An engineered bacterium for producing tetrahydroanthracene compounds and a method for producing anthraquinone compounds

Abstract

The present invention relates to the fields of biotechnology and pharmaceuticals, and discloses an engineered bacterium capable of fermenting and producing tetrahydroanthracene compounds atrochrysone and torosachrysone. By introducing a non-reducing reusable fungal type I PKS (ACAS) and a β-lactamase type thioesterase (ACTE) gene into a host cell, a strain capable of fermenting and producing atrochrysone is obtained; at the same time, the genes of ACAS, ACAT and anthracycline C6-OH methyltransferase are introduced to obtain a strain capable of fermenting and producing torosachrysone. By directly subjecting the fermentation product to simple chemical treatment, important traditional Chinese medicine active ingredients emodin and physcion can be obtained. This method is simple, has a high yield, does not require extraction from medicinal plants or obtaining raw materials from medicinal plants, is suitable for industrial production, can reduce costs, reduce energy, and is conducive to protecting wild resources, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and biotechnology engineering. Specifically, the present invention relates to an engineered strain for producing tetrahydroanthracene compounds atrochrysone and torosachrysone, a method for constructing the same, and a method for preparing emodin and physcion using the fermentation product of this engineered strain. Background Art

[0002] The anthraquinone compound emodin (1,3,8-trihydroxy-6-methylanthraquinone), also known as coptisine, its structural formula is shown in Formula I, is an important active ingredient in various medicinal plants such as rhubarb, polygonum cuspidatum, aloe, and cassia obtusifolia. Research shows that emodin has various activities such as anti-tumor, anti-viral, anti-inflammatory, antibacterial, anti-osteoporosis, antioxidant, liver and kidney protection, lipid-lowering, and laxative effects. Clinically, it is widely used in the treatment of constipation, diabetic nephropathy, epistaxis, damp-heat in the liver and gallbladder, acute pancreatitis, periodontal disease and other diseases.

[0003]

[0004] The anthraquinone compound physcion (1,8-dihydroxy-3-methoxy-6-methylanthraquinone), also known as coptisine B, its structural formula is shown in Formula II, is a derivative of emodin methylated at the 3-position. It exists in traditional Chinese medicine plants such as rhubarb and polygonum multiflorum, and has activities such as antibacterial, anti-cancer, and laxative effects. In the early stage, physcion was mainly used in the pharmaceutical industry. At present, with the in-depth research, the application of physcion in the agricultural field has been gradually developed. Because of its low toxicity to humans and animals, environmental friendliness, low resistance generation, and its ability to effectively promote the growth of new shoots and buds of crops, physcion has been developed into a low-toxic and environmentally friendly biological pesticide for the prevention and control of powdery mildew, downy mildew, gray mold, and anthracnose in the production of green and organic vegetables.

[0005] At present, the main sources of the anthraquinone compounds emodin and physcion are extracted from the roots and rhizomes containing rhubarb. Since such compounds mainly exist in the underground roots and rhizomes of medicinal plants, the sources are restricted by natural conditions, and large-scale excavation is likely to cause the shortage of wild resources.

[0006] Tetrahydroanthracene compounds are mostly microbial secondary metabolites and have cytotoxic activity, antibacterial activity, and drug activity, such as atrochrysone (Formula III) and torosachrysone (Formula IV).

[0007]

[0008] Existing literature reports that atrochrysone can be obtained through engineering bacteria fermentation and can be further biosynthesized into other substances as an intermediate product of fermentation. There is currently no report on obtaining anthraquinone compounds emodin or physcion through engineering bacteria fermentation and further processing. Summary of the Invention

[0009] The object of the present invention is to provide an engineering strain capable of producing tetrahydroanthracene compounds atrochrysone or torosachrysone.

[0010] Another object of the present invention is to provide a method for producing anthraquinone compounds emodin or physcion.

[0011] An engineering bacterium for fermentative production of tetrahydroanthracene compounds, selected from the following strains:

[0012] (A) When the tetrahydroanthraquinone compound is atrochrysone, it contains a gene encoding a fungal non-reducing reusable type I polyketide synthase (ACAS) and a gene encoding a β-lactamase type thioesterase (ACTE).

[0013] (B) When the tetrahydroanthraquinone compound is torosachrysone, it further contains a gene encoding an anthracycline C6-OH methyltransferase (aurJ).

[0014] The type I polyketide synthase is a protein having more than 50% similarity and the same function as the amino acid sequence of SEQ ID No. 2; the β-lactamase type thioesterase is a protein having more than 50% similarity and the same function as the amino acid sequence of SEQ ID No. 4. The anthracycline C6-OH methyltransferase is a protein having more than 50% similarity and the same function as the amino acid sequence of SEQ ID No. 6.

[0015] Preferably, the type I polyketide synthase contains the amino acid sequence shown in SEQ ID No. 2, the β-lactamase type thioesterase contains the amino acid sequence shown in SEQ ID No. 4, and the anthracycline C6-OH methyltransferase contains the amino acid sequence shown in SEQ ID No. 6.

[0016] Preferably, the amino acid sequence of the type I polyketide synthase is as shown in SEQ ID No. 2, the amino acid sequence of the β-lactamase type thioesterase is as shown in SEQ ID No. 4, and the amino acid sequence of the anthracycline C6-OH methyltransferase is as shown in SEQ ID No. 6.

[0017] Furthermore, the gene encoding the non-reducing reusable type I polyketide synthase (ACAS) of the fungus is a nucleotide sequence encoding a protein with more than 50% similarity to SEQ ID No. 1 and having the same function; the gene encoding the β-lactamase type thioesterase (ACTE) is a nucleotide sequence encoding a protein with more than 50% similarity to SEQ ID No. 3 and having the same function; the gene encoding the anthracycline C6-OH methyltransferase is a nucleotide sequence encoding a protein with more than 50% similarity to SEQ ID No. 5 and having the same function.

[0018] Preferably, the gene encoding the type I polyketide synthase (ACAS) contains the nucleotide sequence shown in SEQ ID No. 1, the gene encoding the β-lactamase type thioesterase (ACTE) contains the nucleotide sequence shown in SEQ ID No. 3, and the gene encoding the anthracycline C6-OH methyltransferase contains the nucleotide sequence shown in SEQ ID No. 5.

[0019] More preferably, the nucleotide sequence of the gene encoding the non-reducing reusable type I polyketide synthase (ACAS) of the fungus is as shown in SEQ ID No. 1; the nucleotide sequence of the gene encoding the β-lactamase type thioesterase (ACTE) is as shown in SEQ ID No. 3; the nucleotide sequence of the gene encoding the anthracycline C6-OH methyltransferase is as shown in SEQ ID No. 5.

[0020] The host cell of the engineered bacterium is a fungus, selected from any one of Aspergillus oryzae, Saccharomyces cerevisiae, Pichia pastoris, Bacillus sp., Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Alternaria alternata or Fusarium sp. More preferably, the host cell is Aspergillus oryzae.

[0021] The construction method of the above-mentioned engineered bacterium is to place the genes involved between a strong promoter and a terminator to construct an expression plasmid; and introduce the constructed expression plasmid into the host cell. Preferably, the host cell is a fungus.

[0022] Preferably, the fungus is the filamentous fungus Aspergillus oryzae; the promoter and terminator are the amylase promoter / terminator P amyB / T amyB 。

[0023] A method for preparing tetrahydroanthraquinone compounds is to induce the expression of the above-mentioned engineered bacterium with starch.

[0024] The above-mentioned engineered bacterium has high fermentation efficiency. The yield of the engineered bacterium for fermenting and producing chrysophanol anthrone can reach 728 ± 94 mg / L; the yield of the engineered bacterium for fermenting and producing atrochrysone can reach 2.21 ± 0.21 g / L.

