Biosynthetic gene cluster of tropolone compound isatropolones and oriented high-yield strain

Abstract

The invention relates to a biosynthesis gene cluster of a tropolone compound, namely isatropolones, and a bacterial strain capable of realizing directional high yield. The gene cluster comprises an isaF gene, an isaJ gene and an isaS gene; the sequence of the isaF gene is as shown in SEQ ID NO.1, the sequence of the isaJ gene is as shown in SEQ ID NO.3, and the sequence of the isaS gene is as shown in SEQ ID NO.5. The strain capable of realizing the directional high yield is (1) a strain capable of realizing the directional high yield of isatropolone A, namely a strain which takes streptomyces sp.CGMCC (China General Microbiological Culture Collection Center) No.15540 as a host and is used for blocking an isaJ gene of the host or blocking an isaS gene of the host and is also used for realizing the overexpression of isaF at the same time, and the strain capable of realizing the directional high yield of the isatropolone A is a strain capable of realizing the directional high yield of the isatropolone A. And (2) the strain of the oriented high-yield isatropolone C is a strain which takes streptomyces sp.CGMCC (China General Microbiological Culture Collection Center) No.15540 as a host and is used for overexpressing the genes of isaF and isaS, or a strain which is used for overexpressing the genes of isaJ and isaS, or a strain which is used for overexpressing the genes of isaF, isaJ and isaS, or a strain which is used for overexpressing the genes of isaF, isaJ and isaS.

Description

Isatropolones biosynthetic gene cluster and targeted high-yielding strain Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a biosynthetic gene cluster of tropone compounds isatropolones and a directed high-yield strain. Background Art

[0002] Streptomyces sp. CGMCC No. 15540 (CPCC 204095) was isolated from soil in Miyun District, Beijing, my country, by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Microbiological chemical characterization revealed that this strain produces secondary metabolites, isatropolones, during fermentation. Isatropolones belong to the tropone family of compounds, with a chemical structure consisting of a tropone ring, a cyclopentanone, and a deoxysugar, representing a class of polycyclic fused compounds (Figure 1).

[0003] Isatropolones have been found to have a variety of pharmacological or biological activities, indicating that they have important commercial application prospects. For example:

[0004] (1) Leishmania donovani is the causative agent of the zoonotic disease visceral leishmaniasis (also known as kala-azar), which can be transmitted to humans by insects such as sand flies and sand flies. Cutaneous and mucous membrane leishmaniasis can cause permanent scarring or severe disability. If visceral leishmaniasis is not promptly intervened, the mortality rate can be greater than 95%. Every year, there are approximately 50,000 to 90,000 new cases of visceral leishmaniasis and approximately 600,000 to 1,000,000 new cases of cutaneous leishmaniasis worldwide. The World Health Organization (WHO) has listed it as a "neglected tropical disease". Isatropolones have significant inhibitory activity against Leishmania donovani, and the activity of isotropolone A is comparable to that of miltefosine, a clinically approved anti-leishmaniasis drug. [1] Miltefosine's gastrointestinal toxicity, hemolytic side effects, and reproductive impairment limit its clinical use. [2&3] In contrast, isatropolones have extremely low cytotoxicity and possess the potential and value to be developed as a drug for the treatment of leishmaniasis.

[0005] (2) Potato scab is a common disease in potato cultivation. It is caused by potato tubers infected with pathogenic actinomycetes in the soil, resulting in a deterioration in the appearance of the tubers, poor storage resistance, and reduced commercial value. Isatropolone C has a good inhibitory effect on Streptomyces scabies, the pathogen of potato scab. [4] .

[0006] (3) Autophagy is a mechanism by which cells maintain their own physiological homeostasis. It is a basic physiological process in which unused components and damaged organelles in cells are degraded and reused. Isatropolones have the biological activity of activating cell autophagy. [5] .

[0007] However, the production of isatropolones in native strains is low and they are easily converted into other products (e.g., non-enzymatically to isarubrolones), making it difficult to accumulate them in large quantities. Based on the excellent activity and development potential of isatropolones in various aspects, constructing strains that can produce high-yield isatropolone A or isatropolone C, thereby addressing the issue of low fermentation titers, would have considerable application value and economic benefits.

[0008] Based on this, the present invention is proposed.

[0009] [References]

[0010] 1.Cai X, Shi YM, Pohlmann N, et al. Structure and Biosynthesis of Isatropolones, Bioactive Amine-Scavenging Fluorescent Natural Products from Streptomyces [J].Angew Chem Int Ed,2017,56(18):4945-4949.

