Genes for the biosynthesis of oxaborneane-type polyene polyketides and their applications

Abstract

This invention belongs to the field of genetic engineering and biosynthesis, and specifically relates to the biosynthesis of a class of polyketide compounds. This invention provides genes, named pruA, pruB, and pruC, and their encoded polypeptides, that play a key role in the formation of oxabicyclo[2.2.1]heptane structures in the biosynthesis of a class of polyketide compounds. The application of these polypeptides in the biosynthesis of this class of polyketide compounds is also provided.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biosynthesis, specifically to a class of genes for the biosynthesis of polyene polyketide compounds and the enzymes they encode. Background Technology

[0002] Formulas (I) and (II) are derived by Lin et al. from fungi in 2024. Talaromyces The polyene polyketide compound isolated from sp. JNU18266-01, with oxabicyclo[2.2.1]heptane and dihydropyran rings on both sides of its polyene chain, exhibits inhibitory activity against respiratory syncytial virus (RSV). IC50 50 The values ​​were 12 and 25 μM, respectively. These compounds have currently only been isolated from fungi, with only about 30 compounds identified. Therefore, expanding the structural diversity of these compounds and achieving their efficient preparation is of great significance for their further development and application. Biosynthetic methods are simple to operate, environmentally friendly, and have great application potential in synthesizing active compounds with complex structures. However, the biosynthesis of these polyketide compounds remains unclear. Summary of the Invention

[0003] The present invention provides a polypeptide or enzyme comprising the polypeptide sequence shown in any of the following: (a) polyketide synthase PruA, the amino acid sequence of which is shown in SEQ ID NO: 1; (b) flavoprotein monooxygenase PruB, the amino acid sequence of which is shown in SEQ ID NO: 2; (c) thioesterase PruC, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0004] Furthermore, the present invention provides a nucleic acid molecule capable of expressing the aforementioned enzyme. In one specific embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO: 4-6, wherein: the nucleic acid sequence shown in SEQ ID NO: 4 encodes polyketide synthase PruA; the nucleic acid sequence shown in SEQ ID NO: 5 encodes flavoprotein monooxygenase PruB; and the nucleic acid sequence shown in SEQ ID NO: 6 encodes thioesterase PruC.

[0005] This invention provides a vector comprising the aforementioned nucleic acid molecule. Preferably, the vector is an expression vector, a cloning vector, a plasmid, or a recombinant virus.

[0006] This invention provides a recombinant cell comprising the aforementioned nucleic acid molecules, expression cassettes, or vectors. In one specific embodiment, the cell is a fungal cell. In a preferred embodiment, the cell is... Talaromyces Fungi, for example Talaromycessp. JNU18266-01. In another preferred embodiment, the cells are Aspergillus oryzae cells.

[0007] Furthermore, the present invention provides a composition, which is an enzyme composition or an expression cassette composition. In one specific embodiment, the enzyme composition comprises three enzymes: PruA, PruB, and PruC. In another specific embodiment, the expression cassette composition comprises an expression cassette capable of expressing the three enzymes: PruA, PruB, and PruC. The expression cassette comprises a nucleic acid sequence encoding the enzyme and a transcriptional regulatory element, the transcriptional regulatory element including a promoter and a terminator; preferably, the expression cassette further comprises a selection marker gene and / or a tag sequence.

[0008] This invention provides the use of the aforementioned enzymes, enzyme compositions, expression cassette compositions, nucleic acid molecules, expression cassettes, vectors, or recombinant cells in the preparation of polyketide compounds or synthetic intermediates thereof, said polyketide compounds comprising an oxabicyclo[2.2.1]heptane structure. The six-membered lactone ring.

[0009] In one specific embodiment, the polyketide compound is preferably an oxabicyclo[2.2.1]heptane structure. It has a structure that connects with polyolefin chains to form bridged rings; or it is called "oxabornane-type polyene polyketide compound".

