CN113832123B - Polyketide synthase Preu 3-delta CMeT and application thereof in preparation of orchromatic acid - Google Patents

Abstract

The invention relates to polyketide synthase Preu 3-delta CMeT and application thereof in preparation of orchromatic acid, belonging to the technical fields of molecular biology and biochemistry. The amino acid sequence of polyketide synthase Preu 3-CMeT is shown as SEQ ID NO.1, and the polyketide synthase Preu3 is constructed by taking polyketide synthase Preu3 from fungus of the genus Leucoblack as a material and knocking out the CMeT structural domain based on a combined biosynthesis technology; transferring the strain into saccharomyces cerevisiae to successfully obtain mutant strains capable of efficiently producing the orcein; the prepared orcein is taken as an object, the inhibition activity of the orcein on clinical drug-resistant bacteria and crop pathogenic fungi is researched, and the orcein has strong antagonism on carbapenem-resistant pseudomonas aeruginosa, and the MIC is 12.5μg/mL. The invention greatly enriches the production sources of the orcein, and has important scientific value and application prospect for expanding the derivatization path of the orcein and developing novel antibacterial agents.

Description

Polyketide synthase Preu 3-delta CMeT and application thereof in preparation of orchromatic acid

Technical Field

The invention belongs to the technical field of molecular biology and biochemistry, and particularly relates to a novel unnatural polyketide synthase Preu 3-delta CMeT and application thereof in preparation of orchromatic acid.

Background

Fungal polyketidespolyketidesPKs) is a large group of important natural products, has chemical structure and biological activity diversity, and is a high-quality resource library for discovery of novel drug lead compounds. Representative fungal PKs include lovastatin (lovastatin), the fungal inhibitor griseofulvin (griseofulvin), the bacterial inhibitor rifamycin (rifamycin), the immunosuppressant mycophenolic acid (mycophenolic acid), actin, and the angiogenesis inhibitor cytochalasin E (cytochalasin E), among others. Fungal polyketide synthases, key enzymes for PKs biosynthesispolyketide synthase, PKS) is an iterative type I PKS, the domains typically comprising: initiation unit ACP transacylasestarter unit ACP transsacylase, SAT), beta-ketoacyl synthase (beta-ketoacylsynthase, KS), acyltransferaseacyltTransfer enzyme, AT), product templateproduct tDisplacement, PT), acyl carrier proteinacyl carrier pProtein, ACP), ketoreductaseketorReducing enzyme, KR), dehydratasedehydracase, DH), alkenyl reductaseenoylrReducing enzyme, ER, methyltransferasemethyl tTransfer enzyme, CMeT) and thioesterase [ (], thioesterasethioesterase, TE) or reductaserReducing, R). Wherein CMeT is responsible for introducing methyl groups during extension of the polyketone carbon chain.

The oroselinic acid (structure shown in figure 1) is an aromatic polyketide small molecule compound which is formed by gradually condensing one molecule of acetyl coenzyme A and three molecules of malonyl coenzyme A under the catalysis of polyketide synthase. More than 200 derivatives of orcein, including monomers and polymers, have been identified in herbaceous plants, lichens, fungi and bacteria by isolation. Researches report that the orc and the derivatives thereof have the biological activities of anti-inflammatory, antibacterial, anti-tumor, antioxidant, antidiabetic and the like, so the orc can be used as potential biological resources of natural antibacterial agents, antioxidants, novel medicines and the like in the food and medicine industries. However, currently, the commercial orc (CAS: 480-64-8) is produced by chemical synthesis, and has a selling price of about 1000 yuan/100 mg, low yield and high selling price, so that the application of the acid in the fields of foods, medicines and the like is limited. Therefore, how to overcome the defects of the prior art for preparing the orcinol is a problem which needs to be solved in the technical field of the microbial chemistry at present.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides the construction of a novel unnatural polyketide synthase Preu 3-delta CMeT and application thereof in the preparation of the orchromatic acid. The invention takes polyketide synthase Preu3 (synthetic 3-methyl sequoyic acid) from fungus of the genus Leucoblack as a material, and based on the combined biosynthesis technology, the CMeT structural domain is knocked out to construct a novel non-natural polyketide synthase Preu 3-delta CMeT; transferring it into Saccharomyces cerevisiae to obtain mutant strain capable of producing orcein with high efficiency. The invention greatly enriches the production sources of the orcein, and has important scientific value and application prospect for expanding the derivatization path of the orcein and developing novel antibacterial agents.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the polyketide synthase Preu 3-delta CMeT is characterized in that the amino acid sequence of the polyketide synthase Preu 3-delta CMeT is shown as SEQ ID NO. 1.

A gene encoding the polyketide synthase Preu3- Δcmet of claim 1.

Further, it is preferable that the coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO. 2.

The invention also provides a recombinant vector containing the gene.

The invention also provides a recombinant genetically engineered bacterium obtained by transforming the recombinant vector.

The invention further provides the use of the polyketide synthase Preu3- ΔCMeT according to claim 1 for the preparation of orchromatic acids.

The invention also provides a construction method of a yeast mutant strain capable of efficiently producing the orcein, which comprises the following steps:

the recombinant vector of claim 4 is used for transforming saccharomyces cerevisiae by adopting a LiAc/PEG4000 mediated transformation method, the transformation solution is uniformly coated on an SC-Ura solid flat plate, and the saccharomyces cerevisiae mutant strain is obtained by culturing in a 30 ℃ incubator.

The invention further provides a preparation method of the orcein, and the yeast mutant strain constructed by the construction method is characterized by comprising the following steps:

step (1), inoculating a yeast mutant strain into an SC-Ura liquid culture medium, and performing shake culture at 30 ℃; culturing for 2d, adding 1% YPD culture medium, and culturing for 3-4d to obtain fermentation broth;

wherein, the volumes of the 1% YPD culture medium and the SC-Ura liquid culture medium are the same;

step (2), extracting the fermentation broth obtained by culturing in the step (1) with ethyl acetate with equal volume, and concentrating the extract to obtain crude extract;

and (3) purifying the crude extract obtained in the step (2) to obtain the orcein.

Further, in the step (3), the crude extract obtained in the step (2) is preferably purified by the following method: dissolving the crude extract with methanol, and passing through ODS C ₁₈ Performing reverse phase silica gel column chromatography, and performing gradient elution by using methanol-water as flowing relative crude extract to obtain five component segments Fr.A-Fr.E; concentrating the target metabolite, namely the orcein, in the Fr.E component, and preparing the Fr.E component into the orcein through a high-efficiency liquid phase;

wherein, methanol used in the gradient elution mobile phase: the volume ratio of water is 5:95, 10:90, 15:85, 20:80 and 25:75 in sequence, and the total volume of the mobile phase used by each gradient elution is one tenth of the volume of the SC-Ura liquid culture medium.

The invention finally provides application of the orcein prepared by the preparation method of the orcein in preparing medicines for resisting crop pathogenic fungi or medicines for clinically resisting bacteria, which is characterized in that the crop pathogenic fungi are alternaria mali, fusarium wilt of cotton, alternaria corn, verticillium solani of potato, sclerotium of rape, banded sclerotial blight of rice and black spot of strawberry; the clinical drug-resistant bacteria are methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli, carbapenem-resistant pseudomonas aeruginosa, carbapenem Acinetobacter baumannii, multi-drug-resistant enterococcus faecium, multi-drug-resistant enterococcus faecalis, carbapenem-resistant klebsiella pneumoniae and multi-drug-resistant staphylococcus epidermidis.

The orcein and its derivatives have anti-inflammatory, antibacterial, antitumor, antioxidant and antidiabetic activities, so that they can be used as potential biological resources for natural antibacterial agents, antioxidants and novel drugs in food and pharmaceutical industries. However, currently, the commercial orc (CAS: 480-64-8) is produced by chemical synthesis, and has a selling price of about 1000 yuan/100 mg, low yield and high selling price, so that the application of the acid in the fields of foods, medicines and the like is limited.