[0011] 2. Reimao JQ, Pita Pedro DP, Coelho AC. The Preclinical Discovery and Development of Oral Miltefosine for the Treatment of Visceral Leishmaniasis: A Case History[J]. Expert Opin Drug Discov, 2020, 15(6): 647-658.

[0012] 3. Soto JA, Berman JD. Miltefosine Treatment of Cutaneous Leishmaniasis[J]. Clin Infect Dis, 2021, 73(7):e2463-e2464.

[0013] 4.Sarwar A,LatifZ,Zhang S,et al.Biological Control of Potato Common Scab with Rare Isatropolone C Compound Produced by Plant Growth Promoting Streptomyces A1RT[J].Front Microbiol,2018,9:1126.

[0014] 5.Li L, Li S, Jiang B, Zhang M, et al.Isarubrolones Containing a Pyridooxazinium Unit from Streptomyces as Autophagy Activators[J].J Nat Prod.2019;82(5):1149-1154.

[0015] Summary of the Invention

[0016] The present invention first relates to the biosynthetic genes of the tropolone compounds isatropolones. Preferably, the isatropolones are isatropolone A and / or isatropolone C. The genes are:

[0017] (1) isaF, full length 891 bp, encoding 296 amino acids of IsaF protein

[0018] The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2;

[0019] or (2) isaJ, 792 bp in length, encoding the 263 amino acid IsaJ protein

[0020] The gene sequence is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4;

[0021] or (3) isaS, 1230 bp in length, encoding the 409 amino acid IsaS protein

[0022] The gene sequence is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.6;

[0023] The present invention also relates to the isa gene cluster of Streptomyces sp. CGMCC No. 15540 (i.e., CPCC 204095) comprising the aforementioned genes, wherein the gene cluster comprises the aforementioned isaF, isaJ, and isaS genes;

[0024] Preferably, the position of the isa gene cluster in the genome of Streptomyces sp. CGMCC No. 15540 (i.e., CPCC 204095) is: 6,151,891 to 6,159,911 nt + 6,451,081 to 6,476,781 nt.

[0025] The present invention also relates to the use of the isaF, isaJ, isaS genes and / or gene clusters in the biosynthesis of isatropolones, a tropone compound; preferably, the isatropolones include isatropolone A and / or isatropolone C.

[0026] The present invention also relates to the use of the proteins encoded by the isaF, isaJ, isaS genes and / or gene clusters in the biosynthesis of isatropolones, a tropone compound; preferably, the isatropolones include isatropolone A and / or isatropolone C.

[0027] The present invention also relates to the use of the isaF, isaJ, isaS genes and / or their encoded proteins in regulating the production of isatropolones in host bacteria, wherein the regulation refers to,

[0028] (1) overexpressing the isaF gene and / or its encoded protein to increase the biosynthesis of isatropolones; and / or

[0029] (2) blocking the isaJ and / or isaS genes and / or their encoded proteins to increase the biosynthesis of isatropolone A; and / or

[0030] (3) overexpressing the isaJ and / or isaS genes and / or their encoded proteins to increase the biosynthesis of isatropolone C;

[0031] Preferably, the regulation refers to,

[0032] (1) while blocking the isaJ gene or the isaS gene, overexpressing the isaF gene and / or its encoded protein to increase the biosynthesis of isatropolone A; more preferably, using PrpsL (XC) The overexpression of isaF was driven by the promoter or kasO*p promoter;

[0033] The PrpsL (XC) The sequence of the promoter is shown in SEQ ID NO.9, and the sequence of the kasO*p promoter is shown in SEQ ID NO.10;

[0034] or (2) overexpression:

[0035] a) isaF, isaS genes and / or their encoded proteins, or

[0036] b) isaJ, isaS genes and / or their encoded proteins, or

[0037] c) isaF, isaJ, isaS genes and / or their encoded proteins;

[0038] To increase the biosynthesis of isatropolone C.

[0039] The host bacteria include, but are not limited to, Streptomyces and Escherichia coli; preferably, Streptomyces sp. CGMCC No. 15540.

[0040] The present invention also relates to a strain for directional high-yield isotropolone A, wherein the strain is: Streptomyces sp. CGMCC No. 15540 is used as a host, the host isaJ gene or the host isaS gene is blocked, and PrpsL is used to generate the isatropolone A. (XC) isaF overexpression driven by the promoter or the kasO*p promoter.