[0025] A method for preparing anthraquinone compounds, the steps including:

[0026] Treating the tetrahydroanthracene compounds with a base and reacting for 2 - 6 hours.

[0027] The base is NaOH or KOH, and the final concentration in the fermentation broth is 0.3 - 2 mol / L; preferably 0.8 - 1.5 mol / L.

[0028] It further includes: neutralizing and acidifying the mixture after the base treatment with an acid, the acid being hydrochloric acid, and the molar ratio of the acid to the base is 0 - 5:1, preferably 0.8 - 3:1, more preferably 0.9 - 1.5:1.

[0029] Preferably, an organic solvent is added to the product after the base treatment or neutralization and acidification for extraction. More preferably, the organic solvent is ethyl acetate.

[0030] The above method performs base dehydration and oxidation treatment on the fermentation broth or the fermentation broth extract, obtains emodin from the fermentation broth of the engineering bacteria producing atrochrysone, and obtains physcion from the fermentation broth of the engineering bacteria producing torachrysone.

[0031] Specifically, when preparing the anthraquinone compound emodin, the tetrahydroanthracene compound is atrochrysone; when the tetrahydroanthraquinone compound is physcion, the tetrahydroanthracene compound is torachrysone.

[0032] The tetrahydroanthracene compounds are prepared by the following method: inducing the expression of the above engineering bacteria producing tetrahydroanthracene compounds with starch, and taking the fermentation broth or the fermentation broth extract. That is, when preparing the anthraquinone compound emodin, fermenting with the engineering bacteria producing the tetrahydroanthracene compound atrochrysone, and performing acid or base dehydration and oxidation treatment on the fermentation broth or its extract to obtain emodin. When producing the anthraquinone compound physcion, fermenting with the engineering bacteria producing the tetrahydroanthracene compound torachrysone, and performing acid or base dehydration and oxidation treatment on the fermentation broth or its extract to obtain physcion.

[0033] After treating the fermentation products by the above method, the yields of emodin and physcion obtained can reach 15 mg / L and 336 mg / L respectively (calculated based on the volume of the fermentation broth).

[0034] The present invention constructs an engineered bacterium for fermentative production of atrochrysone and chrysoobtusin through bioengineering, and large-scale production of atrochrysone and chrysoobtusin can be achieved by induction with starch; the fermentation broth or crude extract of the fermentation broth is treated with alkali and acid, and emodin and physcion are obtained respectively after dehydration and oxidation. The preparation method is simple, the yield is high, and the important traditional Chinese medicine active ingredients emodin and physcion can be obtained through simple chemical treatment of the fermentation products, without extracting from medicinal plants or obtaining raw materials from medicinal plants, which is suitable for industrial production, can reduce costs, reduce energy, and is conducive to protecting wild resources, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings are used to illustrate the specific embodiments of the present invention, and are not used to limit the scope of the present invention.

[0036] Figure 1 Pathways for biosynthesis of atrochrysone and chrysoobtusin and their respective conversions to emodin and physcion.

[0037] Figure 2 Maps of plasmids pAdeA-ACAS / ACTE and pAdeA-ACAS / ACTE / aur J for heterologous expression.

[0038] Figure 3 HPLC detection map of the crude extract of the engineered strain for production of atrochrysone and chrysoobtusin (detection wavelength 390 nm).

[0039] Figure 4 HPLC detection map of the fermentation broth and crude extract of the atrochrysone engineered strain after treatment with acid or alkali (detection wavelength 390 nm).

[0040] Figure 5 HPLC detection map of the fermentation broth and crude extract of the chrysoobtusin engineered strain after treatment with acid or alkali (detection wavelength 390 nm).

[0041] Figure 6 NMR spectrum of atrochrysone produced by the engineered strain fermentation. (A) 1 1H NMR spectrum; (B) 13 13C NMR spectrum.

[0042] Figure 7 NMR spectrum of chrysoobtusin produced by the engineered strain fermentation. (A) 1 1H NMR spectrum; (B) 13 13C NMR spectrum. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments in terms of specific techniques or conditions, the techniques or conditions described in the literature in the art or according to the product specifications shall be followed. For those reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0044] It should be understood that within the scope of the present invention, all the technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0045] Example 1 Construction of expression vector

[0046] The gene sequence of SEQ ID No.1 was amplified by the primer pair shown in SEQ ID No.7 and SEQ ID No.8 in the sequence listing, encoding the protein ACAS (fungal non-reducing re-usable type I polyketide synthase) of SEQ ID No.2.

[0047] The gene sequence of SEQ ID No.3 was amplified by the primer pair shown in SEQ ID No.9 and SEQ ID No.10 in the sequence listing, encoding the protein ACTE (β-lactamase type thioesterase) of SEQ ID No.4.

[0048] The gene sequence of SEQ ID No.5 was amplified by the primer pair shown in SEQ ID No.11 and SEQ ID No.12 in the sequence listing, encoding the protein aurJ (anthracycline C6-OH methyltransferase) of SEQ ID No.6.

[0049] All genes were obtained by PCR amplification, and two main methods were used for plasmid construction: Gibson assembly method.

[0050] The specific construction method is as follows:

[0051] Based on the information of predicted coding regions, primers acas-for / acas-rev (SEQ ID No.7 and SEQ ID No.8), acte-for / acte-rev (SEQ ID No.9 and SEQ ID No.10) were used to amplify the ACAS gene (SEQ ID No.1) and the ACTE gene (SEQ ID No.3) from the genome of Talaromyces sp. F08Z-0631, encoding a fungal non-reducing re-usable type I polyketide synthase (the protein of SEQ ID No.2) and a β-lactamase type thioesterase (the protein of SEQ ID No.4), respectively; primers aurJ-for / aurJ-rev (SEQ ID No.11 and SEQ ID No.12) were used to amplify the anthracycline C6-OH methyltransferase AurJ gene (SEQ ID No.5) from the genome of Fusarium graminearum J1-012, encoding an anthracycline C6-OH methyltransferase (the protein of SEQ ID No.6). The amplified ACAS, ACTE and AurJ genes were inserted into the pTAex3 vector digested with EcoRI / KpnI by Gibson assembly method respectively.

[0052] 1) Construction of pAdeA-ACAS / ACTE plasmid: Primers pAdeA-PstI-for / Tamy-R1 (SEQ ID No.13 and SEQ ID No.14) and PAmy-F1 / pAdeA-SpeI-rev (SEQ ID No.15 and SEQ ID No.16) were used to amplify the amylase promoter / terminator P amyB / T amyB ACAS and ACTE gene fragments containing, and inserted into the pAdeA vector digested with PstI / SpeI by Gibson assembly method to obtain the heterologous expression plasmid shown in Figure 2 Figure A;

[0053] 2) Construction of pAdeA-ACAS / ACTE / aurJ plasmid: Primers pAdeA-PstI-for / Tamy-R1, Pamy-F1 / Tamy-R2 and Pamy-F2 / pAdeA-SpeI-rev (SEQ ID No.17 and SEQ ID No.18) were used to amplify the amylase promoter / terminator P amyB / T amyBThe ACAS, ACTE, and aurJ gene fragments were inserted into the PstI / SpeI double-digested pAdeA vector by the Gibson assembly method to obtain the heterologous expression plasmid shown in Figure 2 Figure B.

[0054] Example 2 Construction of Aspergillus oryzae Mutant Strains for Heterologous Expression of Atrochrysone and Cassia Anthrone

[0055] The plasmids pAdeA-ACAS / ACTE and pAdeA-ACAS / ACTE / aurJ were introduced into the Aspergillus oryzae host by the protoplast transformation method to construct Aspergillus oryzae strains heterologously expressing the tetrahydroanthracene compound atrochrysone (hereinafter referred to as strain A) and the strain expressing cassia anthrone (hereinafter referred to as strain B), respectively. After the transformants grew out, 5-6 transformants were picked to extract the genome, and the target gene was verified by PCR. The strains with positive PCR results were subcultured for expansion and used for subsequent preservation and fermentation experiments.