[0010] In one specific embodiment, the polyketide compound is shown in the following formula: R1 is selected from any one of the following: oxygen atom, carbon atom, nitrogen atom, and sulfur atom; R2 is selected from any one of the following: hydroxyl, acetoxy, hydroxymethyl, hydroxyethyl, and hydrogen atom; Alternatively, R2 can be any other group.

[0011] In one specific embodiment, the polyketide compound is or .

[0012] In one specific embodiment, the present invention provides a method for preparing polyketide compounds, using the polypeptide PruB and a polyene precursor compound containing an lactone ring structure. touch. In one specific embodiment, the present invention provides a method for preparing polyketide compounds, using PruA and PruC or an expression cassette containing the thereof to co-catalyze the formation of acetyl-CoA, malonyl-CoA, and S-adenosylmethionine. The compound shown; the compound shown in PruB catalytic formula (III) is generated. and The compound shown.

[0013] The present invention provides a kit comprising the aforementioned enzymes, enzyme compositions, expression cassette compositions, nucleic acid molecules, vectors, or recombinant cells.

[0014] In one specific implementation, the nucleotide sequences of PruA, PruB, and PruC are expressed in Aspergillus oryzae to obtain recombinant Aspergillus oryzae fermentation yields formulas (I) and (II).

[0015] Beneficial effects: This invention... Talaromyces A synthetic gene for oxabornene-type polyene polyketides, represented by formulas (I) and (II), was discovered in sp. JNU18266-01. pruA , pruB and pruC .Will pruA , pruB and pruC Expression in Aspergillus oryzae yields oxaborneol-type polyene polyketide compounds of formula (I) / (II), which have advantages such as simple process, high stereoselectivity specificity, and low environmental pollution. Attached Figure Description

[0016] Figure 1 The structural formula of the oxabornene-type polyene polyketide compound and the gene for the biosynthesis of the oxabornene-type polyene polyketide compound are shown. Figure 1 A shows the structures of oxabornene-type polyene polyketides of formulas (I) and (II); Figure 1 B shows the identification from the present invention. Talaromyces The biosynthesis gene of oxaborneol-type polyene polyketide compounds sp. JNU18266-01.

[0017] Figure 2 The analysis of metabolites from heterologous expression of the pru gene in Aspergillus oryzae is shown. Figure 2 A shows the HPLC detection chromatogram of the extract expressing the pru gene in Aspergillus oryzae strain; Figure 2 The structure was isolated from a B strain of Aspergillus oryzae expressing the pru gene. Detailed Implementation

[0018] Example 1: Obtaining candidate genes Talaromycessp. JNU18266-01 was isolated in our laboratory. It was first activated on potato agar (PDA) medium, and after two days of culture, a small amount of mycelium was inoculated into potato liquid (PDB) medium and cultured at 28 °C with shaking at 220 rpm for 3 days. The mycelium was collected by filtration, ground in liquid nitrogen, and total DNA was extracted using the phenol-chloroform method. Sequencing was performed using the Illumina HiSeq 2500 sequencing platform. Sequence analysis was performed using the SOAPdenovo software (version 2.04, http: / / soap.genomics.org.cn / soapdenovo.html). A total of 655 contigs were obtained, with a total length of approximately 34.5 Mb. Gene prediction was performed using the AUGUSTUS software (http: / / bioinf.uni-greifswald.de / augustus / ).

[0019] Genes associated with biosynthesis of formula (I) / (II) were obtained through gene function prediction using AUGUSTUS software. Figure 1 B), of which pruA (The polypeptide sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 4.) pruB (The polypeptide sequence is shown in SEQ ID NO: 2, and the nucleic acid sequence is shown in SEQ ID NO: 5.) pruC (The polypeptide sequence is shown in SEQ ID NO: 3, and the nucleic acid sequence is shown in SEQ ID NO: 6) is used as the target gene for research.