[0041] The present invention also relates to a strain with a directional high yield of isatropolone C, which is a strain that uses Streptomyces sp. CGMCC No. 15540 as a host and overexpresses the isaF and isaS genes, or overexpresses the isaJ and isaS genes, or overexpresses the isaF, isaJ and isaS genes.

[0042] The present invention also relates to a method for fermenting the directed high-yield isatropolone A strain to produce isatropolone A. The method comprises: culturing and fermenting the host bacteria in an M5 culture medium to obtain the isatropolone A; the M5 culture medium comprises a formula of 2.5% malt extract, 0.4% glucose, 0.4% yeast extract, 0.6% soy flour, 2% agar, and deionized water.

[0043] Preferably, when fermenting the strain producing high-yield isotropolone A, the inoculum size is 10 10 The spores were inoculated into 35 ml of M5 solid medium and fermented for 24 to 45 h.

[0044] The beneficial effects of the present invention are:

[0045] Isatropolones have important biological functions and their structures are shown in the following formula:

[0046] Among them, isatropolone A has anti-Leishmania donovani activity and low cytotoxicity, and has the potential to be developed into a drug for the treatment of leishmaniasis; isatropolone C has significant antibacterial activity against the pathogen of potato scab, Streptomyces scab, and has great application prospects in potato cultivation and production.

[0047] Isatropolone A and isatropolone C only differ in the hydroxyl group at the C-3 position, and isatropolone A has better inhibitory activity against Leishmania donovani than isatropolone C. This work elucidates the biosynthetic regulatory mechanism of isatropolones and the synthetic mechanism responsible for C-3 hydroxylation, and obtains targeted high-yield strains of isatropolone A and isatropolone C through synthetic biology strategies, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1. Structure of isatropolones.

[0049] Figure 2. The isatropolones biosynthetic gene cluster predicted by bioinformatics analysis after whole-genome sequencing of Streptomyces sp. CGMCC No. 15540 (CPCC 204095).

[0050] Figure 3. Domain structure and amino acid homology analysis of IsaF. The N-terminus contains an OmpR-type DBD domain, with arrows indicating β-sheet regions and boxes indicating α-helical regions. The C-terminus contains a BTAD domain, with seven predicted α-helical structures. IsaF is aligned with the amino acid sequences of MoaR1 from Streptomyces sp. ADI95-17, PapR1 and PapR2 from Streptomyces evorotatoria, and OtcR from Streptomyces cremasterii. Identical sites are marked in black, and similar sites are marked in gray.

[0051] Figure 4. Schematic diagram of the construction of the isaF overexpression plasmid.

[0052] Figure 5. Enzyme digestion identification of plasmid pL-isaF:

[0053] Lane M, 1kb plus DNA ladder (10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1,000,800,500,300bp); Lanes 1~2, pL-isaF / KpnI&XhoI(4.4kb+1.6kb+0.6kb+0.2kb).

[0054] Figure 6. PCR identification of isaF overexpressing strains and control strains

[0055] A, Lane M, 1 kb plus DNA ladder (10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 800, 500, 300 bp); Lanes 1-3, PCR product of the attB from Streptomyces sp. CPCC 204095 / pL-isaF; Lane 4, Streptomyces sp. CPCC 204095 genomic DNA as a negative control;

[0056] B, Lane M, 1 kb plus DNA ladder (10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 800, 500, 300 bp); Lanes 1-6, PCR product of the attB from Streptomyces sp. CPCC 204095 / pSET152; Lane 7, Streptomyces sp. CPCC 204095 genomic DNA as a negative control.

[0057] Figure 7. HPLC analysis of fermentation products of isaF overexpression strain (pSET152 is an empty plasmid control, pL-isaF is an overexpression strain).

[0058] Figure 8. Transcriptional expression of related genes in the isaF overexpression strain (pSET152 is the empty plasmid control, and pL-isaF is the overexpression strain).

[0059] Figure 9. Transcriptional expression of related genes in isaF recombinant strains (blocking strain FKO, complementing strain FKO / pL-isaF).

[0060] Figure 10. HPLC analysis of fermentation products of the isaF-blocking strain and the reverting strain (blocking strain FKO, complementing strain FKO / pL-isaF).