The inventors have previously cloned from a fungus of the genus Leuconostoc (Preussia isomera, genBank accession MK 300824.1) a polyketide synthase Preu3 (ZL 202110910678.3) related to the synthesis of polyketide molecule 3-methylorotic acid (3-methylorsellinic acid, structure see FIG. 1), whose domain composition is SAT-KS-AT-PT-ACP-CMeT-TE. In terms of chemical structure, 3-methyl sequoyiate is the product of methylation of sequoyiate at position 3C, i.e., the CMeT domain of polyketide synthase Preu3 has a methyl group introduced at position 3C during synthesis of 3-methyl sequoyiate. Therefore, the inventor intends to take polyketide synthase Preu3 as a target, and knock out the CMeT domain of the polyketide synthase based on a combined biosynthesis technology to construct a novel 'non-natural' polyketide synthase capable of producing the orcein with high yield.

In the invention, polyketide synthase Preu3 (synthetic 3-methyl sequoyic acid) from fungus of the genus Photonigella is used as a material for the first time, and a novel unnatural polyketide synthase Preu 3-delta CMeT is constructed based on the combination biosynthesis technology to knock out the CMeT structural domain; transferring the strain into saccharomyces cerevisiae to successfully obtain mutant strains capable of efficiently producing the orcein; and the prepared orcein is taken as a target, and the inhibition activity of the orcein on eight clinical drug-resistant bacteria and seven crop pathogenic fungi is studied. The research shows that the orcein has different degrees of inhibition effects on four crop pathogenic fungi of carbapenem-resistant pseudomonas aeruginosa, alternaria mali, alternaria corn, verticillium potato and sclerotium brassicae, wherein the carbapenem-resistant pseudomonas aeruginosa has strong antagonism (MIC, 12.5 mug/mL). The invention greatly enriches the production sources of the orcein, and has important scientific value and application prospect for expanding the derivatization path of the orcein and developing novel antibacterial agents.

Compared with the prior art, the invention has the beneficial effects that:

1. at present, the commercial orc (CAS: 480-64-8) is produced by chemical synthesis, and has the selling price of about 1000 yuan/100 mg, low yield and high selling price. The invention constructs a novel non-natural polyketide synthase Preu 3-delta CMeT based on a combined biosynthesis technology, successfully obtains a yeast mutant strain (the yield of the orotic acid is about 0.1 g/L) capable of efficiently producing the orotic acid based on a heterologous expression technology, provides a novel microbial fermentation way for producing the orotic acid, has simple process, high yield and environmental protection, and has considerable application prospect.

2. The invention also researches the inhibitory activity of the orcein on seven crop pathogenic fungi (apple ring rot germ, cotton fusarium wilt germ, corn small spot germ, potato verticillium, rape sclerotium germ, rice sheath blight germ and strawberry black spot germ) and eight clinical drug-resistant bacteria (methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli, carbapenem-resistant pseudomonas aeruginosa, carbapenem-resistant acinetobacter baumannii, multi-drug-resistant enterococcus faecium, multi-drug-resistant enterococcus faecalis, carbapenem-resistant klebsiella pneumoniae and multi-drug-resistant staphylococcus epidermidis) for the first time. The research shows that the orcein has strong antagonism (MIC, 12.5 mug/mL) to carbapenem-resistant pseudomonas aeruginosa, and is expected to develop novel antibiotics.

Drawings

FIG. 1 is a chemical structural formula of orcinolic acid (ORselinic acid) and 3-methyl orcinolic acid (3-methylorsellinic acid); wherein, (a) is a orcein; (b) is 3-methyl orcinolic acid;

FIG. 2 shows polyketide synthase Preu 3-. DELTA.CMeT and its heterologous expression vector YEpADH2p-URA-Preu 3-. DELTA.CMeT; wherein (a) is a domain composition of polyketide synthase Preu3- ΔCMeT; (b) Plasmid map for heterologous expression vector YEpADH2p-URA-Preu 3-delta CMeT;

FIG. 3 is a diagram showing the cleavage verification of polyketide synthase Preu 3-. DELTA.CMeT heterologous expression vector YEpADH2p-URA-Preu 3-. DELTA.CMeT; wherein M, trans 2K plus marker; line 1, vector YEpADH2p-URA-Preu3- ΔCMeT;

FIG. 4 is a metabolic analysis of a variant of polyketide synthase Preu 3-. DELTA.CMeT s.Saccharomyces cerevisiae; wherein (a) is an empty vector Saccharomyces cerevisiae; (b) is a polyketide synthase Preu3 Saccharomyces cerevisiae mutant; (c) is a polyketide synthase Preu3- ΔCMeT Saccharomyces cerevisiae mutant;

FIG. 5 is an ultraviolet absorbance graph of the target metabolite of polyketide synthase Preu 3-. DELTA.CMeT s.cerevisiae mutant;

FIG. 6 is a high resolution mass spectrum of a target metabolite of a mutant strain of Saccharomyces cerevisiae of polyketide synthase Preu3- ΔCMeT; wherein, (a) is a negative ion mode and (b) is a positive ion mode;

FIG. 7 is a compound of the acid form of the tongue with color ¹ H nuclear magneto-optical spectrum (deuterated methanol, 500 MHz);

FIG. 8 shows the compound orchromatic acid ¹³ C nuclear magneto-optical spectrum (deuterated methanol, 125 MHz).

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.

In the invention, polyketide synthase Preu3 (synthetic 3-methyl sedge acid, ZL 202110910678.3) from fungus of genus Leucoblack (Preussia isomera, genBank number MK 300824.1) is taken as a material, and a novel non-natural polyketide synthase Preu 3-delta CMeT is constructed based on the combined biosynthesis technology to knock out the CMeT domain; the strain is transferred into Saccharomyces cerevisiae, and a yeast mutant strain capable of efficiently producing orcinolic acid (structure shown in figure 1) is successfully obtained. The experiment comprises the following steps: firstly, knocking out a CMeT domain of Preu3 based on combined biosynthesis and homologous recombination technology, constructing polyketide synthase Preu 3-delta CMeT, and constructing a heterologous expression vector YEpADH2p-URA-Preu 3-delta CMeT (a plasmid map is shown in figure 2); then, a PEG4000/LiAc conversion method is adopted to convert the constructed heterologous expression vector into Saccharomyces cerevisiae to obtain a positive yeast transformant, metabolic products of the Saccharomyces cerevisiae are analyzed and structural identification is carried out, and the constructed yeast mutant strain is found to be capable of producing the orcein (about 0.1 g/L) at high yield; finally, the inhibitory activity of the prepared sequoyic acid against seven crop pathogenic fungi (Aphanothece, corn small spot, potato verticillium, sclerotinia sclerotiorum, rhizoctonia solani and strawberry black spot) and eight clinical drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Acinetobacter baumannii, multi-drug-resistant enterococcus faecium, multi-drug-resistant enterococcus faecalis, carbapenem-resistant Klebsiella pneumoniae and multi-drug-resistant Staphylococcus epidermidis) was studied (experimental results are shown in tables 3 and 4). The specific experimental procedure is as follows.

1. Construction of polyketide synthase Preu 3-delta CMeT and heterologous expression vector YEpADH2p-URA-Preu 3-delta CMeT thereof

1.1 construction of polyketide synthase gene preu 3-. DELTA.CMeT (domain composition see FIG. 2): knocking out the CMeT domain of polyketide synthase preu3 (V ¹⁷⁸⁹ -G ²¹¹⁴ Corresponding to base G ⁵³⁶⁵ -A ⁶³⁴² ) A novel polyketide synthase gene preu 3-delta CMeT is constructed, the total length of mRNA sequence of the polyketide synthase gene is 6594-bp, SEQ ID NO.2 is shown, and cloning primers are shown in Table 1.

Table 1: cloning-related primer information of polyketide synthase gene preu 3-delta CMeT

/>

PCR reaction system configuration (100 μl): ddH ₂ O55.5. Mu.L; 5X Phusion HF buffer. Mu.L; dNTPs (2.5 mmol/L) 8. Mu.L; 5. Mu.L of forward primer (10 pmol/. Mu.L); reverse primer (10 pmol/. Mu.L) 5. Mu.L; DMSO 3 μl; phusion DNA Ploymerase (2U/. Mu.L) 1. Mu.L; cDNA 2.5. Mu.L. The PCR procedure was: 98 ℃ for 3min;98 ℃ for 30s,58 ℃ for 30s,72 ℃ for 2min, and 34cycles total; and at 72℃for 10min.

