichia coli. Cloning and functional verification of the promoter litIP lay a reliable early foundation for improving biosynthesis efficiency of lithocanol and obtaining novel derivatives through a biosynthesis strategy and transcriptional regulation in the later period.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a novel lithocanol biosynthesis gene promoter of a deep sea fungus phomopsis FS508.
Background
Phomopsis (Phomopsis lithocarpus) FS508 is a filamentous fungus separated from deep sea mud, and is separated from the filamentous fungus to obtain the polyisoprenyl benzophenone compound lithocarols and tenellone heterozygous macrolide dimer lithocarpins with stronger antitumor activity, so that the preparation method has the potential of being developed into a novel antitumor drug lead compound. The fungal source of the polyprenyl benzophenone is a subset of aromatic polyketides with unique structures, which are heteroterpene compounds composed of benzophenone (or ketone) and at least two C5 units (isopentenyl), the specific chemical structure rendering them biologically active.
The poly-isopentenyl benzophenone compound lithocarosa-G (Jianlin Xu, zhaoming Liu, yuchan Chen, haibo Tan, haohua Li, saini Li, heng Guo, zilei Huang, xiaoxagao, hongxin Liu, weimin Zhang. Lithocarpols A-F, six tenellone derivatives from the deep-sea derived fungus Phomopsis lithocarpus FS508.Bioorganic Chemistry 87 (2019) 735) with certain antitumor activity and antibacterial activity was obtained from P.lithocarpus FS508 in the early stage, but the yield was low, so that the research on the promoter of the key gene for the biosynthesis of lithocarols is helpful for elucidating the transcriptional regulation mechanism of the biosynthesis of the lithocarols and for improving the biosynthesis efficiency of the lithocarols through transcription and the expression of the key genes.
Disclosure of Invention
The first object of the invention is to provide a promoter litIP, the nucleotide sequence of which is shown as SEQ ID NO. 2.
The second object of the present invention is to provide an expression vector comprising the promoter litIP described above.
It is a third object of the present invention to provide a host cell containing the above-described expression vector.
Preferably, the host cell is E.coli Trans5α, E.coli BL21 (DE 3) or Phomopsis (Phomopsis lithocarpus) FS508.
It is a fourth object of the present invention to provide the use of the promoter litIP described above to initiate expression of a downstream gene in a host cell.
Preferably, the downstream gene is the lithocarols biosynthesis gene litI, the ampicillin resistance gene AmpR or the luciferase reporter gene.
Preferably, the host cell is E.coli Trans5α, E.coli BL21 (DE 3) or Phomopsis (Phomopsis lithocarpus) FS508.
It is a fifth object of the present invention to provide an expression cassette comprising the promoter litIP described above.
It is a sixth object of the present invention to provide the use of the promoter litIP described above, or the expression vector described above, or the host cell described above, or the expression cassette described above, in the biosynthesis of polyisopentenyl benzophenone compounds lithocarols.
A seventh object of the present invention is to provide a method for regulating transcription of a target gene, which comprises introducing the above promoter litIP, or the above expression vector, or the above expression cassette into a host cell to enhance transcription of the target gene in the host cell.
The phomopsis (Phomopsis lithocarpus) FS508 of this patent was isolated from the submarine sediment at 3606m of Indian ocean (111 DEG 53.335'E,16 DEG 50.508' N).
The invention has the advantages that:
the lithocarols biosynthesis gene promoter litIP has higher transcriptional activity, the activity of the promoter luciferase in escherichia coli is close to that of a strong promoter pgpdA, and the promoter can promote the expression of an ampicillin resistance gene. Cloning and functional verification of the promoter litIP lay a reliable molecular biology foundation for improving biosynthesis efficiency and yield of lithocanol through a biosynthesis strategy and transcriptional regulation in the later period and obtaining novel derivatives.
The deep sea fungus phomopsis (Phomopsis lithocarpus) FS508 of the invention has the preservation number of: GDMCC NO:60433, disclosed in the invention patent CN201810974840.6, entitled: the compound lithocarpinol B, a preparation method thereof and application thereof in preparing antifungal medicaments.