[0061] Figure 11. Domain structure and amino acid homology analysis of IsaJ. This analysis shows an OmpR-like DBD domain at the N-terminus, with arrows indicating β-sheet regions and boxes indicating α-helical regions. The C-terminus contains a BTAD domain with seven predicted α-helices. IsaJ is aligned with the amino acid sequences of RubR from Streptomyces sp. KIB-H033, PapR1 from Streptomyces evorotatoria, and OtcR from Streptomyces schizophylla. Identical sites are marked in black, and similar sites are marked in gray.

[0062] Figure 12. PCR identification of isaJ overexpression strains.

[0063] Lane M, 1 kb plus DNA ladder (10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 800, 500, 300 bp); Lanes 1-3, PCR product of the attB from CPCC 204095 / pL-isaJ; Lane 4, CPCC 204095 genomic DNA as a negative control.

[0064] Figure 13. LC-MS analysis of fermentation products of isaJ overexpressing strains.

[0065] A: HPLC profiles of fermentation products of the control strain CPCC 204095 / pSET152 and the isaJ overexpressing strain CPCC 204095 / pL-isaJ;

[0066] B: Extracted molecular weight of isotropolone A / B m / z 457 [M+H] +Atlas.

[0067] Figure 14. Transcriptional expression of related genes in isaJ overexpression strains.

[0068] Figure 15. Transcriptional expression of related genes in isaJ recombinant strains (blocking strain JKO, complementing strain JKO / pL-isaJ).

[0069] Figure 16. HPLC analysis of fermentation products of the isaJ-blocked strain and the restored strain.

[0070] Figure 17. PCR identification of isaS overexpression strains:

[0071] Lane M, 1 kb plus DNA ladder (10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 800, 500, 300 bp); Lanes 1-3, PCR product of the attB from CPCC 204095 / pL-isaS; Lane 4, CPCC 204095 genomic DNA as a negative control.

[0072] Figure 18. LC-MS analysis of fermentation products of isaS overexpressing strains:

[0073] A: HPLC profiles of fermentation products of the control strain CPCC 204095 / pSET152 and the isaS overexpression strain CPCC 204095 / pL-isaS;

[0074] B: Extracted molecular weight of isotropolone A / B m / z 457 [M+H] + Atlas.

[0075] Figure 19. HPLC analysis of fermentation products of the isaJ blocked strain and the isaS cross-revertant strain.

[0076] Figure 20. HPLC analysis of fermentation products of the isaS-blocked strain.

[0077] Figure 21. Isatropolone A production assay in the first-generation high-yielding strains (JKO, SKO) and the second-generation targeted high-yielding strains (JKO / eF, SKO / eF). Three zygotes (biological replicates) were used for each strain. Fold changes in production compared with the parent strains are shown. *p<0.05, **p<0.01.

[0078] Figure 22. Yield analysis of isotropolone A in the third generation of targeted high-yielding strains (with a strong promoter replaced). Three conjugates (biological replicates) were used for each assay. The fold change in yield was compared with that of the second generation SKO / eF strain. *p<0.05.

[0079] Figure 23. Screening of high-yield culture medium for Isatropolone A.

[0080] FIG24 . Fermentation of different batches confirms that high-yielding fermentation of isatropolone A is reproducible, with two zygotes each (biological replicates).

[0081] FIG25 . Increasing the inoculum spore amount can shorten the high-yield fermentation time of isatropolone A, two zygotes each (biological replicates).

[0082] Figure 26 shows the fold change of yield of three conjugates (biological replicates) of each Isatropolone C directed high-yielding strain compared with the empty plasmid control strain. DETAILED DESCRIPTION

[0083] The Streptomyces sp. CPCC 204095 strain was deposited on April 2, 2018, at the General Microbiology Center of the China Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The taxonomic name of the strain is Streptomyces sp., and the deposit number is CGMCC No. 15540.

[0084] We first sequenced the whole genome of Streptomyces sp. CPCC 204095 and used bioinformatics analysis to predict the isatropolones biosynthetic gene cluster, which we named isa (Figure 2). We also annotated the genes in the isatropolones biosynthetic gene cluster (Table 1).

[0085] Table 1 Gene annotation of the isatropolones biosynthetic gene cluster

[0086] 1. Culture medium

[0087] (1) LB medium, for liquid culture of E. coli. Tryptone 1.0%, yeast extract 0.5%, NaCl 1.0%

[0088] 1.5% agar was added to the pH 7.0 solid culture medium, and antibiotics at appropriate concentrations were added as needed.