Note that: fragment 1 constructed by the gene was purified by the primer Preu3- ΔCMeT-1F, preu3- ΔCMeT-1R; fragment 2 constructed by the gene was purified by the primer Preu3- ΔCMeT-2F, preu3- ΔCMeT-2R; fragment 3 constructed by the gene was purified by the primer Preu3- ΔCMeT-3F, preu3- ΔCMeT-3R; fragment 4 constructed from the gene was primed with primer Preu 3-. DELTA.CMeT-4F, preu 3-. DELTA.CMeT-4R.

Wherein polyketide synthase Preu3 is first divided into three fragments: fragment 1 (2.5-kb, A ¹ -C ²⁴⁹² ) Fragment 2 (2.6-kb, G ²⁴⁶⁶ -C ⁵⁰¹⁹ ) And fragment three (2.6-kb, A ⁴⁹⁹³ -G ⁷⁵⁷² ) Wherein CMeT domain (base G ⁵³⁶⁵ -A ⁶³⁴² ) Is positioned in the third segment; then the CMeT domain fragment within fragment three was knocked out and truncated into fragment 3 (0.4-kb, A ⁴⁹⁹³ -T ⁵³⁶⁴ ) And fragment 4 (1.3-kb, C ⁶³⁴³ -G ⁷⁵⁷² )。

The DNA fragment was recovered using Thermo Scientific GeneJET gel recovery kit (K0692), see the description for specific steps.

1.2 preparation of heterologous expression vector fragments: fragment 5 (6.2-kb) was obtained by restriction enzyme Nde I/Pde I digestion of plasmid YEpADH2 p-FLAG-URA. The plasmid YEpADH2p-FLAG-URA was given away for the professor Istvan Monar at the university of Arizona, academy of life sciences.

Enzyme digestion System configuration (180. Mu.L): ddH ₂ O132. Mu.L, 10X FastDigest Green Buffer. Mu.L, plasmid YEpADH2p-FLAG-URA 18. Mu.L, restriction enzyme (1) (Nde I) 6. Mu.L, restriction enzyme (2) (Pme I) 6. Mu.L. After enzyme digestion for 2h at 37 ℃, thermo Scientific GeneJET gel recovery kit (K0692) is adopted for gel recovery, and specific steps are described in the specification.

1.3 construction and transformation of heterologous expression vectors: a heterologous expression vector YEpADH2p-URA-Preu 3-delta CMeT is constructed by recombining Preu 3-delta CMeT gene fragments 1-4 prepared by 1.1 and vector fragment 5 prepared by 1.2 by adopting SE seamless cloning and assembly kit (Zhuang Meng organism, ZC 231).

Recombination reaction System configuration (10. Mu.L): 5X SE Cloning Buffer. Mu.L; fragment 12. Mu.L; fragment 2 2. Mu.L; fragment 3 1. Mu.L; fragment 4 1. Mu.L; fragment 5 1. Mu.L; SE Recombinase 1. Mu.L. The reaction was carried out at 37℃for 0.5h on ice for 2min, and then T1 Phage resistance competent cells (Zhuang Meng organism, ZC 102) were transformed, see the description for specific procedures.

1.4 extraction and validation of heterologous expression plasmids: the extraction of colibacillus plasmid

AxyPrep Plasmid Miniprep Kit (AP-MN-P-250), see the description for specific steps. Nde I single cleavage verification (7623/4773/460-bp) and sequencing are carried out on the constructed heterologous expression vector, and the correctness of the vector sequence is confirmed (the vector cleavage verification is shown in FIG. 3).

Configuration of the cleavage verification System (10. Mu.L): ddH ₂ O5.5. Mu.L, 10X FastDigest Green Buffer. Mu.L, heterologous expression plasmid 3. Mu.L, restriction enzyme (Nde I) 0.5. Mu.L. And (3) performing enzyme digestion reaction at 37 ℃ for 0.5h, and then running the gel.

2. Construction of polyketide synthase Preu 3-delta CMeT saccharomyces cerevisiae heterologous expression mutant strain and metabolite analysis and identification thereof

2.1 construction of polyketide synthase Preu 3-. DELTA.CMeT Saccharomyces cerevisiae heterologous expression mutant: the vector YEpADH2p-URA-Preu 3-delta CMeT was transformed into Saccharomyces cerevisiae using LiAc/PEG4000 mediated transformation.

100. Mu.L of the prepared yeast competent cells 3600r/min, centrifuging for 5min, discarding the supernatant, and then sequentially adding 78. Mu.L of ddH to the tube ₂ O, 36. Mu.L LiAc (1 mol/L), 240. Mu.L PEG4000 solution (50% (w/v), 3. Mu.L heterologous expression vector (YEpADH 2p-URA-Preu 3-. DELTA.CMeT), and the mixture was mixed uniformly; standing at 30deg.C for 30min, and transferring to 42deg.C for 30min; the transformed solution was centrifuged at 3600r/min for 5min, the supernatant was discarded, the cells were resuspended in 200. Mu.L of sterile water and then spread evenly on SC-Ura solid plates and incubated in an incubator at 30℃for 2-4d. After the yeast transformed strain grows out, it is fermented and analyzed for metabolites.

Configuration of SC-Ura solid plate: YNB 6.7g/L, glucose 20g/L,0.77g/L-ura Do Supplement (Clontech Co., U.S.A.), 15g of agar, and sterilized at 121℃for 15min.

2.2 Small fermentation of Yeast mutants: two clones were randomly picked on transformed plates, streaked on SC-Ura solid plates, and the plates were placed in an incubator at 30℃overnight for cultivation. An appropriate amount of bacterial cells (preferably 1mL OD inoculated) _600nm Bacterial liquid=0.5) was inoculated into 25mL SC-Ura broth (125 mL triangular flask), cultured at 30 ℃,220r/min for 1d; 2d, 25mL of 1% YPD medium was added, and the culture was continued for 3d, followed by secondary metabolite analysis.

Configuration of SC-Ura liquid culture medium: YNB 6.7g/L, glucose 20g/L,0.77g/L-ura Do Supplement (Clontech Co., USA), and sterilized at 121℃for 15min.

Configuration of 1% ypd medium: yeast extract 10g/L, peptone 20g/L, glucose 10g/L, and sterilizing at 121deg.C for 15min.

2.3 analysis of the metabolites of yeast mutants (chromatograms see FIG. 4): the Saccharomyces cerevisiae fermentation broth was extracted with an equal volume of ethyl acetate, and the ethyl acetate phase was taken into a round bottom flask and distilled. After the solvent was suspended, 800. Mu.L of methanol was added to dissolve the metabolite for LC-MS analysis. The chromatographic conditions were: reverse-phase C ₁₈ column (Kromasil 100-5-C18, 4.6X105 mm,5 μm); the mobile phase is water (A)/methanol (B) added with 0.1 percent of glacial acetic acid by volume percent; the flow rate is 0.8mL/min; sample injection volume 5. Mu.L; a DAD detector with a detection wavelength of 300nm; column temperature 25 ℃; gradient elution was used, the procedure being for a starting mobile phase proportion of 5% B to rise linearly from 5% to 100% in 30min, for 100% B10 min, then for 1min B to drop linearly from 100% to 5% and for 5% B4 min. The mass spectrum conditions were: a Q exact detector; an electrospray ion (ESI) source; positive and negative ion switching collection; primary mass spectrum full scan (150-1000 m/z); spray voltage 3.8kv; the temperature of the ion transmission tube is 325 ℃; sheath gas flow rate 40arb; an auxiliary air flow rate of 20arb; the heating temperature of the auxiliary gas is 350 ℃.

2.4 isolation and purification of target metabolites of yeast mutants: fermenting the yeast mutant strain capable of producing the target metabolite in a large scale for 2L,the fermentation and extraction methods are respectively 2.2 and 2.3, and 1.2g of crude extract is prepared. Dissolving the crude extract with small amount (about 1mL, the amount of the crude extract is enough to be dissolved) of methanol, and passing through ODS C ₁₈ Reversed phase silica gel column chromatography using methanol-water (MeOH-H) ₂ O) carrying out gradient elution (the gradient ratio of mobile phases (methanol: water volume ratio) is respectively 5:95, 10:90, 15:85, 20:80 and 25:75 in sequence, the mobile phases used by each gradient elution are 100 mL), and obtaining five component sections (Fr.A-E) altogether, wherein the target metabolites are concentrated in the Fr.E component, and the target metabolites are prepared by high performance liquid phase (chromatographic conditions are the same as 2.3, t _R =24.1 min) gives about 0.2g of the target monomer compound.