[0056] Example 3 Synthesis and Detection of Tetrahydroanthracene Compound Atrochrysone and Cassia Anthrone

[0057] After the constructed mutant strains were expanded in the screening plate, an appropriate amount of mycelia and spores were inoculated into 10 mL of DPY (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.5% KH2PO4) liquid medium and cultured at 28 °C with 150 rpm for 2 d. Then it was transferred to 100 ml of Czapek-Dox (CD) medium (0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% starch, pH 5.5) for starch-induced expression at 28 °C with 150 rpm for 6 d.

[0058] The fermentation product of strain A was directly added with an equal volume of ethyl acetate and extracted three times, and the upper organic layers of the three extractions were combined. After drying with a rotary evaporator, chromatographic-grade ethyl acetate was added to redissolve the sample. After high-speed centrifugation, the supernatant was taken and detected by LC-MS.

[0059] The fermentation product of strain B was directly added with an equal volume of ethyl acetate and extracted three times, and the upper organic layers of the three extractions were combined. After drying with a rotary evaporator, chromatographic-grade ethyl acetate and methanol were added to redissolve the sample. After high-speed centrifugation, the supernatant was taken and detected by LC-MS.

[0060] The results are shown in Figure 3As shown, a peak of atrochysone appears in the fermentation product of strain A, and a peak of torachrysone appears in the fermentation product of strain B.

[0061] Example 4 Enrichment, Purification and Identification of Tetrahydroanthracene Compounds Atrochrysone and Torachrysone

[0062] In order to identify the compound structures of the obtained tetrahydroanthracene compounds atrochrysone and torachrysone products, a large-scale fermentation (10 - 20 L) was carried out on the Aspergillus oryzae mutant strain. An equal volume of ethyl acetate was added to the fermentation broth and extracted 3 - 4 times. The extracted organic layers were combined and concentrated using a rotary evaporator to obtain a crude extract. The crude extract was subjected to normal-phase C18 column chromatography and gradient elution with a methanol - water system. The fractions containing the target compounds were collected; this fraction was purified by semi-preparative HPLC on a C18 column using pure acetonitrile and ultrapure water as the mobile phase to obtain pure compounds. The compounds were analyzed by HR-LCMS to determine the exact molecular weights, and NMR 1 H, 13 C spectra were collected to finally determine the molecular structures. The NMR spectrum results of atrochrysone produced by engineering strain A are as Figure 6 shown, and the NMR spectrum of torachrysone produced by engineering strain B is as Figure 7 shown.

[0063] The results showed that the fermentation product of strain A was atrochrysone with a shake-flask yield of 2.21 ± 0.21 g / L; the fermentation product of strain B was torachrysone with a shake-flask yield of 728 ± 94 mg / L.

[0064] Example 5 Preparation of Anthraquinone Compounds Emodin and Physcion

[0065] Tetrahydroanthracene compounds are prone to dehydration and oxidation reactions to form anthraquinone compounds under both acidic and alkaline conditions. In this invention, three different methods were used to treat the fermentation products of the engineering strains to achieve the transformation of the direct fermentation products into anthraquinone compounds emodin and physcion.

[0066] The process of engineering bacteria fermentation and post-treatment is as Figure 1 shown.

[0067] 1) Acid treatment of fermentation crude extract

[0068] The crude extract obtained by ethyl acetate extraction of 10 ml of fermentation broth was dissolved in 4 ml of distilled water, 1 mL of 12 mol / L concentrated hydrochloric acid was added, and the reaction was carried out at 80 °C for 1 hour. Then, an equal volume of ethyl acetate was added for extraction. After drying the organic phase with a nitrogen blower, it was dissolved in 2 ml of acetonitrile;

[0069] 2) Alkali treatment of fermentation crude extract

[0070] The crude extract obtained by extracting 10 ml of the fermentation broth with ethyl acetate was dissolved in 4 ml of 5% aqueous NaOH solution and reacted at room temperature for 3 hours. Subsequently, 1 mL of 12 mol / L concentrated hydrochloric acid was added for neutralization. An equal volume of ethyl acetate was added for extraction. After the organic phase was dried by a nitrogen blower, it was dissolved in 2 ml of acetonitrile;

[0071] 3) Directly perform alkali treatment on the fermentation broth

[0072] 500 mg of NaOH (final concentration 50 g / L) was added to 10 ml of the fermentation broth and reacted at room temperature for 3 hours. Subsequently, 1 mL of 12 mol / L concentrated hydrochloric acid was added for neutralization. An equal volume of ethyl acetate was added for extraction. After the organic phase was dried by a nitrogen blower, it was dissolved in 2 ml of acetonitrile.

[0073] The fermentation products of engineered strain A treated by three methods were detected by HPLC. Using emodin standard as the standard curve, the content of emodin in the products was quantified. The results are as Figure 4 shown. Detect the emodin therein.

[0074] The fermentation products of strain B were treated by the same method. Using physcion standard as the standard curve, the content of physcion in the products was quantified. The results are as Figure 5 shown.

[0075] After treating the fermentation broths of the two engineered strains by the third method, the contents of emodin or physcion were the highest. Among them, up to 15 mg / L of emodin and 336 mg / L of physcion could be obtained (based on the volume of the fermentation broth).