[0089] (2) MS medium, used for conjugation transfer between E. coli ET12567 / pUZ8002 and Streptomyces sp. CPCC 204095. Mannitol 2.0%, soybean powder 2.0%, agar 2.0%

[0090] Prepare with water and add MgCl2 to a final concentration of 10 mM when using.

[0091] (3) M1 (ISP2) medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0092] (4) M2 medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0093] (5) M3 medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0094] (6) M4 medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0095] (7) M5 medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0096] (8) M6 medium, used for solid fermentation of Streptomyces sp. CPCC 204095 and its derivatives.

[0097] 2. General Methods

[0098] 2.1 Preparation and transformation of competent E. coli

[0099] The competent E. coli was prepared and transformed using methods commonly used in the art.

[0100] 2.2. Extraction of E. coli plasmid DNA

[0101] Plasmid extraction was performed according to the instructions of the plasmid extraction kit of Beijing Quanshijin Biotechnology Co., Ltd.

[0102] 2.3 Extraction of total Streptomyces genomic DNA

[0103] Total DNA was extracted using the Magen bacterial genome extraction kit according to the instructions.

[0104] 2.4. Joint transfer

[0105] The conjugation transfer experiment was performed according to the experimental procedures in the Streptomyces manual.

[0106] 2.5. Extraction of total RNA from Streptomyces

[0107] Total RNA extraction was performed based on TRIzol Reagent (#15596-026, 100 ml) and TRIzol Plus Purification Kit (#12183-555, 50 preps) from Life Technologies.

[0108] 2.6 Reverse transcription and real-time fluorescence quantitative PCR

[0109] The mRNA was reverse transcribed and synthesized into first-strand cDNA using the TransGen AT311 kit.

[0110] Roche Fast Start Universal SYBR Green Master (Rox) was used for real-time quantitative PCR, using cDNA as a template and hrdB primers as an internal control. To exclude genomic DNA contamination, each primer pair was also amplified using a negative control without reverse transcriptase as a template.

[0111] 2.7. Fermentation of strains and HPLC and LC-MS analysis conditions of fermentation products

[0112] 2.7.1 Spread an appropriate amount of spores on solid fermentation medium and culture at 28°C.

[0113] 2.7.2 Collect the fermentation medium and bacteria under different culture conditions and culture time, add 2 volumes of ethyl acetate and extract at room temperature for 24 hours.

[0114] 2.7.3 Take 1 ml of each fermentation extract, evaporate the ethyl acetate using a vacuum concentrator, add 100 μl of chromatography-grade methanol to reconstitute the membrane with a pore size of 0.22 μm, and inject the sample.

[0115] 2.7.4 LC-MS conditions: Agilent Eclipse Plus C18 (4.6×150 mm, 5 μm), phase A: 100% acetonitrile; phase B: 1‰ water (1‰ HAc); gradient elution: 15% to 70% acetonitrile: water (1‰ HAc), 30 min. Column temperature was maintained; flow rate: 1 ml / min; injection volume: 10 μl of fermentation extract. LC-MS was performed in positive ion mode with a fragmentor voltage of 120 V. The extracted isotropolone A molecular ion peak (m / z 457 [M+H] + ), isatropolone C molecular ion peak (m / z473[M+H] + ).

[0116] 2.7.5 Preparation of a Quantitative Standard Curve: Prepare a 6 mg / L stock solution of pure isotropolone A in chromatography-grade methanol. Dilute the solution two-fold to six concentrations. Inject 10 μl of each solution through a 0.22 μm pore size membrane. LC-MS detection conditions are the same as above. Perform a linear regression between the injected volume and the corresponding peak area to generate a standard curve.

[0117] 2.7.6 Quantification of isatropolone A: Substitute the liquid phase peak area of ​​each fermentation sample into the standard curve and calculate the isatropolone A yield in each fermentation sample.

[0118] 3. Antibiotics and other storage solutions

[0119] Table 2 Antibiotics and other storage solutions

[0120] 4. Enzymes, reagents and antibodies

[0121] Restriction endonucleases were purchased from Takara Biotechnology (Dalian) Engineering Co., Ltd.

[0122] High-Fidelity DNA Polymerase, T4 DNA ligase, and proteinase K were purchased from NEB.

[0123] RNase was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0124] DNase I was purchased from Promega.