[0020] Example 2: Construction of Aspergillus oryzae expression strain (1) Construction of Aspergillus oryzae heterologous expression plasmids (pTAex3-pruA, pUSA-pruB and pAdeA-pruC) pruA , pruB and pruC strain Talaromyces Using sp. JNU18266-01 genomic DNA as a template, amplification was performed using the corresponding primer pairs Inf-pruA-F (SEQ ID NO: 7) / Inf-pruA-R (SEQ ID NO: 8), Inf-pruB-F (SEQ ID NO: 9) / Inf-pruB-R (SEQ ID NO: 10), and Inf-pruC-F (SEQ ID NO: 11) / Inf-pruC-R (SEQ ID NO: 12). The amplified DNA was then ligated into Aspergillus oryzae using the Infusion ligation method. A. oryzaeRecombinant plasmids (pTAex3-pruA, pUSA-pruB, and pTAex3-pruC) were formed by combining NSAR1 (provided by Professor Ikuro Abe of the University of Tokyo) expression plasmid pTAex3 and pUSA plasmid (provided by Professor Ikuro Abe of the University of Tokyo). Using recombinant plasmid pTAex3-pruC as a template, the pruC gene expression cassette containing the amylase amyB promoter and terminator was amplified using primers Inf-pAdeA-F (SEQ ID NO: 13) / Inf-pAdeA-R (SEQ ID NO: 14). The cassette was then ligated into pAdeA plasmid (provided by Professor Ikuro Abe of the University of Tokyo) using infusion ligation to construct expression plasmid pAdeA-pruC.

[0021] (2) Preparation and transfection of Aspergillus oryzae protoplasts.

[0022] 1) Add 100 μL of Aspergillus oryzae spore preservation solution to a 50 mL test tube containing 10 mL of DPY medium, and incubate at 30℃ and 220 rpm for 2 days with shaking.

[0023] 2) Add 10 mL of culture medium to a 500 mL Erlenmeyer flask containing 100 mL of DPY culture medium, mix thoroughly, and incubate at 30 ℃ and 220 rpm for 24 hours with shaking.

[0024] 3) Take about 15 mL of bacterial culture, filter it using a sterilized syringe filter, press the bacteria dry, remove the bacteria with a sterilized spatula, put them into a new 50 mL centrifuge tube, and add 10 mL of TF solution 1 (maleic acid 0.058 g, ammonium sulfate 0.79 g, yatalase 0.1 g, pH 5.5).

[0025] 4) Shake in a 30°C incubator for 3 hours until the supernatant is noticeably turbid and pale red. Use a syringe filter to filter the protoplasts into a 50 mL test tube. If blockage occurs, gently pry the surface of the cotton with a bamboo stick.

[0026] 5) Add an equal amount of TF solution 2 (10 mL), invert the test tube, mix gently, and centrifuge at 4 ℃ and 1500 rpm for 10 minutes.

[0027] 6) Remove the supernatant, add 5 mL of TF solution 2, centrifuge at 1500 rpm for 10 minutes at 4 ℃, remove the supernatant, and add an appropriate amount of TF solution 2 to make the protoplast concentration 1. 10 7 / mL, inverted and suspended.

[0028] 7) Using a 1000 μL pipette tip, take 200 μL of protoplast solution into a 15 mL centrifuge tube, add 10 μL of plasmid (pTAex3-pruA and / or pUSA-pruB and / or pAdeA-pruC) at a concentration of 1 μg / μL, and mix gently.

[0029] 8) Let stand on ice for 30 minutes. During this time, melt the Top agar in a microwave oven and keep it warm in a 50°C water bath.

[0030] 9) Add 250 μL, 250 μL and 850 μL of TF solution 3 to the suspension in 7) in three separate portions. After each addition, mix well by pipetting and let stand at room temperature for 20 minutes.

[0031] 10) Add 5 mL of TF solution 2, invert the test tube and mix gently.

[0032] 11) Centrifuge at 4 ℃, 1500 rpm for 10 minutes, remove the supernatant, add 200 μL of TF solution 2, gently resuspend using a 1 mL pipette, and add to the center of the culture dish. Quickly add 5 mL of Topagar (incubated at 50 ℃) around the center of the culture dish and mix rapidly.