Drawings
FIG. 1 shows the amplification of promoters I-1, I-2 of FS508 novel fungal isopentenyl benzophenone lithocarols.
FIG. 2 shows construction of recombinant vectors pGL3-I-1, pGL 3-I-2; m: DL5000 DNAMarker;1-5: colony PCR verification containing I-1 promoter; 6-10: colony PCR verification of the I-2 promoter.
FIG. 3 shows the detection of I-1 and I-2 promoter activity by luciferase.
FIG. 4 is construction of recombinant pET28a-litIP vector: a) Amplifying the promoter fragment litIP; b) Colony PCR verifies pET28a-litIP vector; c) pET28a-litIP plasmid map.
FIG. 5 shows the transcriptional activity of different concentrations of ampicillin resistant plates on spot plates, P being the positive control, N being the negative control and I being Trans5α -pET28a-litIP.
FIG. 6 OD values of Trans5α -pEASY-T1, trans5α -pET28a-AmpR, trans5α -pET28a-litIP in LB medium at 0, 25, 40 μg/mLAmp concentrations, respectively.
FIG. 7 is an element analysis of promoter litIP.
Detailed Description
The invention will be further explained below in connection with specific embodiments with reference to the drawings. The examples themselves do not limit the invention in any way.
The technical route of the invention is as follows:
the invention takes a biosynthesis gene cluster mdp of benzophenone compounds monodimorphenone as a target, and adopts Local blast, anti-SMASH and 2nd finder to locate a lit gene cluster to take charge of biosynthesis of lithocarols. Further adopting PLANTCARE (www.plantcare.co.uk) promoter to online analyze website to predict the promoter sequence of key biosynthesis gene litI (coding Baeyer-Villiger oxidase) of lithaoles, then adopting luciferase reporter gene vector and ampicillin resistance gene expression in colibacillus to make functional verification so as to lay foundation for improving biosynthesis efficiency of lithaoles and obtaining more high-activity lithaoles derivatives through transcriptional regulation, over-expression and heterologous expression.
The novel promoter for the biosynthesis genes of the phomopsis FS508 is prepared by performing whole genome sequencing on the phomopsis FS508, positioning the biosynthesis gene cluster litof the phomopsis by adopting Local blast, anti-SMASH and 2nd finder, analyzing key genes litI and litQ, and predicting the promoters litP and litQP in an upstream sequence through a PLANTCARE (www.plantcare.co.uk) promoter online analysis website. And designing a specific primer according to a predicted result, and performing PCR by taking the FS508 genome as a template, thereby obtaining a novel lithocarols biosynthesis gene promoter litIP of FS508.
The PCR amplificationThe increasing agent isMax Super-Fidelity DNAPolymerase (Nanjinozan Biotechnology Co., ltd.).
A function verification method of a novel lithocarols biosynthesis gene promoter of phomopsis FS508 comprises the steps of inserting a promoter sequence into a luciferase expression vector pGL3-Basic through homologous recombination, then introducing the recombinant vector into escherichia coli BL21 (DE 3), performing ultrasonic disruption to obtain a supernatant, and analyzing the transcriptional activity of the supernatant by using luciferase. The promoter sequence was further introduced into the constructed pET28a-AmpR vector by homologous recombination, positive clones were screened using kanamycin resistance plates before inserting the promoter into the ampicillin resistance gene, then positive clones were spotted onto LB plates containing different ampicillin, and the function of the promoter was further verified by monitoring its growth curve by culturing in well plates.
The method comprises the following specific steps: integrating a promoter litIP into pGL3-basic through NdeI and HindIII digestion vectors by using homologous recombinase (ClonExpress IIOne Step Cloning Kit C, wuhan Aibo taek Biotechnology Co., ltd.), converting into escherichia coli, amplifying a litIP sequence through colony PCR to obtain positive clone, extracting recombinant plasmid, converting into escherichia coli BL21 (DE 3), performing ultrasonic disruption to obtain supernatant, and analyzing the fluorescence intensity of a corresponding sample by using a luciferase reporter gene detection kit (Shanghai Biyun Biotechnology Co., ltd.) to verify the in vitro activity of the litIP; the litIP promoter sequence was further inserted into pET28a-AmpR vector using a homologous recombination kit to initiate expression of the ampicillin resistance gene AmpR, and positive clones were screened by kanamycin plate screening and colony PCR amplification of the litIP sequence. Positive clones were spotted onto LB plates containing 0, 15, 25, 30, 40, 50. Mu.g/mL, incubated at 37℃for 12 hours, and the growth of each colony in the medium was observed, thereby verifying the function of litIP.