2.5 structural resolution of target metabolites of yeast mutants: the target metabolite is white crystal, the maximum ultraviolet absorption wavelength is 300nm (figure 5), and the structure is characterized by High Resolution Mass Spectrum (HRMS) and one-dimensional nuclear magnetic resonance spectrum ¹ H-NMR、 ¹³ C-NMR) identification, HRMS found that the molecular ion peak m/z was 167.03467[ M-H ]]169.04955[ M+H ]]++ (FIG. 6), molecular formula C ₈ H ₈ O ₄ The nuclear magnetic data required for the structural analysis are shown in fig. 7-8 and table 2, and the structure is finally analyzed as the orce (molecular structure is shown in fig. 1).

Table 2: the compound is of the acid type ¹ H and ¹³ c Nuclear magnetic data (500 MHz, CD3OD, δin ppm, J in Hz)

3. Test of the antibacterial Activity of Carpesium album acid

3.1 test of the activity of the orcein against pathogenic fungi of crops: the orotic acid and ketoconazole (positive control) were formulated as a 1mg/mL stock solution in DMSO.

Activating target crop pathogenic fungi (apple ring rot, cotton fusarium wilt, corn small spot, potato verticillium, sclerotium bacteria, rice sheath blight bacteria and strawberry black spot bacteria) in a PDB (potato dextrose broth) culture medium for 2-3d, and adding 1mL of bacterial liquid into 100mL of PDB culture medium after activation to obtain diluted bacterial liquid. 2 mu L of target compound is added into 198 mu L of target diluted bacterial liquid by taking ketoconazole as a positive control, and the antagonistic activity of the oroxylic acid on the seven crop pathogenic fungi is tested by adopting a double dilution method (the activity results are shown in Table 3).

Preparing a PDB culture medium: 23g of PDB powder (Beijing obucing Biotechnology Co., ltd.) was taken, 1000mL of distilled water was added, and the mixture was autoclaved at 121℃for 15 minutes.

3.2 test of the activity of the orcein against clinically resistant bacteria: the orotic acid and ciprofloxacin (positive control) were formulated in DMSO at a concentration of 1 mg/mL.

Target clinical drug-resistant bacteria (methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli, carbapenem-resistant pseudomonas aeruginosa, carbapenem acinetobacter baumannii, multi-drug-resistant enterococcus faecium, multi-drug-resistant enterococcus faecalis, carbapenem-resistant klebsiella pneumoniae and multi-drug-resistant staphylococcus epidermidis) are activated in LB (lysis broth) culture medium for 8 hours, and 50 mu L of bacterial liquid is taken after the activation and added into 50mL of LB culture medium to obtain diluted bacterial liquid. Taking ciprofloxacin as a positive control, adding 2 mu L of target compound into 198 mu L of target diluted bacterial liquid, and testing antagonistic activity of the oroxylic acid on the eight clinical drug-resistant bacteria by adopting a double dilution method (the activity results are shown in Table 4).

Preparing an LB culture medium: 25g of LB powder (Beijing cool Lei Corp.) was added with 1000mL of distilled water and autoclaved at 121℃for 20min.

Table 3: inhibitory Activity of Fuscoporia acid against seven crop pathogenic fungi (Ketoconazole as positive control)

Table 4: inhibitory Activity of Carpesium sedge acid against eight clinically resistant bacteria (ciprofloxacin as positive control)

4. Conclusion(s)

Experimental results show that the novel non-natural polyketide synthase Preu 3-delta CMeT is constructed based on a combined biosynthesis technology by taking polyketide synthase Preu3 (synthesized 3-methyl sequoyic acid) from fungus of the genus Leucoblack as a material for the first time, and is transferred into Saccharomyces cerevisiae to successfully obtain mutant strain (-0.1 g/L) capable of producing sequoyic acid with high efficiency; the prepared orcein has different degrees of inhibition effects on four crop pathogenic fungi of carbapenem-resistant pseudomonas aeruginosa, alternaria mali, alternaria corn, verticillium potato and sclerotium brassicae, wherein the carbapenem-resistant pseudomonas aeruginosa has strong antagonism (MIC, 12.5 mug/mL).

The orcein and its derivatives have anti-inflammatory, antibacterial, antitumor, antioxidant and antidiabetic activities, so that they can be used as potential biological resources for natural antibacterial agents, antioxidants and novel drugs in food and pharmaceutical industries. However, currently, the commercial orc (CAS: 480-64-8) is produced by chemical synthesis, and has a selling price of about 1000 yuan/100 mg, low yield and high selling price, so that the application of the acid in the fields of foods, medicines and the like is limited. In the invention, polyketide synthase Preu3 (synthesized 3-methyl sequoyic acid) from fungus of the genus Photonigella is used as a material for the first time, a novel unnatural polyketide synthase Preu 3-delta CMeT is constructed by knocking out a CMeT structural domain based on a combined biosynthesis technology, and is transferred into saccharomyces cerevisiae to successfully obtain a mutant strain capable of efficiently producing sequoyic acid; the prepared sequoyic acid is taken as an object, and the inhibition activity of sequoyic acid on seven crop pathogenic fungi (apple ring rot germ, cotton fusarium wilt, corn small spot germ, potato verticillium, rape sclerotium germ, rice sheath blight germ and strawberry black spot germ) and eight clinical drug resistant bacteria (methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli, carbapenem-resistant pseudomonas aeruginosa, carbapenem-resistant acinetobacter baumannii, multi-drug resistant enterococcus faecium, multi-drug resistant enterococcus faecalis, carbapenem-resistant klebsiella pneumoniae and multi-drug resistant staphylococcus epidermidis) is studied. The research shows that the orcein has different degrees of inhibition effects on four crop pathogenic fungi of carbapenem-resistant pseudomonas aeruginosa, alternaria mali, alternaria corn, verticillium potato and sclerotium brassicae, wherein the carbapenem-resistant pseudomonas aeruginosa has strong antagonism (MIC, 12.5 mug/mL). The invention greatly enriches the production sources of the orcein, and has important scientific value and application prospect for expanding the derivatization path of the orcein and developing novel antibacterial agents.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> university of national south China

<120> polyketide synthase Preu 3-fate CMeT and its use in the preparation of Cardioic acid

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2197

<212> PRT

<213> Artificial sequence ()