[0033] 12) After the surface of the plate is fully dry, wrap it with sealing film and invert it for 4 days. 7 days.

[0034] 13) The obtained transformants were passaged for 1 hour on a selection medium without sorbitol. Repeat 3 times to stabilize the transformant.

[0035] (3) The Aspergillus oryzae transformant strain expressing the target gene was inoculated into 5 mL of DPY medium and cultured at 28 ℃ and 220 rpm for 2 days as seed culture. The seed culture was then inoculated into a 500 mL culture flask containing 100 mL of CD-Starch medium and cultured at 28 ℃ and 200 rpm for 3 days. After fermentation, the culture was filtered to separate the mycelia and bacterial culture. The bacterial cells were extracted twice with an equal volume of ethanol, and the ethanol extracts were combined, evaporated under reduced pressure, and the extract was dissolved in 2 mL of methanol for HPLC analysis. The results are as follows: Figure 2 As shown.

[0036] The HPLC conditions are as follows: Instruments: Dionex UltiMate 3000 equipped with UltiMate 3000 Diode Array Detector (DIONEX, USA) and Amazon SL ion trap electrospray mass spectrometer (BRUKER, Germany); Alltech (Grace) 2000ES evaporative light scattering detector (ELSD) (Alltech Co., Ltd., Portland, USA). Liquid chromatography column: COSMOSIL 3μ C18 column (4.6 × 150 mm) Mobile phase A: Water; Mobile phase B: Acetonitrile Gradient setting: 0 15 min 65% B, 15 30 min 70% B, 30 40 min 100% B, Flow rate: 0.8 mL / min Example 3: Isolation, purification, and structural identification of compounds The methods and results of compound analysis and purification involved in Example 2 above are summarized as follows: 3.1 Compound Isolation and Purification Isolation of compounds of formula (I) and formula (II): Following the plasmid construction method in "Example 2: Construction of Aspergillus oryzae expression strain", pTAex3-pruA, pUSA-pruB and pAdeA-pruC plasmids were constructed, and the three plasmids were co-introduced into a blank Aspergillus oryzae strain. A. oryzae NSAR1 was used to obtain a transgenic Aspergillus oryzae strain containing the pruABC gene (hereinafter referred to as pruABC). The gene introduction method is described in Part (2) of "Example 2: Construction of Aspergillus oryzae expression strain" above, "Preparation and transfection of Aspergillus oryzae protoplasts". Then, the transgenic Aspergillus oryzae strain (pruABC) was fermented in 10 L CD-starch medium for 3 days. After the cells were filtered through gauze, they were extracted three times by soaking in ethanol at room temperature. The ethanol layer was concentrated under reduced pressure and then extracted with ethyl acetate. Subsequently, silica gel column chromatography was performed, using a gradient elution of cyclohexane-ethyl acetate (10:1, 7:1, 5:1, 4:1 and 1:1) to obtain 5 fractions. The fraction containing compounds of formula (I) and formula (II) (5:1) was concentrated and further purified by semi-preparative high performance liquid chromatography (YMC-PackODS-A column, 3 mL / min) with 80% methanol-water to obtain compound (I) (9.3 mg) and compound (II) (4.6 mg).

[0037] Isolation of compound (III): Following the plasmid construction method in "Example 2: Construction of Aspergillus oryzae expression strain", pTAex3-pruA and pAdeA-pruC plasmids were constructed, and these two plasmids were jointly introduced into a blank Aspergillus oryzae strain. A. oryzae NSAR1 was used to obtain a transgenic Aspergillus oryzae strain containing the pruAC gene (hereinafter referred to as pruAC). The gene introduction method is described in Part (2) of "Example 2: Construction of Aspergillus oryzae expression strain" above, "Preparation and transfection of Aspergillus oryzae protoplasts". The transgenic Aspergillus oryzae strain (pruAC) was fermented in 20 liters of CD-starch medium for 2 days. After the bacterial cells were filtered through gauze, they were extracted twice by soaking in ethanol at room temperature. The extract was concentrated and then extracted again with ethyl acetate. The extract was separated by silica gel column chromatography using a gradient elution of cyclohexane:ethyl acetate (10:1, 8:1, 7:1 and 5:1) to obtain four fractions. The (8:1) fraction containing formula (III) was concentrated and further purified by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A column, 3 mL / min) with isocratic elution using 90% acetonitrile-water solution to finally obtain compound (III) (6.5 mg).