The plasmid pET28a-AmpR is an escherichia coli expression vector with a kanamycin resistance gene KanR and an ampicillin resistance gene AmpR, and is a recombinant vector constructed by the invention. The specific method comprises the following steps: primers AmpR-F cgcggcagccatatggatgagtattcaacatttccg (SEQ ID NO. 8) and AmpR were designed containing the pET28a vector homology arm: aaagttaaacaa aattatttttaccaatgcttaatcagtgag (SEQ ID NO. 9) the AmpR fragment was obtained by PCR using pBARGE1 vector (purchased from Wohan vast, biotechnology Co., ltd.) as a template, and simultaneously EcoRI and BamHI double-digested pET28a vector was used to recover the product. Connecting the Amp R fragment with the digested pET28a by using a ClonExpress IIOne Step Cloning Kit C homologous recombination kit, transforming into a Trans5 alpha competent cell, coating on a kanamycin resistance plate, screening positive clones, carrying out bacterial liquid PCR amplification of the AmpR fragment, and carrying out sequencing verification, and amplifying and culturing plasmids by the positive clones with correct verification to obtain the pET28a-AmpR vector with ampicillin resistance.
The formula of the LB culture medium is as follows: peptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1L.
The escherichia coli is Trans5α and BL21 (DE 3), is a well-known commodity which can be purchased from Beijing full-scale gold biotechnology Co., ltd.
Example 1: luciferase promoter Activity analysis of novel lithocanol biosynthesis Gene promoter of Phomopsis FS508
Amplification of the litIP promoter sequence:
first, the Phomopsis FS508 was subjected to whole genome sequencing, the biosynthetic gene cluster litof lithocarols was located using Local blast, anti-SMASH and 2nd finder, and the key gene litI encoding BV oxidase was analyzed (its encoding amino acid sequence is shown as SEQ ID NO.1, and its promoter litIP was predicted in the upstream sequence by online analysis of the website through the PLANTCARE (www.plantcare.co.uk) promoter.
The promoter sequence was analyzed using a bioinformatics database and primer design was performed on the litI promoter sequence by SnapGene. The gene of about 500bp upstream of litI is divided into two sections according to a promoter prediction website (http:// www.fruitfly.org/seq_tools/promoter. Html), and is respectively marked as I-1, and the nucleotide sequence of the gene is shown as SEQ ID NO.3 [ the used primer is I-1F: acgcgtgctagcccgggctgttgggtgccccaagcc(SEQ ID NO.4)，I-1R：aacagtaccggaatgccaggtggtgattgt gattgaattg(SEQ ID NO.5)]I-2, the nucleotide sequence of which is shown as SEQ ID NO.2 [ litIP, the primer used is I-2F: acgcgtgctagcccgggcgttcaggtgagaatgaacagatag(SEQ ID NO.6)，I-2R：aacagtaccggaatgcca gcttgaatgcagcaagcag(SEQ ID NO.7)]And carrying out PCR amplification on the DNA, extracting FS508 genome DNA as an amplification template, wherein a PCR amplification reagent isMax Super-Fidelity DNA Polymerase (Nanjinozan Biotechnology Co., ltd.). The PCR product was purified and recovered to obtain litI promoter fragment with homology arm (FIG. 1). pGL3-basic vector (purchased from Wohan vast Ling Biotechnology Co., ltd.) was double digested with restriction enzymes XhoI and HindIII, and the digested product was subjected to agarose gel electrophoresis to identify and then the digested product was recovered and its concentration was measured. Fragments I-1 and I-2 (litIP) of the order of the promoters and pGL3-basic vectors were subjected to homologous recombination by ClonExpres s IIOne Step Cloning Kit C homologous recombination kit (Hitachi Biotechnology (Shanghai) Co., ltd.) and transformed into Trans5α competent cells, which were plated on LB plates containing 100. Mu.g/mL ampicillin (sigma, USA) resistant, positive clones were screened, I-1 and I-2 (litIP) fragments were amplified by bacterial liquid PCR and sequenced and verified (FIG. 2), and the positive clones were amplified to culture and extract plasmids, thereby obtaining pGL3-I-1 and pGL3-litIP (pGL 3-I-2) recombinant plasmids. The recombinant plasmids were transformed into BL21 (DE 3) competent cells with pGL3-pgpd-basic (positive control, DOI: 10.13560/j.cnki.bioechnech.bull.1985.2020-1386, pgpd is a strong promoter known in fungi) and pGL3-basic plasmid vector (negative control), respectively, and the monoclonal was selected and the strain was stored. Inoculating the preserved monoclonal bacterial liquid into an EP tube containing 1mL of 100 mug/mL Amp/LB liquid medium, culturing for 12h at 37 ℃ at 200r/min, inoculating into 15mL of Amp/LB liquid medium according to a ratio of 1:50, continuously culturing for 3-5h, determining an OD value to be 0.8-1.0, taking 10mL of bacterial liquid, centrifuging, washing for 3-4 times by PBS, adding 1mL of PBS into the precipitate obtained by the bacterial liquid obtained by the last centrifugation, resuspending, crushing (amplitude of 35%, stop of 3s for 5 s) by using an ultrasonic cell crusher, centrifuging for 5min at 10000r/min, and sucking the supernatant into the EP tube. 100. Mu.L of supernatant and 100. Mu.L of firefly luciferase were taken separatelyDetection reagent (RG 06, shanghai Biyun biotechnology Co., ltd.) was used in 96-well plates to detect RU values of each reaction using a Tecan Spark multifunctional microplate reader, and the reaction time was 10s. The results showed that the strains containing pgpd promoter had higher luciferase activity than the recombinant strain containing I-1 and I-2 (litIP) promoter fragments, and the recombinant strain containing litIP promoter fragment had higher luciferase activity than the recombinant strain containing I-1 promoter fragment (FIG. 3), indicating that the litIP promoter fragment had higher efficiency of promoting luciferase gene than the I-1 promoter fragment. The in vitro luciferase activity analysis result shows that the litIP promoter region has higher transcriptional activity, is close to the promoter activity of the strong promoter pgpdA and is far higher than that of a blank vector, and suggests that the litIP is a strong promoter (figure 3), so that the in vivo transcriptional activity of the litIP promoter in the escherichia coli resistant gene needs to be further verified.
Example 2: activity verification of the litIP transcribed ampicillin resistance Gene AmpR
Primers [ I2-28a-F: actttaagaaggagatattgttgggtgccccaagcc(SEQ ID NO. 10) and I2-28a-R aaatgttgaatactcatcgttcaggtgagaatgaacagatag(SEQ ID NO.11)]The litIP promoter sequence containing homology arms was amplified (FIG. 4A), pET28a-AmpR was double digested with NcoI and NdeI restriction enzymes, and the digested product was identified by agarose gel electrophoresis, and the product was recovered and its concentration was determined. The litIP promoter fragments were recombined and ligated with empty pET28a-AmpR respectively by using a homologous recombination kit (Hitachi Biotechnology (Shanghai), inc., C112), transformed into Trans 5. Alpha. Competent cells, plated on LB plates containing 50. Mu.g/mL kanamycin resistance to screen positive clones, and PCR-amplified on bacterial solutions to obtain positive clones (FIG. 4B). Sequencing verification was performed and pET28a-litIP plasmid was extracted (FIG. 4C).