<400> 1

Met Asn Pro Pro Ser Ala Leu Ala Phe Gly Pro Glu Glu Arg Ile Pro

1 5 10 15

Thr Ala Ser Asn Leu Arg Leu Leu Lys Asp Val Leu Gln Asp Asp Pro

20 25 30

Thr Phe Ala Gly Ile Thr Ala Cys Leu Lys Gln Leu Pro Asp Thr Trp

35 40 45

Lys Ala Leu Leu His Gln Asp Ala Gln Leu Gln Ser Leu Ala Ser Glu

50 55 60

Arg Arg Ala Ala Val Leu Ser Asn Ser Leu Leu Asn Asp Glu Gln His

65 70 75 80

Glu Gly Asp Met Asp Thr Asn Gln Val Ile Met Pro Met Thr Val Leu

85 90 95

Val His Met Val Gln Tyr Arg Gln Phe Leu Gln Gln Ser Ser Pro Ser

100 105 110

Ser His Ala Thr Val Met Gln Ser Val Ala Ala Gly Gly Val Gln Gly

115 120 125

Phe Cys Ala Gly Leu Leu Ser Ala Phe Ala Val Cys Ser Met Thr Asn

130 135 140

Glu Asp Asp Phe Asp Ala Cys Ala Thr Tyr Ala Ile Lys Leu Ala Met

145 150 155 160

Cys Val Gly Ala Tyr Val Asp Leu Ala Met Glu Ser Glu Lys Gly Asp

165 170 175

Met Ala Ser Ala Ile Val Arg Trp Ser Ile Pro Asp Gly Arg Asn Arg

180 185 190

Val Asn Lys Ala Val Gly Arg Tyr Gln Ser Ala Tyr Ile Ser Ala Ile

195 200 205

Ser Asp Glu Asp Asn Val Thr Val Thr Ala Ser Arg Pro Asp Leu Asp

210 215 220

Ala Ile Cys Thr Ser Leu Gly Ser Thr Gly Met Ser Ser Lys Ile Leu

225 230 235 240

Ala Met Thr Gly Ser Phe His His Pro Lys Asn Phe Asp Leu Leu Gln

245 250 255

Arg Met Ile Ser Leu Leu Arg Ala Pro Gln Leu Ala Pro Ser Thr Lys

260 265 270

Phe Thr Asn Ala Leu Leu Arg Ser Asn Ser Thr Gly Glu Leu Leu Thr

275 280 285

Gly Ala Lys Thr Ile Glu Asn Ile Leu Glu Asp Ile Leu Cys Lys Thr

290 295 300

Ala Asp Trp Arg Leu Thr Met Ala Asn Thr Ser Lys Ala Leu Arg Ser

305 310 315 320

Gly Asn Gly Ser Arg Pro Asn Ile His Thr Phe Gly Leu Val Glu Phe

325 330 335

Ile Pro Ser Phe Val Lys Asn Glu Phe Asn Ile Leu Thr Gln Arg Leu

340 345 350

Ala Pro Thr Ala Lys Glu Gln Thr Gly Ala Ser Pro Ser Lys Ser Thr

355 360 365

Leu Gln Tyr Asn Asp Asn Ala Val Ala Val Val Gly Met Ala Cys Arg

370 375 380

Phe Pro Gly Ala Asp Asp Leu Asp Glu Phe Trp Glu Leu Leu Gln Ser

385 390 395 400

Gly Lys Ser Met His Glu Arg Met Pro Ala Asp Arg Phe Ser Thr Thr

405 410 415

Gly Leu Arg Arg Ser Asn Asp Gly Ala Pro Phe Trp Gly Asn Phe Leu

420 425 430

Lys Asp Ile Asp Ala Phe Asp His Gln Phe Phe Lys Lys Ser Ser Arg

435 440 445

Glu Ala Ala Ala Met Asp Pro Gln Gln Arg Leu Leu Leu Gln Cys Ala

450 455 460

Tyr Val Ala Met Glu Asn Ala Gly Tyr Phe Asp Pro Ser Val Gln His

465 470 475 480

Lys Ile Arg Asp Thr Gly Val Tyr Leu Gly Ala Cys Ser Ser Asp Tyr

485 490 495

Asn Asp Asn Val Ala Ser His Lys Pro Thr Ala Tyr Ser Thr Leu Gly

500 505 510

Thr Leu Arg Ala Phe Leu Thr Gly Arg Ile Ser His Tyr Phe Asp Trp

515 520 525

Thr Gly Pro Ser Val Val Tyr Asp Thr Ala Cys Ser Ser Ser Ala Val

530 535 540

Ala Ile Asp Ala Ala Cys Lys Ala Ile Leu Ala Gly Asp Cys Gln Gln

545 550 555 560

Ala Leu Ala Gly Gly Val Ser Leu Tyr Thr Ser Pro Asn Phe Tyr Gln

565 570 575

Asn Leu Asp Ala Ala Ser Phe Leu Ser Gln Thr Gly Pro Cys Lys Pro

580 585 590

Phe Asp Ala Asn Ala Asp Gly Tyr Cys Arg Gly Glu Gly Val Gly Leu

595 600 605

Val Val Leu Lys Lys Leu Ser Asp Ala Ile Arg Cys Gly Asp Lys Ile

610 615 620

Val Ala Val Ile Ala Ser Thr Gly Val Asn Gln Asn Arg Asn Cys Thr

625 630 635 640

Gly Ile Thr Val Pro His Gly Gly Ser Gln Ala Asp Leu Tyr Arg Arg

645 650 655

Val Val Ala Lys Ser Gly Leu Asn Ala Ser Gln Val Ser Tyr Val Glu

660 665 670

Ala His Gly Thr Gly Thr Pro Val Gly Asp Pro Ile Glu Phe Thr Ser

675 680 685

Ile Lys Ser Val Phe Ala Asn Pro Asp Ile Thr Arg Asp Glu Pro Leu

690 695 700

Thr Ile Ala Ser Val Lys Gly Ser Ile Gly His Leu Glu Gly Ala Ala

705 710 715 720

Gly Val Ala Ser Leu Ile Lys Val Cys Leu Met Leu Gln His Ser Ala

725 730 735

Ile Pro Pro Gln Ala Asn Phe Thr Lys Pro Asn Pro Asn Leu Gly Gly

740 745 750

Val Asp Met Arg Asn Ile Val Ile Pro Thr Ser Ser Ile Pro Trp Lys

755 760 765

Ala Arg Asn Lys Val Ala Cys Ile Asn Asn Tyr Gly Ala Ala Gly Ser

770 775 780

Asn Gly Ala Met Ile Val Cys Gln Pro Ser Glu Pro Ala Ser Lys Thr

785 790 795 800

Gln Thr Arg Leu Pro Ser Gln Ser Leu Ser Tyr Pro Leu Phe Ile Ser

805 810 815

Gly Asp Gly Thr Asp Ala Val Glu Ala Asn Cys Arg Ala Ile Ala Lys

820 825 830

Tyr Ala Arg Gln Leu Gln Gln Lys Arg Ala Pro Ser Val Val Ala Ser

835 840 845

Leu Ala Tyr Arg Leu Ala Thr Ser Gln Asn Gln Asn Leu Ser Tyr Ala

850 855 860

Met Val Thr Thr Ile Ser Glu Asn Gly Asp Ile Glu Ser Thr Leu Thr

865 870 875 880

Lys Ala Ser Ala Thr Leu Thr Gln Pro Arg Ser Lys Ala Lys Gln Ser

885 890 895

Val Val Leu Cys Phe Gly Gly Gln Val Lys Ala Phe Val Gly Leu Asp

900 905 910

Gln Gln Leu Phe Asp Ser Ser Ser Ile Leu Gln Lys His Leu Arg Leu

915 920 925

Cys Asp Ser Thr Met Arg Asp Leu Gly Tyr Pro Ser Ile Phe Pro Ala

930 935 940

Ile Phe Gln Ser Glu Pro Leu Lys Asp Pro Val Gln Leu His Gly Val

945 950 955 960

Leu Phe Ala Met Gln Tyr Ser Ser Ala Lys Ala Trp Leu Asp Cys Ser

965 970 975

Leu Gln Val Asp Ala Val Val Gly His Ser Phe Gly Gln Leu Thr Ala

980 985 990

Leu Thr Val Ala Gly Val Leu Ser Leu Lys Asp Gly Leu Arg Leu Val

995 1000 1005

Cys Gly Arg Ala His Leu Ile Lys Thr Lys Trp Gly Ser Ala Thr Gly

1010 1015 1020

Ala Met Ile Ala Ile Glu Ala Pro Leu Ile Arg Val Gln Glu Ile Leu

1025 1030 1035 1040

Ser Lys Ile Ser Val Ala Gly His Glu Ala Glu Ile Ala Cys Tyr Asn

1045 1050 1055

Ala Arg Glu Ser His Val Leu Val Gly Thr Thr Thr Ala Ile Asp Ala

1060 1065 1070

Val Arg Thr Phe Val Leu Glu Ser Gly Ile Lys His Lys Arg Leu Pro

1075 1080 1085

Val Thr His Gly Phe His Ser Thr Phe Thr Glu Ala Leu Leu Pro Gly

1090 1095 1100

Leu Arg Glu Leu Ala Lys Gly Leu Gln Phe Lys Ser Pro Ile Ile Pro

1105 1110 1115 1120