[0076] By using the first method for treatment, the yield of emodin was 5.8 mg / L and no physcion was produced; by using the second method for treatment, the yield of emodin was 12.4 mg / L and the yield of physcion was 88 mg / L. SEQUENCE LISTING <110> Shanghai University of Traditional Chinese Medicine <120> An engineered bacterium for producing tetrahydroanthracene compounds and a method for producing anthraquinone compounds <130> <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 5801 <212> DNA <213> Talaromyces sp. F08Z-0631 <400> 1 atgactggac aggccaagga tatcgagatg gagtactcgg agccttccaa catgaatatc 60 tcgtcatgct ccgcaacccc ttcggtcaca gagtcaagtt tggttgagcc tgcccaaatg 120 atgctctgct attttggcaa cgagttcccg caggataacc tcaaggacct tttccgtcgc 180 ctacacagcc acagcaaaga tagacaacat cctttgcttg ctcaattcat ccaggaggcc 240 acattggctg tgcgtaacga agtgcagctg ctgccagcca cattgaagtc ccttgttcct 300 ccatttgaga ctattttcga cttgacagat gatgtggagc tgcgtaatgg ccccctatct 360 gggtcagtag atggaatgct gctctgtgta gttcagattg caaccttcat tgggtatgta 420 cacttaatca ttgatatgtc cacgggtgaa ctctagagtt gatcttttct tactactgca 480 gatactacga ggataacact gagatcacat atgactttca gagtgcagag gcttgcctcg 540 ctggtttggg cacggggctt ttgtcaactg ctgctgtttc gatttcatca actttggcga 600 acctcccgct tgccggtgct gcggtgattg ctgtcgcctt cagacttggt gtcattgtcg 660 atgaagtttc ccagaacctt cagcctcgct caattgataa tggccctcga gacagttggg 720 cctatgtggt cccagatgtt gtggcggaag aggttcagca tgagctagac atagtccagg 780 ccgcagaagt aagttcccgg ctgccaggcg tataggttgg aaatatacta aggaccttta 840 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agctatagtg acagcttacg aagcactgga acgagctggc tatgtgccaa accgtaccgc 1740 ctctactaac cttcatcgca tcggtacctt ttatggccaa gcaagcgatg actatcgtga 1800 agtcaataca gctcaagaga tcggcaccta tttcatcact ggaggctgca gagccttcgg 1860 accagggcgc gttaattatt tcttcaagtt ctccggacct agctacagta ttgatacggc 1920 gtgttcttcg ggattggcgg cagtacatgt aagtgattat tcctttttac acgccgcaag 1980 attattctaa ttcaaaaaaa agcttgcctg caactccctc tggactggtg atacagacat 2040 ggcagtcaca ggaggactga acgtgttgac caactcagac gcattcgccg gtcttggaaa 2100 cggccacttt ctatccaaga caccgaacgc atgcaaaaca tgggactctg aagccgatgg 2160 gtactgtcgt gctgatggtg tagtctcact cgtgttgaaa cgacttgagg atgcagagct 2220 cgacaacgat aatatattag gcgtcatcct cgcagctggc accaatcact ccgccgaggc 2280 catctccatt acacaccctc acgcaggcca tcaggccgac cttacaagac agatactcaa 2340 tcaggccgcc gtggatccgc ttgatgtcag ctacgtggaa atgcatggga caggtacgca 2400 ggcaggtgac ctgcaggaga tccaatctgt catgaatgta ttcgctcctg ttacgaactc 2460 aaaacgtcgc agtcccaaac gcccattgca tatcggtgcc gtgaaagcaa acgtcggtca 2520 ttctgaggca gcagccggct caacagcgtt actcaaggtc ctcctcatgt tcgacaaaga 2580 ggctattccg cctcatgtag gcattaagaa tgctatcaac cccggctttc caaaggattt 2640 cgagaaaaga aatttgcata ttccttataa aaagacacca tggcagcggt ctcctgaaag 2700 aaagcgcatt gctgtcgtga acaatttcag tgcagctggg ggcaactcga gcattgtcat 2760 tgaagagcct ccgatcagac ataccaagga tcttgatata gtagatcctc gttccactca 2820 ggttgtcatt gtttcggcta agagcaaagt ctcattcaaa cgaaaccttg agaatttaat 2880 cgaatatttg gatgcacagc ctaacatatc actgcctaac ctctcataca caaccacagc 2940 tcgacgacat catcataatc accggatttg tgtagctaca tcggacgttg caaacttgcg 3000 gagtcgactg acttcatgtc tcacatcagc tgaaacccac aagccaattc cgtcgtctgg 3060 atcgccacct atcgtctttg ccttcactgg ccaaggtgcc tcacaccgat catccaacct 3120 caaactattt catgattcac cctatttccg aggccagctt ttgcacctag attcactatg 3180 ccaggcccaa ggatttccgt ctatcatccc agtaatcgat ggcagccacc cacatgatta 3240 cgcgcatcca cctactccga cgcagctcgc cttggtttgt acggagatgg cactggcaca 3300 gtactggaag tcactcggag ttcgaccaga cgccgtcatc ggccacagct tgggcgagta 3360 cgctgcgcta tacgttgcgg gagttctatc tgctgccgat gccatctttc tagtgggaca 3420 gcgtgctctc atgctggagc gcaagtgcac gaccaacagt cacaaaatgc tagcagtccg 3480 ggcctcaatc gaggagattc gcgccaaagc cggcgacaag tcgtttgagg ttgcatgtat 3540 aaatgggcct aaagacactg tgctcagtgg ctcggccgat gaaatcaagg agttagccga 3600 tgtgctgcag gcgagcagca gtgatgatag cgaggttggc ggtttcaagt gctttccatt 3660 agatgtggca ttcgcattcc attctgcgca gatggatccc attctgaatg aactggagga 3720 aactgcgaac actggagctc tttaccaccc acctagtctg cctgtcattt cgccgctact 3780 tggcaaggtt atttttgacg agaagactgt gaatggaaac tacatcaagc gggccaccag 3840 ggaaacagtc aacttcgtgg cagccgtcca gtcggctctc gacatgtcaa tcgtggacga 3900 taccacagta tggattgaga taggtcctca tcccgtatgc atgggtttcg tgcgttttat 3960 tttaccttct gtgaatatag caatgccttc tatgcgacga ggtgaagatg attggcggac 4020 gctgtcatcc gccctagctg cagtccattc tgcaggcgtt ccagtggatt ggaatgagtt 4080 ccatcgcccc ttcgagcgcg gtctggagct attggatctt cctacgtatt cgtggaatga 4140 caagactttc tggatccaat acaatggcga ctgggccctt acaaagggta acactttcta 4200 tgaagccgag aaagcagaga tggcagcaaa gaacaatagc gtttcagctg cacccatttc 4260 tagtctccgg acatcctcag tccaacgtgt cattgaagag acattctcgg gctctgcagg 4320 tcgcgttgtt gcgcagtctg atatcacgca gtcaaacttt ttagctgctg cgtggggaca 4380 caagatgaac ggagcagctg tggtgacatc ggtacgtaac atttttaata tttacctagc 4440 atttcaacta attccaaaga aaacaacctt gctaacacta tgtatcttga aactggcagt 4500 ctatccacgc agacattgcc tggacactcg gcgagtacct aattcaaaaa ctcaaaccac 4560 acgacaagaa ggacaactat gtcaccaaca tggaaatatc caacctcgtc gtccgtgagg 4620 gcctcgtagt gcagaagaac accaaagttc cccagctcat ccaagtctca attactacgg 4680 aagacatcga tgccggtgta gcttatctcg agtggcacaa cgtcgccaac gatggtatct 4740 ccctaatcga tccggagccc atagttacag cacgagtcgt ttatggaaag cccgatgcct 4800 ggatttccag ctgggcacct acgcgacatt ttgtccaagg ccgtatcgag acattatcac 4860 ggctcaatga ggagggcgtc gcgaacaagc tcagtcacag catgacgtac ctgctatttg 4920 caaacaacct agtcgactac gcagacaagt atcgtggcat gcaaagtgta gtcatgcacg 4980 ggcttgaagc cttcgcgaaa gttatcatca aaagcggtga caagggaggc atttggacag 5040 ttcctccatt tttcatcgat agcgtatgtc atctagcggg cttcgtaatg aatgtctcgg 5100 atgatctaga taccaagaaa aacttctctg tcacgcctgg ttggggctca ctgcgaattg 5160 ccaggccact ggtacctgga gtggaataca catcctacgt caaaatgatt cccacgccgg 5220 cagacccaac aatttaccta ggtgatgtgt atattctaca