The Trans5 alpha strain (negative control) containing pET28a-AmpR, the Trans5 alpha strain containing pET28a-litIP transformant and the Trans5 alpha strain (positive control) containing pEASY-T1 are respectively inoculated into 5mL LB liquid culture medium containing kanamycin for culturing for 12h at 37 ℃ and 200r/min, 1mL of bacterial liquid is taken to determine that the OD value is about 1.0, 1mL of bacterial liquid is taken, and a proper amount of non-antibiotic LB liquid culture medium is addedUntil the liquid was 1mL. 100. Mu.L of the mixture was diluted 100-fold by adding to 900. Mu.L of the antibiotic-free LB liquid, which was designated as 10 -1 Then from 10 -1 100. Mu.L of the culture medium was taken out and added to 900. Mu.L of the antibiotic-free LB liquid medium, which was designated as 10 -2 Then from 10 -2 100. Mu.L of the culture medium was taken out and added to 900. Mu.L of the antibiotic-free LB liquid medium, which was designated as 10 -3 Repeating until the liquid concentration is 10 -5 . mu.L of each of the bacterial solutions of different concentrations was incubated on LB plates with ampicillin concentrations of 0, 15, 25, 30, 40 and 50. Mu.g/mL at 37℃for 12 hours, and the growth of each colony in the medium was observed and recorded by photographing. As a result, only the Trans5α -pEASY-T1 and Trans5α -pET28a-litIP grew out of colonies on the ampicillin resistance plate of 40. Mu.g/mL (FIG. 5), further demonstrating that litIP was able to initiate expression of the ampicillin resistance gene AmpR in E.coli BL21 (DE 3).
According to the results of the data plate, 2 mu L of Trans5 alpha-pET 28a-litIP transformant bacterial liquid is respectively added into 198 mu L of LB liquid culture medium with ampicillin concentration of 0, 25 and 40 mu g/mL, bacterial liquids with different resistance concentrations are added into a 96-well plate, meanwhile, 200 mu L of LB liquid culture medium with ampicillin concentration of 0 mu g/mL is added into the 96-well plate, three repetitions are arranged for each resistance concentration bacterial liquid and each no resistance liquid culture medium, three blank wells are designed to be used as a blank, and the culture is carried out for 48 hours at 37 ℃. OD of strain was measured with Thermo multifunctional enzyme-labeled instrument 600 The values were measured once at 12h, 24h, 36h, 48h, and the experiment was repeated 3 times. Significance analysis was performed on growth OD values for different ampicillin resistance concentrations between the same strains using GraphPad Prism 8.0.1. The growth curves and significance analysis of the promoters at 0, 25 and 40 mug/mL ampicillin resistance concentrations show that the OD value of the Trans5 alpha-pEASY-T1 bacterial liquid at the same dilution concentration is larger than that of the Trans5 alpha-pET 28a-litIP, and the OD value corresponding to the Trans5 alpha-pET 28a-litIP is significantly larger than that of a blank vector negative control (figure 6), so that the function of the litIP promoter is further verified.
The functional components of the promoter were predicted using the planta care database, and analysis in combination with the characteristics of the promoter sequence revealed that on the litI promoter fragment litP, there was a TATAbox at-138 to precisely initiate transcription, and a CAAT box at-182 to control the transcription initiation frequency (fig. 7). By combining with the analysis of the dot-matrix results of the promoter, it is shown that the promoter litIP has more forward regulated promoter functional components, so that the promoter has stronger transcriptional activity.