Ile Glu Thr Cys Thr Glu Tyr Lys Ser Trp Glu Gln Ala Thr Ala Asp

1125 1130 1135

Met Ile Thr Lys His Thr Arg Glu Pro Val Tyr Phe Val His Ala Ile

1140 1145 1150

Glu Arg Leu Ser Ala Arg Leu Gly Pro Cys Thr Trp Val Glu Ala Gly

1155 1160 1165

Thr Gly Ala Ser Thr Pro Ala Met Ile Lys Arg Cys Leu Pro Asp Ser

1170 1175 1180

Cys Ala Asp Ser Phe Ile His Ala Thr Leu Glu Ser Asn Lys Ala Phe

1185 1190 1195 1200

Gly Ser Leu Ala Asp Ala Thr Ala Asn Leu Trp Arg Cys Ser Gln Pro

1205 1210 1215

Val Gln Phe Trp Pro Phe His Ala Ser Asp Arg Gly Arg Tyr Val Pro

1220 1225 1230

Leu Asn Leu Pro Gly Tyr Gln Phe Arg Lys Thr Lys His Trp Leu Glu

1235 1240 1245

Trp Gln Asp Thr Val Ala Leu Pro Ala Phe Leu Glu Lys Glu Pro Ser

1250 1255 1260

Thr Ser Glu Pro Lys Gly His Glu Leu Leu Thr Phe Ser Ser Phe Glu

1265 1270 1275 1280

Asp Thr Ser Lys Ser Val Ala Ala Phe Lys Val Asp Pro Glu Ser Asp

1285 1290 1295

Glu Phe Met Met Leu Val Lys Gly His Ala Val Val Ala Gln Pro Leu

1300 1305 1310

Cys Pro Ala Pro Leu Tyr Cys Glu Leu Ala Leu Arg Ala Ile Lys His

1315 1320 1325

Leu Ser Pro Glu Thr Ala Ser Asn Ala Pro Asp Ile Arg Asp Leu Gln

1330 1335 1340

Ile His Ala Pro Leu Gly Leu Lys Thr Asn Arg Asn Ile Arg Leu Val

1345 1350 1355 1360

Val Gln Lys Asn Ser Ile Pro Gly His Trp Thr Phe Thr Val Lys Ser

1365 1370 1375

Ser Met Gly Ser Asp Asp Glu Leu Thr His Ala His Gly Leu Val Ala

1380 1385 1390

Phe Gly Gly Thr Val Glu Gln Glu Leu Ala Ser Tyr Gln Arg Leu Ile

1395 1400 1405

Gly His Gln Lys Ile Gln Ser Leu Met Thr Asp Pro Glu Cys Asp Ala

1410 1415 1420

Leu Arg Gly Ser Ala Thr Tyr Lys Ala Phe Asn Arg Val Val Thr Tyr

1425 1430 1435 1440

Ser Ser Tyr Tyr Lys Gly Val Gln Ala Ile Tyr Gly Arg Gln Asn Glu

1445 1450 1455

Ala Cys Gly Lys Ile Glu Leu Ser Ser Gly Glu Glu Gln Met Ala Gln

1460 1465 1470

Ala Arg Gly Ile Leu Thr Pro Leu Leu Ala Asp Asn Phe Ile Gln Ile

1475 1480 1485

Ala Gly Leu Gln Ile Asn Val Leu Gly Asp Cys Glu Asp His Leu Val

1490 1495 1500

Phe Val Cys Thr Glu Thr Gln Arg Ile Ile Tyr Gly Pro Gly Leu His

1505 1510 1515 1520

Gln Gln Pro Ala Ala Arg Tyr Glu Val Tyr Ser Thr Ile Ser Gln Asn

1525 1530 1535

Gly Pro Lys Glu Val Met Ser Asp Val Val Val Phe Asp Pro Ala Thr

1540 1545 1550

Lys Asn Val Glu Phe Val Ala Leu Gly Cys Arg Phe Thr Arg Val Thr

1555 1560 1565

Val Pro Gly Leu Arg Asn Ala Leu Gln Ala Ala Asn Gly Asp Ala Arg

1570 1575 1580

Ala Gln Glu Arg Pro Ser Gly Ser Arg Ile Ser Pro Ser Pro Leu Ala

1585 1590 1595 1600

Pro Glu Leu Pro Ala Lys Ile Gln Ile Gln Ser Arg Glu Asn Leu Asp

1605 1610 1615

Ile Thr Glu Lys Ser Gly Arg Gly Lys Pro Pro Arg Val Glu Asn Ile

1620 1625 1630

Gln Ile Ala Thr Pro Lys Val Asp Tyr Leu Ala Gln Val Lys Ala Leu

1635 1640 1645

Leu His Lys Val Ser Asp Val Pro Ile Asp Thr Ile Gln Lys Asp Ser

1650 1655 1660

Thr Leu Asp Asp Leu Gly Ile Asp Ser Leu Met Val Met Glu Val Gln

1665 1670 1675 1680

Thr Glu Val His Ser Glu Phe Gln Leu Thr Ile Pro Asn Lys Asp Trp

1685 1690 1695

Ala Thr Leu Glu Thr Pro Gly Lys Leu Ala Glu Tyr Leu Ala Lys Thr

1700 1705 1710

Leu Gly Gly Ser Val Pro Asp Ser Ala Pro Pro Gly Val Gln Arg Val

1715 1720 1725

Pro Ala Leu Val Ile Ser Asp Ala Glu Gln Ser Ser Asp Glu Ser Pro

1730 1735 1740

Tyr Asp Ser Thr Asp Asp Ser Ala Ser Gly Tyr Gly Asp Leu Asp Ile

1745 1750 1755 1760

Asp Thr Ala Ala Thr Thr Pro Gly Ile Phe Ala Thr Arg Asp Ser Ser

1765 1770 1775

Pro Phe Arg Lys Ala Ala Leu Asp Ser Pro Asn Pro Arg Pro His Val

1780 1785 1790

Leu Ala Asp Glu Asn Leu Trp Asp Gln Ser Leu Arg Ala Ala Gly Tyr

1795 1800 1805

Gly Asp Val Gln Trp Thr Glu Gly Gln Ser Glu Glu Ser Lys Thr Leu

1810 1815 1820

Arg Leu Ile Ala Ala Phe Asn Val Ser Asn Glu Asp Ala Lys Ala Ala

1825 1830 1835 1840

Asn Ala Leu Ala Ser Ala Leu Ala Val Pro Gly Arg Lys Gly Arg Thr

1845 1850 1855

Ser Ala Thr Thr Ile Arg Trp Lys Gln Glu Gly Asp Leu Asp Leu Met

1860 1865 1870

Ala Asp Val Tyr Leu Pro Ser Asp Leu Asp Ala Ser Thr Val Ser Arg

1875 1880 1885

Pro Val Ala Leu Ile Leu His Gly Gly Gly His Val Leu His Thr Arg

1890 1895 1900

Lys His Ile Asn Pro Arg His Ile Lys Met Leu Gln Asp Leu Gly Phe

1905 1910 1915 1920

Leu Pro Val Ser Val Asp Tyr Arg Leu Cys Pro Glu Val Asn Ile Arg

1925 1930 1935

Asp Gly Pro Met Thr Asp Ala Cys Glu Ala Val Asp Trp Ala Arg Asn

1940 1945 1950

Ile Leu Pro Cys Leu Pro Val Cys Ser Glu Leu Arg Val Asp Lys Glu

1955 1960 1965

His Val Val Val Ile Gly Tyr Ser Thr Gly Gly His Leu Ala Leu Thr

1970 1975 1980

Thr Ala Phe Thr Thr Arg Val Arg Gly Phe Lys Pro Pro Ser Ala Ile

1985 1990 1995 2000

Leu Gly Phe Tyr Cys Pro Thr Asn Tyr Ser Ala Asp Trp Trp Arg Ser

2005 2010 2015

Pro Ile Tyr Pro Glu Leu Ala Gln Gln Ser Ser Ser Glu Thr Phe Asp

2020 2025 2030

Leu Leu Glu Gly Val Asn Glu His Ala Ile Ala Gly Tyr Thr Pro Thr

2035 2040 2045

Val Asn Asn Asn Val Ala Ala Leu Leu Met Ser Leu Asp Asp Pro Arg

2050 2055 2060

Trp Arg Phe Val Leu His Ala Asn Trp Arg Ala Gln Thr Leu Pro Met

2065 2070 2075 2080

Leu Ile Asn Gly Leu Pro Ser Lys Ser Arg Leu Ala Arg Ser Gly Gln

2085 2090 2095

Thr Val Asp Ser Val Ile Asn Arg Glu Ile Pro Asp Ala Glu Asp Val

2100 2105 2110

Ala Ser Ile Ser Pro Tyr Asp Gln Ile Val Arg Gly Ser Tyr Ser Thr

2115 2120 2125

Pro Thr Phe Leu Leu His Gly Thr Lys Asp Asp Leu Ile Pro Trp Gln

2130 2135 2140

Gln Ser Ile Ala Thr Val Asp Ala Leu Ala Arg Arg Gly Val Asn Ala

2145 2150 2155 2160

Arg Val Glu Ile Ile Glu Gly Ala Glu His Cys Phe Asp Val Trp Ser

2165 2170 2175

Asp Lys Tyr Asp Gly Met Ile Gly Arg Ala Leu Glu Trp Leu Val Glu

2180 2185 2190

Gln Cys Arg Asn Ala

2195

<210> 2

<211> 6594

<212> DNA/RNA

<213> Artificial sequence ()