[0125] Example 1: Study on antibiotic susceptibility of Streptomyces sp. CPCC 204095

[0126] First, the antibiotic sensitivity of Streptomyces sp. CPCC 204095 was studied. A spore suspension of Streptomyces sp. CPCC 204095 was prepared, and the concentration of the prepared spore suspension was calculated to be 3×10 9 50 μl of each solution was spread on MS plates containing a certain concentration of antibiotics and cultured at 28°C for 5 days. The colonies formed were counted and the ratio of the number of colonies to the number of spores spread was used as the colony formation rate (CFR). If CFR < 10 -7 , it can be considered that Streptomyces sp. CPCC 204095 is sensitive to the antibiotic at that concentration; if CFR>10%, it can be considered that Streptomyces sp. CPCC 204095 is resistant to the antibiotic at that concentration.

[0127] The experimental results showed that Streptomyces sp. CPCC 204095 was sensitive to 50 μg / ml apramycin and 20 μg / ml thiostrepton. Therefore, these two antibiotics can be used for subsequent screening of genetic manipulation.

[0128] Example 2: IsaF positively regulates the biosynthesis of isatropolones

[0129] 1. Bioinformatics analysis of IsaF

[0130] The isaF gene is located near the left boundary of the isa gene cluster (nt 6,469,578–6,470,468) (Figure 2). It is 891 bp long and encodes the 296-amino acid IsaF protein. The gene sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0131] Analysis of the IsaF amino acid sequence using the Xtalpred-RF tool revealed that the protein contains an OmpR-like DNA binding domain (DBD) at its N-terminus. The OmpR domain is an HTH motif flanked by β-sheets and often has DNA binding activity. The C-terminus of the IsaF protein contains a typical bacterial transcriptional activator domain (BTAD) composed of seven α-helices (Figure 3).

[0132] 2. Overexpression analysis of isaF

[0133] Overexpression analysis of target genes is a common method for determining gene function. We overexpressed the isaF gene in a primary strain and analyzed changes in fermentation products and transcriptional levels of various structural genes to preliminarily determine the function of the IsaF protein.

[0134] 2.1 Construction of isaF overexpression plasmid

[0135] The plasmid pICLSet containing the bacteriophage ΦC31 attP integration site was used as a vector, the isaF gene was inserted into the wild-type strain Streptomyces sp. CPCC 204095, and the isaF gene was overexpressed.

[0136] Plasmid pICLSet does not contain a Streptomyces replicon. The attP integration site it contains can be integrated into the attB site of the Streptomyces genome under the action of ΦC31 integrase. The erythromycin strong promoter ermE*p and SD sequence tuf1 are introduced upstream of the multiple cloning site region of this plasmid.

[0137] Plasmid construction was accomplished using the Gibson assembly method, specifically using the NEBuilder HiFi DNA Assembly Cloning Kit. The plasmid construction is shown in Figure 4. isaF-F and isaF-R were used to amplify the coding region of the isaF gene. The upstream primer isaF-F of the target gene overlaps with the end of pICLSet digested with NdeI by 18 bp, while the downstream primer isaF-R overlaps with the end of pICLSet digested with BamHI by 18 bp.

[0138] We first used the genome of Streptomyces sp. CPCC 204095 as a template and primers isaF-F and isaF-R to amplify the 903-bp coding region of the isaF gene. PCR products were recovered by agarose gel electrophoresis. pICLSet was digested with NdeI and BamHI to recover the large fragment. The recovered PCR product and the linearized vector were ligated at appropriate ratios using NEBuilder HiFi DNA Assembly Master Mix. The ligated product was transformed into E. coli DH5α, and the plasmid was extracted. Since the assembled plasmid no longer contained NdeI and BamHI restriction sites, we selected the KpnI and XhoI restriction sites contained in the vector and insert for restriction analysis. The expected band sizes were 4.4 kb, 1.6 kb, 0.6 kb, and 0.2 kb (Figure 5), which were consistent with the expected band sizes. The plasmid that matched the expected band was sequenced and the recombinant plasmid that was sequenced correctly was named pL-isaF.