Sequence listing
<110> institute of microorganisms at the academy of sciences of Guangdong province
<120> a novel lithocanol biosynthesis Gene promoter of the deep sea fungus Phomopsis FS508
<160>11
<210>1
<211>456
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<213> coding sequence
<400>1
MGSTMIASSLKLQSSHLGMVHRDQDPEPGSVEECGRLLQKNHEVYHMFFRDIAGMNHISHSLLSCLALGATPDDLQARYQDEGVNQRPMPPIDAELLEKMYCDAEVLYANLCVKPLYQTFLRLFERLIEEKGWKAVVQEYLFSRTKTAERMLASMFDGLYHPFIHLGLGVEFQLPGLVAEALAQAASNDDSHLPKLFGACETAAATTGAIPARQKSLLDLMQEVRANDKIRNAPQWSDLGLKLKNGLVGRACDEFAQVAGQFIIENTEEALARRTAEMISTVAFVSGAAQQKGRKTKIDFFVMHSVTSSIFCSVMAQQGDWISLEDRIRLMEWKARADLAWYAVVGSPEFDGSYISEYGGQASQNSVEWSDIFAAVVKEHDDGHAAKYIRALKNGELVGCQYENDSNWAGYFPMKGDMWLKLANMCQDTTTNRPIPTKWVMFTGYPEAWKRPDLAS
<110> institute of microorganisms at the academy of sciences of Guangdong province
<120> a novel lithocanol biosynthesis Gene promoter of the deep sea fungus Phomopsis FS508
<160>11
<210>2
<211>313
<212>DNA
<213> amplification sequence
<400>1
GTTCAGGTGAGAATGAACAGATAGTATGTCGTCTCAGTATCTGCGAATAATCGTATGGAGCCGGTGAAGACCGACTTGTGGCACTGAATATTATTTGCAAACGGTTTCGATCGAATTACCTTCTATTAACATTTTTATAGCGGCTAACTGCGCAGAAAACGTTTGTCGAACATGCTCAGCAATATGGTCTGATCATCAGCTGTGATTTATCTTGGGAGGTGGCCGTATGCATGGCTACATGGGTACATGTACCTCCGCCTGTCCCAAATTGGTCAACTCCGATGCCCGAGTTGCCTGCTT GCTGCATTCAAGC
<110> institute of microorganisms at the academy of sciences of Guangdong province
<120> a novel lithocanol biosynthesis Gene promoter of the deep sea fungus Phomopsis FS508
<160>11
<210>3
<211>167
<212>DNA
<213> amplification sequence
<400>1
TGTTGGGTGCCCCAAGCCAACATTGCACGGCGTGCATTTCAGAGACCAGTTGGAAGATATTTTTGAAATGCTTTGGCCTCGAGTTTGCAATTACAAATCTGAACAATTTCTCTGGCTAGTCCCGCCCTCATCACATCCTTCTCACC AATTCAATCACAATCACCACC
SEQ ID NO.4-11 shows the submitted sequence Listing in detail.
Claims (10)
1. A promoter litIP is characterized in that the nucleotide sequence of the promoter litIP is shown as SEQ ID NO. 2.
2. An expression vector comprising the promoter litIP of claim 1.
3. A host cell comprising the expression vector of claim 2.
4. A host cell according to claim 3, wherein said host cell is e.coli Trans5 a, e.coli BL21 (DE 3) or phomopsis (Phomopsis lithocarpus) FS508.
5. Use of the promoter litIP of claim 1 for promoting downstream gene expression in a host cell.
6. The use according to claim 5, wherein the downstream gene is the lithocarols biosynthesis gene litI, the ampicillin resistance gene AmpR or the luciferase reporter gene.
7. The use according to claim 5, wherein the host cell is E.coli Trans5α, E.coli BL21 (DE 3) or Phomopsis (Phomopsis lithocarpus) FS508.
8. An expression cassette comprising the promoter litIP of claim 1.
9. Use of the promoter litIP of claim 1, or the expression vector of claim 2, or the host cell of claim 3, or the expression cassette of claim 8 in the biosynthesis of polyisopentenyl diphenyl ketones lithocarols.
10. A method for regulating transcription of a target gene, characterized in that the promoter litIP according to claim 1, or the expression vector according to claim 2, or the expression cassette according to claim 8 is introduced into a host cell, whereby transcription of the target gene in the host cell is enhanced.
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| CN202311806887.9A CN117778391A (en) | 2023-12-26 | 2023-12-26 | Novel lithocanol biosynthesis gene promoter of deep sea fungus phomopsis FS508 |
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| CN202311806887.9A CN117778391A (en) | 2023-12-26 | 2023-12-26 | Novel lithocanol biosynthesis gene promoter of deep sea fungus phomopsis FS508 |
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| CN202311806887.9A Pending CN117778391A (en) | 2023-12-26 | 2023-12-26 | Novel lithocanol biosynthesis gene promoter of deep sea fungus phomopsis FS508 |
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| Country | Link |
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| CN (1) | CN117778391A (en) |
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2023
- 2023-12-26 CN CN202311806887.9A patent/CN117778391A/en active Pending
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