<400> 2

atgaaccctc ctagcgcttt ggcctttggt cccgaagagc gcatcccaac tgcttcaaac 60

ttgcgacttc taaaggatgt tttgcaagac gatccgacat tcgccggcat tactgcctgc 120

ctgaagcagt taccagatac ctggaaagcc ttgctccatc aagatgcaca attacaatcg 180

cttgcaagcg agaggcgtgc tgctgttctg tcgaactcgc ttctcaatga cgagcagcat 240

gaaggtgaca tggacaccaa ccaggtcatc atgcccatga cagtcctcgt ccacatggtg 300

caataccgtc agttcttgca gcagagctct ccttccagcc atgctacagt catgcagagc 360

gtcgcagccg gaggtgtgca aggcttctgt gcgggtcttc tttccgcctt tgctgtgtgt 420

tcgatgacca acgaggacga ctttgacgca tgtgctacat atgcgatcaa gctagccatg 480

tgcgtgggtg cctatgttga cctggccatg gagtcagaga agggggacat ggcatcagct 540

attgttcgct ggtcaatacc cgatggtcga aaccgtgtca acaaagccgt tggacgctat 600

cagagtgctt atatttctgc tatatctgac gaggacaacg ttacagtaac agccagtcga 660

cccgatctcg atgcgatttg cacctccctt ggaagcacag gcatgtccag caagattttg 720

gccatgactg ggagtttcca ccaccccaag aattttgacc tgctccaacg tatgatatcg 780

ctccttaggg ctccacagct cgccccgtcc acgaaattca ctaatgcgct gctccgctcc 840

aatagtactg gggaattact cactggtgcg aagacaattg agaatatcct agaagacatt 900

ctctgcaaga ctgctgattg gcggttgact atggccaaca cctcaaaagc gttgagatca 960

ggaaacggca gccgacccaa cattcataca ttcgggttgg tcgaattcat accttccttc 1020

gtaaaaaatg aattcaacat ccttacacag cggttggcgc cgaccgcgaa agagcagact 1080

ggagcttctc caagcaagtc tactctccag tacaacgata atgcagtcgc cgtggtcgga 1140

atggcctgcc gtttccccgg tgcagatgac ttggatgagt tttgggaact ccttcagtcc 1200

gggaagtcga tgcacgagcg tatgccagca gatcgtttca gcactacagg tctccgacgg 1260

tccaacgatg gtgctccgtt ttggggtaac ttcctcaaag atatcgacgc cttcgatcac 1320

caattcttca agaagtctag ccgcgaggca gctgcgatgg atccccaaca gcgtcttctc 1380

ctccagtgcg cttacgtggc catggaaaac gcgggatact ttgatccatc tgttcagcac 1440

aagatcagag acactggagt ttacctcgga gcatgctcta gcgactacaa tgacaatgtg 1500

gctagccata agcccacagc ttattccacg ctggggacgc tcagggcgtt tctgactggt 1560

cgtatcagtc actactttga ttggaccgga ccctccgttg tctacgacac agcttgttcg 1620

tcgtctgccg tggctatcga cgcagcttgc aaagccatcc ttgctggtga ttgtcaacag 1680

gctttggcag gtggagtctc tttgtacaca tcgcccaact tctatcagaa cttggacgct 1740

gcttcattcc tcagccagac cggtccctgc aagccctttg atgcgaacgc tgacggctac 1800

tgtcgaggcg agggcgtggg attggtggtc ctcaagaagc tgagcgatgc tattcggtgc 1860

ggcgataaga ttgtagccgt cattgctagc acgggggtca accaaaaccg caattgcacg 1920

ggcatcaccg ttcctcatgg tgggtcgcaa gctgatcttt accggagagt tgtagccaag 1980

agtggcttga atgcctcaca ggtttcctat gtggaagcgc atggaacggg cacccccgtc 2040

ggcgatccaa tcgagtttac gagcatcaaa tctgtcttcg caaatcctga cataacccga 2100

gacgagccgt tgactattgc atcggtgaag ggcagcatcg ggcatctcga aggcgcagca 2160

ggagttgcat cgctgatcaa ggtttgcctg atgctacagc actctgctat tcctccccag 2220

gcaaacttta cgaagcctaa ccccaacctt gggggggtcg atatgcgcaa catcgtcata 2280

ccaaccagca gcatcccgtg gaaggcgagg aacaaggttg cgtgcatcaa caactatggt 2340

gcagctggca gcaatggcgc catgattgta tgccagcctt ccgagccagc atcaaagaca 2400

caaacgagac tcccatcgca aagcctttcg tatcctctgt ttatatccgg agatggcact 2460

gatgcggtgg aagccaattg tcgggccatt gccaaatatg ctcgccaact ccagcaaaag 2520

cgagcaccgt ctgtggtggc cagtctcgcg tacaggttgg caacgtccca gaatcaaaat 2580

ctgtcgtatg caatggtgac tacaatctct gaaaatgggg atatcgaaag cacattgacc 2640

aaagcgtccg ctaccttgac gcagcctcgc tcgaaggcaa aacaatcagt tgtgctgtgt 2700

tttggcggcc aagtaaaagc ttttgtgggt ctggaccagc agctgtttga ttcctccagt 2760

attcttcaga aacatctgcg gctctgcgac tcgaccatgc gtgatctggg ctatcccagc 2820

atattcccag ccatcttcca gtcggagcct ctgaaggacc cagtacagct ccatggtgta 2880

ctattcgcga tgcagtattc atctgcgaaa gcttggctgg attgcagtct ccaggttgat 2940

gctgtcgttg gtcacagttt tggacagctt actgctctta ctgttgccgg cgtccttagt 3000

ctgaaggatg gcttgaggct tgtatgcggc cgggcacacc taattaagac gaagtggggc 3060

tcagccactg gggcaatgat tgcaatcgaa gcgcctctaa tccgtgtgca ggaaatcctc 3120

tcgaaaatct ccgtcgccgg acatgaagca gagatcgcgt gctacaatgc tcgagagagc 3180

catgtcctgg tcggtaccac tactgcgatt gatgctgtcc ggacattcgt acttgagagt 3240

ggtatcaagc ataagcgttt gccggtaacc catggatttc actctacctt cacggaagcc 3300

ctgttacctg gtctccgtga gctggcgaaa ggtctacagt tcaagtcccc aatcattccg 3360

atagagacat gcacagaata caagagctgg gagcaggcta cagctgacat gatcacgaaa 3420

cacacgaggg agcctgttta cttcgtccat gcgatagaaa gactctcggc tcggttgggg 3480

ccatgcacat gggttgaggc aggtaccggc gcatcgactc ctgcaatgat caagagatgc 3540

cttccggatt cttgtgcaga cagcttcata cacgcgaccc ttgaatccaa caaagctttc 3600

ggatcgctag ccgatgccac tgcgaatcta tggcgatgca gccagcccgt ccagttctgg 3660

cctttccatg caagcgacag agggagatat gtcccactta acttgccggg ctaccaattc 3720

cgtaagacga agcattggct cgagtggcaa gacaccgtcg ctttgcctgc ttttctagaa 3780

aaagaaccat cgacctctga gcctaaaggg catgagctgc taaccttctc ttcgttcgag 3840

gacacttcaa aatctgttgc agcattcaaa gtggaccctg aaagcgatga attcatgatg 3900

ctggtcaaag gtcatgctgt ggttgctcag ccgctttgcc cggcccccct gtactgcgag 3960

cttgccctac gagctatcaa gcatctctcg ccggaaactg cgtcaaatgc cccagatatc 4020

cgtgatctgc agatccatgc tccccttgga ctgaaaacta atcgaaacat ccgccttgta 4080

gtccagaaga acagtatccc aggtcactgg actttcaccg tgaagagctc aatgggatcc 4140

gacgacgaac ttactcacgc tcatgggctt gtagcattcg gtggaactgt ggaacaggag 4200

ttggcatcgt atcagcgact tatcggtcac cagaagatac agtctctgat gacagacccg 4260

gaatgtgatg ctttgagggg ctccgcgaca tacaaagcgt tcaaccgagt cgtcacatat 4320

tcttcctact acaaaggtgt ccaagcgata tatggtcgac agaacgaagc ttgtgggaag 4380

attgagttgt catcaggcga agaacaaatg gcccaggcac gcggaatact gactcccctc 4440

cttgccgata atttcataca gattgccggt ttgcagatca atgtgctggg agattgcgag 4500

gatcacttgg tcttcgtgtg tacggaaaca caacggatca tttatgggcc aggactccat 4560

caacaaccgg ctgctcgata tgaggtctat tcgacgattt ctcagaacgg ccccaaggaa 4620

gtcatgagtg acgttgtcgt tttcgatccc gccaccaaga atgttgagtt cgtcgcactg 4680

ggttgccgct tcacgcgagt gacagtacca ggccttcgca acgcacttca agccgccaat 4740

ggagacgctc gagctcagga gcggcctagc ggatctcgca tcagccccag tccgcttgca 4800