[0038] By constructing a recombinant Aspergillus oryzae containing only the pruA and pruC genes (pruAC), the key synthetic precursor shown in formula (III) can be prepared in a targeted and efficient manner; the acquisition of this intermediate provides direct evidence for clarifying the catalytic function of PruB and fully elucidating the biosynthetic pathway of formula (I) / (II).

[0039] 3.2 Structures and NMR confirmation data of the compounds involved in the examples NMR assignments in CD3OD for compounds of formula (I) a Assignment may be interchanged in each group. b Indiscernible signals from overlap or complex multiplicity are reported without designating multiplicity. NMR assignments in CD3OD for compounds of formula (II) a Assignment may be interchanged in each group. b Indiscernible signals from overlap or complex multiplicity are reported without designating multiplicity. NMR assignments in CD3OD for compounds of formula (III) a Assignment may be interchanged in each group. b Indiscernible signals from overlap or complex multiplicity are reported without designating multiplicity.

Claims

1. An isolated polypeptide or combination thereof, characterized in that, The polypeptide is selected from at least one of: a) the PruA polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, b) the PruB polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, or c) the PruC polypeptide containing the amino acid sequence shown in SEQ ID NO:

3.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.

3. The nucleic acid molecule according to claim 2, wherein the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO: 4-6, wherein the nucleic acid sequence shown in SEQ ID NO: 4 encodes the PruA polypeptide; the nucleic acid sequence shown in SEQ ID NO: 5 encodes the PruB polypeptide; and the nucleic acid sequence shown in SEQ ID NO: 6 encodes the PruC polypeptide.

4. A vector comprising the polynucleotide of claim 2 or 3.

5. A recombinant cell comprising the vector of claim 4; wherein the recombinant cell is a fungal cell or a bacterial cell; preferably, the fungal cell is selected from... Talaromyces It belongs to the fungal and Aspergillus oryzae cell family; the bacterial cell is Escherichia coli cell.

6. A composition, wherein the composition is an enzyme composition or an expression cassette composition, the enzyme composition comprising PruA, PruB and PruC; the expression cassette composition comprising an expression cassette capable of expressing nucleic acid molecules of PruA, PruB and PruC.

7. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 4, the recombinant cell of claim 5, and the composition of claim 6 in the preparation of polyketide compounds or synthetic intermediates thereof; preferably, the polyketide compound comprises an oxabicyclo[2.2.1]heptane structure. The six-membered lactone ring; more preferably, the polyketide compound is an oxabicyclo[2.2.1]heptane structure. A structure that connects with polyolefin chains to form bridged rings; More preferably, the polyketide compound is shown in the following formula. in, R1 is selected from any one of the following: oxygen atom, carbon atom, nitrogen atom, and sulfur atom; R2 is selected from any one of the following: hydroxyl, acetoxy, hydroxymethyl, hydroxyethyl, and hydrogen atom; Alternatively, R2 can be any other group; More preferably, the polyketide compound is or .

8. A method for preparing polyketide compounds, using a polypeptide PruB or an expression cassette containing the polypeptide PruB and a polyene precursor compound containing an lactone ring structure. touch.

9. A method for preparing polyketide compounds, comprising using PruA and PruC or an expression cassette containing the thereof to co-catalyze the formation of acetyl-CoA, malonyl-CoA, and S-adenosylmethionine. The compound shown; PruB or an expression cassette containing it catalyzes the formation of the compound shown in formula (III). and The compound shown.

10. A reagent kit, characterized in that, It comprises at least one of the following: the polypeptide of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 4, the recombinant cell of claim 5, and the composition of claim 6.

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