gggagatgat attattggtg 5280 ttatgcatgc tatgaagttt cgccaatacc cccgtctttt gctcaatcgc tttttctctg 5340 ccgttgacat caaaaacgtg cttcctaaca caactcccgc aactattcat gccctcaaaa 5400 ccaccatgcc agttgtggtg gcaccttcta cggctaataa agatgacaag ccatcaaagt 5460 ccaagtcagt gcccgaggta ttgccagcac ctgcgcacac aacgatggta gttacttctc 5520 ccattgagac aatacagaaa actaagacag ccatcgtggc accagacacc gacagtgtgg 5580 cagccaaagc cctgggactt gttgctgcgg aggcaggcgt cgaggtatct gatcttatcg 5640 acgaggccat cttctcagaa ttaggaatag acagtctaat gagtctagta attgctgaaa 5700 aactacgcga tcaactcaat gtgacagtca ctgggagtct gttcttggag tatccgacgg 5760 ttggggattt acgacagtgg ttggtggagt attacagcta g 5801 <210> 2 <211> 1842 <212> PRT <213> Talaromyces sp. F08Z-0631 <400> 2 Met Thr Gly Gln Ala Lys Asp Ile Glu Met Glu Tyr Ser Glu Pro Ser 1 5 10 15 Asn Met Asn Ile Ser Ser Cys Ser Ala Thr Pro Ser Val Thr Glu Ser 20 25 30 Ser Leu Val Glu Pro Ala Gln Met Met Leu Cys Tyr Phe Gly Asn Glu 35 40 45 Phe Pro Gln Asp Asn Leu Lys Asp Leu Phe Arg Arg Leu His Ser His 50 55 60 Ser Lys Asp Arg Gln His Pro Leu Leu Ala Gln Phe Ile Gln Glu Ala 65 70 75 80 Thr Leu Ala Val Arg Asn Glu Val Gln Leu Leu Pro Ala Thr Leu Lys 85 90 95 Ser Leu Val Pro Pro Phe Glu Thr Ile Phe Asp Leu Thr Asp Asp Val 100 105 110 Glu Leu Arg Asn Gly Pro Leu Ser Gly Ser Val Asp Gly Met Leu Leu 115 120 125 Cys Val Val Gln Ile Ala Thr Phe Ile Gly Tyr Leu Ile Phe Ser Tyr 130 135 140 Tyr Cys Arg Tyr Tyr Glu Asp Asn Thr Glu Ile Thr Tyr Asp Phe Gln 145 150 155 160 Ser Ala Glu Ala Cys Leu Ala Gly Leu Gly Thr Gly Leu Leu Ser Thr 165 170 175 Ala Ala Val Ser Ile Ser Ser Thr Leu Ala Asn Leu Pro Leu Ala Gly 180 185 190 Ala Ala Val Ile Ala Val Ala Phe Arg Leu Gly Val Ile Val Asp Glu 195 200 205 Val Ser Gln Asn Leu Gln Pro Arg Ser Ile Asp Asn Gly Pro Arg Asp 210 215 220 Ser Trp Ala Tyr Val Val Pro Asp Val Val Ala Glu Glu Val Gln His 225 230 235 240 Glu Leu Asp Ile Ile Phe Ile Ser Ala Leu Ser Gln Ser Ser Val Thr 245 250 255 Ile Ser Gly Pro Pro Ala Arg Leu Lys His Leu Phe His Val Ser Asp 260 265 270 Phe Phe Arg Asp His Lys Cys Val Ala Leu Pro Val Tyr Gly Gly Leu 275 280 285 Cys His Ala Lys His Ile Tyr Ser Lys Asn His Val Asp Glu Ile Ile 290 295 300 Gln Thr Ser Ser Leu Asp Ala Leu Glu Asp Ala Ser Phe Ala Pro Arg 305 310 315 320 Ile Pro Val Phe Ser Thr Ser Ser Gly Lys Pro Phe Gln Val Asn Gly 325 330 335 Pro Arg Ser Leu Phe Phe Lys Ile Val Glu Glu Ile Leu Thr Arg Ser 340 345 350 Ile Gln Trp Asp Arg Val Ile Asp His Val Ile Glu Arg Ala Asn Leu 355 360 365 Val Lys Pro Ser Glu Tyr Leu Val Leu Thr Leu Arg Thr Ser Leu Pro 370 375 380 Ile Arg Glu Leu Val Lys Gly Leu Asn Thr Glu Ala Gln Pro Leu His 385 390 395 400 Val Lys Thr Asn Asp Leu Ile Pro Trp Ile Thr Lys Pro Asp Thr His 405 410 415 Pro Gln Gly Pro Arg Thr Ser Lys Gln Ala Lys Ile Ala Val Val Gly 420 425 430 Met Ser Cys Arg Leu Pro Gly Gly Ala Thr Asp Thr Glu Lys Phe Trp 435 440 445 Glu Val Leu Asp Gln Gly Leu Asp Val His Arg Lys Val Pro Pro Asp 450 455 460 Arg Phe Asp Val Asp Ser His Cys Asp Pro Thr Gly Lys Arg Val Asn 465 470 475 480 Thr Ser Ile Thr Pro Tyr Gly Cys Phe Ile Glu Glu Pro Gly Leu Phe 485 490 495 Asp Ala Ala Phe Phe Asn Met Ser Pro Arg Glu Ala Leu Gln Thr Asp 500 505 510 Pro Met Gln Arg Leu Ala Ile Val Thr Ala Tyr Glu Ala Leu Glu Arg 515 520 525 Ala Gly Tyr Val Pro Asn Arg Thr Ala Ser Thr Asn Leu His Arg Ile 530 535 540 Gly Thr Phe Tyr Gly Gln Ala Ser Asp Asp Tyr Arg Glu Val Asn Thr 545 550 555 560 Ala Gln Glu Ile Gly Thr Tyr Phe Ile Thr Gly Gly Cys Arg Ala Phe 565 570 575 Gly Pro Gly Arg Val Asn Tyr Phe Phe Lys Phe Ser Gly Pro Ser Tyr 580 585 590 Ser Ile Asp Thr Ala Cys Ser Ser Gly Leu Ala Ala Val His Leu Ala 595 600 605 Cys Asn Ser Leu Trp Thr Gly Asp Thr Asp Met Ala Val Thr Gly Gly 610 615 620 Leu Asn Val Leu Thr Asn Ser Asp Ala Phe Ala Gly Leu Gly Asn Gly 625 630 635 640 His Phe Leu Ser Lys Thr Pro Asn Ala Cys Lys Thr Trp Asp Ser Glu 645 650 655 Ala Asp Gly Tyr Cys Arg Ala Asp Gly Val Val Ser Leu Val Leu Lys 660 665 670 Arg Leu Glu Asp Ala Glu Leu Asp Asn Asp Asn Ile Leu Gly Val Ile 675 680 685 Leu Ala Ala Gly Thr Asn His Ser Ala Glu Ala Ile Ser Ile Thr His 690 695 700 Pro His Ala Gly His Gln Ala Asp Leu Thr Arg Gln Ile Leu Asn Gln 705 710 715 720 Ala Ala Val Asp Pro Leu Asp Val Ser Tyr Val Glu Met His Gly Thr 725 730 735 Gly Thr Gln Ala Gly Asp Leu Gln Glu Ile Gln Ser Val Met Asn Val 740 745 750 Phe Ala Pro Val Thr Asn Ser Lys Arg Arg Ser Pro Lys Arg Pro Leu 755 760 765 His Ile Gly Ala Val Lys Ala Asn Val Gly His Ser Glu Ala Ala Ala 770 775 780 Gly Ser Thr Ala Leu Leu Lys Val Leu Leu Met Phe Asp Lys Glu Ala 785 790 795 800 Ile Pro Pro His Val Gly Ile Lys Asn Ala Ile Asn Pro Gly Phe Pro 805 810 815 Lys Asp Phe Glu Lys Arg Asn Leu His Ile Pro Tyr Lys Lys Thr Pro 820 825 830 Trp Gln Arg Ser Pro Glu Arg Lys Arg Ile Ala Val Val Asn Asn Phe 835 840 845 Ser Ala Ala Gly Gly Asn Ser Ser Ile Val Ile Glu Glu Pro Pro Ile 850 855 860 Arg His Thr Lys Asp Leu Asp Ile Val Asp Pro Arg Ser Thr Gln Val 865 870 875 880 Val Ile Val Ser Ala Lys Ser Lys Val Ser Phe Lys Arg Asn Leu Glu 885 890 895 Asn Leu Ile Glu Tyr Leu Asp Ala Gln Pro Asn Ile Ser Leu Pro Asn 900 905 910 Leu Ser Tyr Thr Thr Thr Ala Arg Arg His His His Asn His Arg Ile 915 920 925 Cys Val Ala Thr Ser Asp Val Ala Asn Leu Arg Ser Arg Leu Thr Ser 930 935 940 Cys Leu Thr Ser Ala Glu Thr His Lys Pro Ile Pro Ser Ser Gly Ser 945 950 955 960 Pro Pro Ile Val Phe Ala Phe Thr Gly Gln Gly Ala Ser His Arg Ser 965 970 975 Ser Asn Leu Lys Leu Phe His Asp Ser Pro Tyr Phe Arg Gly Gln Leu 980 985 990 Leu His Leu Asp Ser Leu Cys Gln Ala Gln Gly Phe Pro Ser Ile Ile 995 1000 1005 Pro Val Ile Asp Gly Ser His Pro His Asp Tyr Ala His Pro Pro 1010 1015 1020 Thr Pro Thr Gln Leu Ala Leu Val Cys Thr Glu Met Ala Leu Ala 1025 1030 1035 Gln Tyr Trp Lys Ser Leu Gly Val Arg Pro Asp Ala Val Ile Gly 1040 1045 1050 His Ser Leu Gly Glu Tyr Ala Ala Leu Tyr Val Ala Gly Val Leu 