cctgaattgc ccgcgaagat tcaaattcag tcacgtgaga atctggacat cacggaaaag 4860

tctggacgtg gaaagcctcc tcgtgttgag aatattcaga tcgccacacc aaaggtcgac 4920

tacttggctc aggtgaaggc gcttctccac aaagtttccg atgtccccat cgataccata 4980

caaaaggatt cgacgcttga cgatctgggc attgattcgc ttatggtcat ggaggtgcaa 5040

actgaggtac actccgaatt ccaactgacc atacccaaca aagactgggc tacgttggag 5100

actcctggga agctcgccga atatcttgca aagacgctag ggggttctgt ccctgatagc 5160

gcacctccag gtgtacaacg tgtccctgcg ttagtaatct cggatgctga acagagttcc 5220

gacgaaagcc catatgacag cacagatgat tcagctagcg gttacggtga tctagacatc 5280

gatactgccg cgaccacgcc tggaatcttc gccactcgtg atagttcgcc attccggaaa 5340

gccgcgttgg actcccctaa ccctcgacca cacgttcttg ctgacgaaaa tctctgggac 5400

cagagcttac gagcagctgg ttatggagac gtgcagtgga ctgaagggca gtctgaggaa 5460

tccaagacgc tacgactcat tgctgcgttc aacgtaagca acgaggatgc gaaggcagcg 5520

aacgctctgg cgagcgcctt ggccgtaccc gggcgtaaag gcaggaccag tgcgacgacc 5580

atacgctgga agcaagaggg cgatctggat ctcatggcgg atgtatacct accgtctgat 5640

ctggatgcat caactgtaag cagacccgtc gctctgatcc ttcacggtgg cggccatgtc 5700

ttgcacacta gaaaacacat caacccgcga cacatcaaaa tgctacaaga tcttggcttc 5760

ttaccggttt cagtcgatta tcgtctttgt ccagaggtca acattcgcga tggacctatg 5820

acggacgcat gtgaggcggt ggactgggca aggaatattc taccatgtct gcctgtgtgc 5880

tccgagttgc gtgtcgacaa agagcacgta gtggtgattg gctactctac aggcggacac 5940

cttgctctga caactgcatt cacaacacgt gtcaggggat tcaagccccc ctctgccatt 6000

ctgggattct actgcccgac caactatagc gccgactggt ggcggtctcc catctatccg 6060

gagcttgctc agcagtctag ctcggagact ttcgacttgc tggaaggtgt caatgagcat 6120

gcgattgctg gatacacacc aacagtgaat aataatgtcg ctgcgcttct gatgtctctt 6180

gacgacccac gctggcgctt cgtcctacat gccaactggc gagctcagac tctcccgatg 6240

cttatcaacg ggctgccctc taagtctcgg cttgcgcgca gtggacaaac ggtggacagt 6300

gtcatcaaca gggagattcc agacgctgaa gatgttgcgt ctatcagccc gtatgatcaa 6360

atcgtcagag gaagctacag tacccctaca ttccttctcc atggaacgaa ggacgatctc 6420

attccttggc agcagagcat agcgacagtt gatgctctgg cacgacgtgg ggtgaatgcg 6480

agagtggaga ttattgaggg tgcggagcac tgttttgatg tttggtctga taagtatgat 6540

gggatgattg ggagggcact ggagtggttg gtggagcagt gtcggaatgc ttag 6594

<210> 3

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 3

gactacaaag acgatgacga caagcttcat atgaaccctc ctagcgcttt ggcctttgg 59

<210> 4

<211> 54

<212> DNA

<213> Artificial sequence ()

<400> 4

gcaatggccc gacaattggc ttccaccgca tcagtgccat ctccggatat aaac 54

<210> 5

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 5

ggtggaagcc aattgtcggg ccattgccaa at 32

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 6

cgaatcaatg cccagatcgt caagcgt 27

<210> 7

<211> 54

<212> DNA

<213> Artificial sequence ()

<400> 7

acgcttgacg atctgggcat tgattcgctt atggtcatgg aggtgcaaac tgag 54

<210> 8

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 8

agggttaggg gagtccaacg cggcttt 27

<210> 9

<211> 57

<212> DNA

<213> Artificial sequence ()

<400> 9

cggaaagccg cgttggactc ccctaaccct cgaccacacg ttcttgctga cgaaaat 57

<210> 10

<211> 58

<212> DNA

<213> Artificial sequence ()

<400> 10

agtgatggtg atggtgatgt ccgtttaaac ctaagcattc cgacactgct ccaccaac 58

Claims (9)

1. The polyketide synthase Preu 3-delta CMeT is characterized in that the amino acid sequence of the polyketide synthase Preu 3-delta CMeT is shown as SEQ ID NO. 1.

2. A gene encoding the polyketide synthase Preu3- Δcmet of claim 1.

3. The gene according to claim 2, characterized in that the sequence of the gene is the nucleotide sequence shown in SEQ ID No. 2.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 4.

6. Use of the polyketide synthase Preu3- Δcmet according to claim 1 for the preparation of orchromatic acid.

7. The construction method of the yeast mutant strain capable of efficiently producing the orcein is characterized by comprising the following steps of:

the recombinant vector of claim 4 is used for transforming saccharomyces cerevisiae by adopting a LiAc/PEG4000 mediated transformation method, the transformation solution is uniformly coated on an SC-Ura solid flat plate, and the saccharomyces cerevisiae mutant strain is obtained by culturing in a 30 ℃ incubator.

8. A method for preparing orcein, the yeast mutant strain constructed by the construction method of claim 7, comprising the steps of:

step (1), inoculating a yeast mutant strain into an SC-Ura liquid culture medium, and performing shake culture at 30 ℃; culturing for 2d, adding 1% YPD culture medium, and culturing for 3-4d to obtain fermentation broth;

wherein, the volumes of the 1% YPD culture medium and the SC-Ura liquid culture medium are the same;

step (2), extracting the fermentation broth obtained by culturing in the step (1) with ethyl acetate with equal volume, and concentrating the extract to obtain crude extract;

and (3) purifying the crude extract obtained in the step (2) to obtain the orcein.

9. The method for preparing the orc according to claim 8, wherein:

in the step (3), the crude extract obtained in the step (2) is purified by the following steps: dissolving the crude extract with methanol, and passing through ODS C ₁₈ Performing reverse phase silica gel column chromatography, and performing gradient elution by using methanol-water as flowing relative crude extract to obtain five component segments Fr.A-Fr.E; concentrating the target metabolite, namely the orcein, in the Fr.E component, and preparing the Fr.E component into the orcein through a high-efficiency liquid phase;

wherein, methanol used in the gradient elution mobile phase: the volume ratio of water is 5:95, 10:90, 15:85, 20:80 and 25:75 in sequence, and the total volume of the mobile phase used by each gradient elution is one tenth of the volume of the SC-Ura liquid culture medium.

Images