1055 1060 1065 Ser Ala Ala Asp Ala Ile Phe Leu Val Gly Gln Arg Ala Leu Met 1070 1075 1080 Leu Glu Arg Lys Cys Thr Thr Asn Ser His Lys Met Leu Ala Val 1085 1090 1095 Arg Ala Ser Ile Glu Glu Ile Arg Ala Lys Ala Gly Asp Lys Ser 1100 1105 1110 Phe Glu Val Ala Cys Ile Asn Gly Pro Lys Asp Thr Val Leu Ser 1115 1120 1125 Gly Ser Ala Asp Glu Ile Lys Glu Leu Ala Asp Val Leu Gln Ala 1130 1135 1140 Ser Ser Ser Asp Asp Ser Glu Val Gly Gly Phe Lys Cys Phe Pro 1145 1150 1155 Leu Asp Val Ala Phe Ala Phe His Ser Ala Gln Met Asp Pro Ile 1160 1165 1170 Leu Asn Glu Leu Glu Glu Thr Ala Asn Thr Gly Ala Leu Tyr His 1175 1180 1185 Pro Pro Ser Leu Pro Val Ile Ser Pro Leu Leu Gly Lys Val Ile 1190 1195 1200 Phe Asp Glu Lys Thr Val Asn Gly Asn Tyr Ile Lys Arg Ala Thr 1205 1210 1215 Arg Glu Thr Val Asn Phe Val Ala Ala Val Gln Ser Ala Leu Asp 1220 1225 1230 Met Ser Ile Val Asp Asp Thr Thr Val Trp Ile Glu Ile Gly Pro 1235 1240 1245 His Pro Val Cys Met Gly Phe Val Arg Phe Ile Leu Pro Ser Val 1250 1255 1260 Asn Ile Ala Met Pro Ser Met Arg Arg Gly Glu Asp Asp Trp Arg 1265 1270 1275 Thr Leu Ser Ser Ala Leu Ala Ala Val His Ser Ala Gly Val Pro 1280 1285 1290 Val Asp Trp Asn Glu Phe His Arg Pro Phe Glu Arg Gly Leu Glu 1295 1300 1305 Leu Leu Asp Leu Pro Thr Tyr Ser Trp Asn Asp Lys Thr Phe Trp 1310 1315 1320 Ile Gln Tyr Asn Gly Asp Trp Ala Leu Thr Lys Gly Asn Thr Phe 1325 1330 1335 Tyr Glu Ala Glu Lys Ala Glu Met Ala Ala Lys Asn Asn Ser Val 1340 1345 1350 Ser Ala Ala Pro Ile Ser Ser Leu Arg Thr Ser Ser Val Gln Arg 1355 1360 1365 Val Ile Glu Glu Thr Phe Ser Gly Ser Ala Gly Arg Val Val Ala 1370 1375 1380 Gln Ser Asp Ile Thr Gln Ser Asn Phe Leu Ala Ala Ala Trp Gly 1385 1390 1395 His Lys Met Asn Gly Ala Ala Val Val Thr Ser Ser Ile His Ala 1400 1405 1410 Asp Ile Ala Trp Thr Leu Gly Glu Tyr Leu Ile Gln Lys Leu Lys 1415 1420 1425 Pro His Asp Lys Lys Asp Asn Tyr Val Thr Asn Met Glu Ile Ser 1430 1435 1440 Asn Leu Val Val Arg Glu Gly Leu Val Val Gln Lys Asn Thr Lys 1445 1450 1455 Val Pro Gln Leu Ile Gln Val Ser Ile Thr Thr Glu Asp Ile Asp 1460 1465 1470 Ala Gly Val Ala Tyr Leu Glu Trp His Asn Val Ala Asn Asp Gly 1475 1480 1485 Ile Ser Leu Ile Asp Pro Glu Pro Ile Val Thr Ala Arg Val Val 1490 1495 1500 Tyr Gly Lys Pro Asp Ala Trp Ile Ser Ser Trp Ala Pro Thr Arg 1505 1510 1515 His Phe Val Gln Gly Arg Ile Glu Thr Leu Ser Arg Leu Asn Glu 1520 1525 1530 Glu Gly Val Ala Asn Lys Leu Ser His Ser Met Thr Tyr Leu Leu 1535 1540 1545 Phe Ala Asn Asn Leu Val Asp Tyr Ala Asp Lys Tyr Arg Gly Met 1550 1555 1560 Gln Ser Val Val Met His Gly Leu Glu Ala Phe Ala Lys Val Ile 1565 1570 1575 Ile Lys Ser Gly Asp Lys Gly Gly Ile Trp Thr Val Pro Pro Phe 1580 1585 1590 Phe Ile Asp Ser Val Cys His Leu Ala Gly Phe Val Met Asn Val 1595 1600 1605 Ser Asp Asp Leu Asp Thr Lys Lys Asn Phe Ser Val Thr Pro Gly 1610 1615 1620 Trp Gly Ser Leu Arg Ile Ala Arg Pro Leu Val Pro Gly Val Glu 1625 1630 1635 Tyr Thr Ser Tyr Val Lys Met Ile Pro Thr Pro Ala Asp Pro Thr 1640 1645 1650 Ile Tyr Leu Gly Asp Val Tyr Ile Leu Gln Gly Asp Asp Ile Ile 1655 1660 1665 Gly Val Met His Ala Met Lys Phe Arg Gln Tyr Pro Arg Leu Leu 1670 1675 1680 Leu Asn Arg Phe Phe Ser Ala Val Asp Ile Lys Asn Val Leu Pro 1685 1690 1695 Asn Thr Thr Pro Ala Thr Ile His Ala Leu Lys Thr Thr Met Pro 1700 1705 1710 Val Val Val Ala Pro Ser Thr Ala Asn Lys Asp Asp Lys Pro Ser 1715 1720 1725 Lys Ser Lys Ser Val Pro Glu Val Leu Pro Ala Pro Ala His Thr 1730 1735 1740 Thr Met Val Val Thr Ser Pro Ile Glu Thr Ile Gln Lys Thr Lys 1745 1750 1755 Thr Ala Ile Val Ala Pro Asp Thr Asp Ser Val Ala Ala Lys Ala 1760 1765 1770 Leu Gly Leu Val Ala Ala Glu Ala Gly Val Glu Val Ser Asp Leu 1775 1780 1785 Ile Asp Glu Ala Ile Phe Ser Glu Leu Gly Ile Asp Ser Leu Met 1790 1795 1800 Ser Leu Val Ile Ala Glu Lys Leu Arg Asp Gln Leu Asn Val Thr 1805 1810 1815 Val Thr Gly Ser Leu Phe Leu Glu Tyr Pro Thr Val Gly Asp Leu 1820 1825 1830 Arg Gln Trp Leu Val Glu Tyr Tyr Ser 1835 1840 <210> 3 <211> 942 <212> DNA <213> Talaromyces sp. F08Z-0631 <400> 3 atggcaaccc aagaaggggg ctatcgccag atcaacaagg ctttgaacat ttgtgctttt 60 gaagattatc tggaaagcca acaagtacgc cttcccaaga tactagacgt ggagcaaatc 120 agtccgcgag tcatccgggt tcttggacag aatccaggaa agttcacact acaaggaacc 180 aacacgtata ttatcggtac aggagcaaac cgtctcatta ttgacaccgg tcaaggtgtt 240 ccatattggg ctgatctgat ctcggagacc ctgtcaaaag aaggattctc attgtcgcat 300 gtcctcctga cgcattggca cggcgaccac acaggcggcg tccccgatct catccgcatg 360 tatccaggcc tatctagctc gatcttcaaa aacacgccaa gcaagaccca gcagcccatc 420 gtcgatggcc agaaatttga gatcgaaggg gccaccattc gcgcggttca cgctcccggc 480 cactcacacg accacatgtg tttcattctc gaggaagaac aggccatgtt caccggggac 540 aatgtgctgg gacacggtac agccgcagta gagcatttga gcacttggat gaagacttta 600 cgcgaaatgc aatcccataa ctgcacgaca ggctatccag cgcacggggt tgtaatcgca 660 gacctcaatg ctaagatcag cggtgagctc tccatgaagg cgcggaggga gaaacaggtc 720 atgcaggtgt tacaagatca gaagagaggt acaaatggca gagccaggct cactatcaaa 780 gagctcgtgg ccactttgta tggggaacgg atagacaatt ctgtcaagga actggcgcta 840 gaaccattta tagatgaggt ccttcgaaag ctagctgagg atggaatagt aggattcgac 900 atgagaggag gtatcaagcg ctggttcgcc cttgtaattt ga 942 <210> 4 <211> 313 <212> PRT <213> Talaromyces sp. F08Z-0631 <400> 4 Met Ala Thr Gln Glu Gly Gly Tyr Arg Gln Ile Asn Lys Ala Leu Asn 1 5 10 15 Ile Cys Ala Phe Glu Asp Tyr Leu Glu Ser Gln Gln Val Arg Leu Pro 20 25 30 Lys Ile Leu Asp Val Glu Gln Ile Ser Pro Arg Val Ile Arg Val Leu 35 40 45 Gly Gln Asn Pro Gly Lys Phe Thr Leu Gln Gly Thr Asn Thr Tyr Ile 50 55 60 Ile Gly Thr Gly Ala Asn Arg Leu Ile Ile Asp Thr Gly Gln Gly Val 65 70 75 80 Pro Tyr Trp Ala Asp Leu Ile Ser Glu Thr Leu Ser Lys Glu Gly Phe 85 90 95 Ser Leu Ser His Val Leu Leu Thr His Trp His Gly Asp His Thr Gly 100 105 110 Gly Val Pro Asp Leu Ile Arg Met Tyr Pro Gly Leu Ser Ser Ser Ile 115 120 125 Phe Lys Asn Thr Pro Ser Lys Thr Gln Gln Pro Ile Val Asp Gly Gln 130 135 140 Lys Phe Glu Ile Glu Gly Ala Thr Ile Arg Ala Val His Ala Pro Gly 145 150 155 160 His Ser His Asp His Met Cys Phe Ile Leu Glu Glu Glu Gln Ala Met 165 170 175 Phe Thr Gly Asp Asn Val Leu Gly His Gly Thr Ala Ala Val Glu His 180 185 190 Leu Ser Thr Trp Met Lys Thr Leu Arg Glu Met Gln Ser His Asn Cys 195 200 205 Thr Thr Gly Tyr Pro Ala His Gly Val Val Ile Ala Asp Leu Asn Ala 210 215 220 Lys Ile Ser Gly Glu Leu Ser Met Lys Ala Arg Arg Glu Lys Gln Val 225 230 235 240 Met Gln Val Leu Gln Asp Gln Lys Arg Gly Thr Asn Gly Arg Ala Arg 245 250 255 Leu Thr Ile Lys Glu Leu Val Ala Thr Leu Tyr Gly Glu Arg Ile Asp 260 265 270 Asn Ser Val Lys Glu Leu Ala Leu Glu Pro Phe Ile Asp Glu Val Leu 275 280 285 Arg Lys Leu Ala Glu Asp Gly Ile Val Gly Phe Asp Met Arg Gly Gly 290 295 300 Ile Lys Arg Trp Phe Ala Leu Val Ile 305 310 <210> 5 <211> 1320 <212> DNA <213> Fusarium graminearum J1-012 <400> 5 atgggttcaa tttcttctcc atctctcatc attgaccttg cgaacgctgt gtcctcagca 60 gctaagaact tggacacgca attgcaatct caaggttttc cacagccatc atttgaagct 120 gatggtccaa catacgttgt tcccaaggat gcccccaaag cagcccacga agcccgtgtg 180 gcaaccgctg aggcagccct gaaactgttc aatcttgtat ctggacccag cgagcttcta 240 cccaacatga cagccagtta tcacaccatc tttgcacttc aatggttaca tcatttcgat 300 gttttctctc acattccgct tgatggttcc ctgtcatatg aaaagctagc caccaaagcc 360 aatgtacctg agtctctact caaaagtgtt gcgcgtatgg caatgacaag caacatcttg 420 gcagaaccaa ccacgggtca agtcgcgcac tctgccaatt ctgccatgtt tgtcaaattt 480 cccaacatgc gcgactgggc ctcgtacatg ttcactgcct caattccgac cgctgctgcc 540 atggtccaag ctacagaaaa gtggccagga agtgtaaaga agaccgagac agcttacaac 600 atcgcattca atcatgattt gcccttcttt gatcatttgt cccagagtcc tgtcatgaca 660 aagcagttct ctggatacat gagaagtgtg actgacggtc aaggcatgga tctctcccat 720 ctcgtcaacg ggtttgactg ggccagcctg ccagacaagt ccctcattgt tgacattgga 780 ggctctgcgg gccatgcaag ctatgctcta gccgctgctt atccacacct ccgctttgag 840 gtacaagacc ttgacaccgt ggtcaacgga gaaaaagcag ccaaggagca tgaagaagca 900 gtaagcaagc atgtgattgg caccgataac agggtgacct tcaaagcgca caacttcttt 960 gaggcacagc caaccaaaga cgcaaccgtt tacatgctgc gaatgataat ccacgactgg 1020 ccggacgccg aagcaaagac catcctgggt aatctcgtcc cagcgctcga gtcagccaaa 1080 gcaaccctcc tcatcatgga tacggttctg ccgtcacccg gaagtatccc gtcggtacgg 1140 gagcgtgtca tcagaacgcg agacttgact atgcgccagg tgttcaacgc caaggagcgc 1200 ggtgtcgatg actgggaggc gatcttgcgc gaaacggatt cgaggctgac cttgaagaat 1260 ttgaggcagc cggaggggag caacatgtgc ttattgacca tcagcttaca ggatgactag 1320 <210> 6 <211> 439 <212> PRT <213> Talaromyces sp. F08Z-0631 <400> 6 Met Gly Ser Ile Ser Ser Pro Ser Leu Ile Ile Asp Leu Ala Asn Ala 1 5 10 15 Val Ser Ser Ala Ala Lys Asn Leu Asp Thr Gln Leu Gln Ser Gln Gly 20 25 30 Phe Pro Gln Pro Ser Phe Glu Ala Asp Gly Pro Thr Tyr Val Val Pro 35 40 45 Lys Asp Ala Pro Lys Ala Ala His Glu Ala Arg Val Ala Thr Ala Glu 50 55 60 Ala Ala Leu Lys Leu Phe Asn Leu Val Ser Gly Pro Ser Glu Leu Leu 65 70 75 80 Pro Asn Met Thr Ala Ser Tyr His Thr Ile Phe Ala Leu Gln Trp Leu 85 90 95 His His Phe Asp Val Phe Ser His Ile Pro Leu Asp Gly Ser Leu Ser 100 105 110 Tyr Glu Lys Leu Ala Thr Lys Ala Asn Val Pro Glu Ser Leu Leu Lys 115 120 125 Ser Val Ala Arg Met Ala Met Thr Ser Asn Ile Leu Ala Glu Pro Thr 130 135 140 Thr Gly Gln Val Ala His Ser Ala Asn Ser Ala Met Phe Val Lys Phe 145 150 155 160 Pro Asn Met Arg Asp Trp Ala Ser Tyr Met Phe Thr Ala Ser Ile Pro 165 170 175 Thr Ala Ala Ala Met Val Gln Ala Thr Glu Lys Trp Pro Gly Ser Val 180 185 190 Lys Lys Thr Glu Thr Ala Tyr Asn Ile Ala Phe Asn His Asp Leu Pro 195 200 205 Phe Phe Asp His Leu Ser Gln Ser Pro Val Met Thr Lys Gln Phe Ser 210 215 220 Gly Tyr Met Arg Ser Val Thr Asp Gly Gln Gly Met Asp Leu Ser His 225 230 235 240 Leu Val Asn Gly Phe Asp Trp Ala Ser Leu Pro Asp Lys Ser Leu Ile 245 250 255 Val Asp Ile Gly Gly Ser Ala Gly His Ala Ser Tyr Ala Leu Ala Ala 260 265 270 Ala Tyr Pro His Leu Arg Phe Glu Val Gln Asp Leu Asp Thr Val Val 275 280 285 Asn Gly Glu Lys Ala Ala Lys Glu His Glu Glu Ala Val Ser Lys His 290 295 300 Val Ile Gly Thr Asp Asn Arg Val Thr Phe Lys Ala His Asn Phe Phe 305 310 315 320 Glu Ala Gln Pro Thr Lys Asp Ala Thr Val Tyr Met Leu Arg Met Ile 325 330 335 Ile His Asp Trp Pro Asp Ala Glu Ala Lys Thr Ile Leu Gly Asn Leu 340 345 350 Val Pro Ala Leu Glu Ser Ala Lys Ala Thr Leu Leu Ile Met Asp Thr 355 360 365 Val Leu Pro Ser Pro Gly Ser Ile Pro Ser Val Arg Glu Arg Val Ile 370 375 380 Arg Thr Arg Asp Leu Thr Met Arg Gln Val Phe Asn Ala Lys Glu Arg 385 390 395 400 Gly Val Asp Asp Trp Glu Ala Ile Leu Arg Glu Thr Asp Ser Arg Leu 405 410 415 Thr Leu Lys Asn Leu Arg Gln Pro Glu Gly Ser Asn Met Cys Leu Leu 420 425 430 Thr Ile Ser Leu Gln Asp Asp 435 <210> 7 <211> 46 <212> DNA <213> Synthetic primer <400> 7 agcaagctcc gaattcgaaa caatgactgg acaggccaag gatatc 46 <210> 8 <211> 39 <212> DNA <213> Synthetic primer <400> 8 actacagatc cccggtaccc tagctgtaat actccacca 39 <210> 9 <211> 42 <212> DNA <213> Synthetic primer <400> 9 agcaagctcc gaattcgaaa caatggcaac ccaagaaggg gg 42 <210> 10 <211> 41 <212> DNA <213> Synthetic primer <400> 10 actacagatc cccggtacct caaattacaa gggcgaacca c 41 <210> 11 <211> 35 <212> DNA <213> Synthetic primer <400> 11 agcaagctcc gaattatggg ttcaatttct tctcc 35 <210> 12 <211> 34 <212> DNA <213> Synthetic primer <400> 12 actacagatc cccggctagt catcctgtaa gctg 34 <210> 13 <211> 41 <212> DNA <213> Synthetic primer <400> 13 ggaaagcttg catgcctgca gcgactccaa tcttcaagag c 41 <210> 14 <211> 35 <212> DNA <213> Synthetic primer <400> 14 aacgctctcg cgacgaagta ccatacagta ccgcg 35 <210> 15 <211> 35 <212> DNA <213> Synthetic primer <400> 15 cgtcgcgaga gcgttccact gcatcatcag tctag 35 <210> 16 <211> 41 <212> DNA <213> Synthetic primer <400> 16 cgcagaatcc atatgactag tgtaagatac atgagcttcg g 41 <210> 17 <211> 36 <212> DNA <213> Synthetic primer <400> 17 tacggcttac tccggacatt aatccggatc ctttcc 36 <210> 18 <211> 35 <212> DNA <213> Synthetic primer <400> 18 ccggagtaag ccgtacccat catggtgttt tgatc 35

Claims

1. An engineered bacterium for fermentative production of tetrahydroanthraquinone compounds, characterized in that, Selected from the following strains: When the tetrahydroanthraquinone compound is atrochrysone, it contains a gene encoding a fungal non-reducing and reusing type I polyketide synthase and a gene encoding a β-lactamase type thioesterase; When the tetrahydroanthraquinone compound is chrysophanol anthrone, it contains a gene encoding a fungal non-reducing and reusing type I polyketide synthase, a gene encoding a β-lactamase type thioesterase and a gene encoding an anthracycline C6-OH methyltransferase; The amino acid sequence of the type I polyketide synthase is shown in SEQ ID No.2, the amino acid sequence of the β-lactamase type thioesterase is shown in SEQ ID No.4, and the amino acid sequence of the anthracycline C6-OH methyltransferase is shown in SEQ ID No.6; The host cell of the engineered bacterium is Aspergillus oryzae.

2. The genetically engineered strain according to claim 1, characterized in that The gene encoding the fungal non-reducing and reusing type I polyketide synthase is a nucleotide sequence having more than 50% similarity with SEQ ID No.1 and encoding a protein with the amino acid sequence shown in SEQ ID No.2; the gene encoding the β-lactamase type thioesterase is a nucleotide sequence having more than 50% similarity with SEQ ID No.3 and encoding a protein with the amino acid sequence shown in SEQ ID No.4; the gene encoding the anthracycline C6-OH methyltransferase is a nucleotide sequence having more than 50% similarity with SEQ ID No.5 and encoding a protein with the amino acid sequence shown in SEQ ID No.

6.

3. A method for preparing tetrahydroanthraquinone compounds, characterized in that, The engineered bacterium according to claim 1 or 2 is induced to express with starch, maltose or maltodextrin, and the tetrahydroanthraquinone compound is atrochrysone or chrysophanol anthrone.

4. A method for preparing anthraquinone compounds, characterized in that the steps Including: The tetrahydroanthraquinone compound is treated with an alkali and reacted for 2-6 hours; when the anthraquinone compound is emodin, the tetrahydroanthraquinone compound is atrochrysone; when the anthraquinone compound is physcion, the tetrahydroanthraquinone compound is chrysophanol anthrone; the tetrahydroanthraquinone compound is prepared by the following method: the engineered bacterium according to claim 1 or 2 is induced to express with starch, maltose or maltodextrin, and the fermentation broth or the extract of the fermentation broth is taken.

5. The method according to claim 4, wherein Also including: The mixture after alkali treatment is neutralized and acidified with an acid, the acid is hydrochloric acid, and the molar ratio of the acid to the alkali is 0-5:1.

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