[0139] 2.2 Verification of isaF overexpression strains

[0140] The plasmid pL-isaF and the empty vector control plasmid pSET152 were transformed into E. coli ET12567 / pUZ8002 and introduced into Streptomyces sp. CPCC 204095 by conjugation transfer. The genome of the conjugate was extracted and PCR verification was performed. The primers were designed upstream and downstream of the integration site, one end on the genome and the other end on the plasmid. The correctly integrated strain will amplify a 1.6kb band, and the non-integrated strain will not amplify the target band. As shown in Figure 6, the PCR band of the recombinant strain is as expected (Figure 6A), and the original strain has no target band. The correctly verified strain was named the overexpression strain CPCC 204095 / pL-isaF, and the control strain CPCC 204095 / pSET152 band is as expected (Figure 6B), and the original strain has no target band.

[0141] 2.3 Analysis of fermentation product yields of isaF overexpressing strains

[0142] Since isatropolones compounds easily react spontaneously with amine compounds, in order to effectively detect the production of isatropolones compounds, it is necessary to control the fermentation conditions and fermentation time of the strain.

[0143] The overexpression strain CPCC 204095 / pL-isaF and the control strain CPCC 204095 / pSET152 were inoculated on 9 cm diameter plates containing 12 ml of ISP2 solid medium and fermented for 48 h. The fermentation extracts were extracted with 30 ml of ethyl acetate, and the compound yields were analyzed by HPLC. Biological replicates were performed with the same inoculum size and extraction conditions.

[0144] The results showed that compared with the control strain CPCC 204095 / pSET152, the yields of the main products isatropolone (ISA) C and ISA A in the fermentation product of the isaF overexpression strain were significantly increased (Figure 7), indicating that overexpression of isaF can increase the yield of isatropolones and suggesting that isaF is a positive regulator gene of isatropolones biosynthesis.

[0145] 2.4 Transcriptional levels of isaF overexpressing strains

[0146] To further analyze the function of isaF, we examined the relative expression levels of individual genes in the overexpression strain. The overexpression strain CPCC 204095 / pL-isaF and the control strain CPCC 204095 / pSET152 were inoculated onto ISP2 solid medium containing cellophane. After fermentation for 36 hours, the cells were harvested, and RNA was extracted. Real-time fluorescence quantitative PCR was used to analyze the relative expression levels of each gene using the reverse-transcribed cDNA product as a template.

[0147] Using hrdB as the internal reference gene and the transcription level of each gene in the control strain CPCC 204095 / pSET152 as "1", the transcription levels of possible regulatory genes and structural genes of isa in the overexpression strain CPCC 204095 / pL-isaF were investigated.

[0148] The results are as follows (Figure 8). In the overexpression strain CPCC 204095 / pL-isaF, the transcription level of the regulatory gene isaF itself was significantly increased, approximately 15-fold compared to the original strain, further demonstrating that the overexpression strain was successfully constructed. Within the isa gene cluster, the relative expression levels of the core genes isaG, isaH, and isaI, the cyclase gene isaP, and the dehydrogenase gene isaT were approximately 2-3 times upregulated. The relative expression levels of the methyltransferase gene isaC and the cytochrome P450 enzyme isaS did not change significantly. The relative expression level of the transcriptional regulatory factor isaJ was approximately 2-fold upregulated, while the expression levels of isaA and isaB did not change significantly.

[0149] The above results indicate that in the overexpression strain CPCC 204095 / pL-isaF, the transcription levels of the core genes responsible for isatropolones biosynthesis increased to varying degrees, which is consistent with the significantly increased yield of isatropolones fermentation product in the overexpression strain CPCC 204095 / pL-isaF. This further demonstrates that isaF is a pathway-specific positive regulator gene in isatropolones biosynthesis and can positively regulate the transcription levels of various genes in isa.

[0150] 3. Effects of isaF blocking on the transcription level and yield of isatropolones biosynthesis genes

[0151] To further confirm the positive regulatory role of IsaF in the biosynthesis of isatropolones, we constructed the isaF knockout strain CPCC 204095 / FKO and the complemented strain CPCC 204095 / FKO / pL-isaF and analyzed the changes in the transcription levels of isatropolones biosynthesis genes and the changes in compound production in the recombinant strains.

[0152] 3.1 Transcriptional level research results

[0153] The results (Figure 9) showed that compared to the original strain, no isaF mRNA transcription was detected in the isaF-blocked strain, further confirming that the isaF gene was blocked and the blocked strain was successfully constructed. Introduction of the pL-isaF plasmid into the isaF-blocked strain increased the transcription level of the isaF gene, confirming the successful construction of the complementation strain. Transcription levels of the isatropolones biosynthesis-related genes, isaG, isaH, isaI, isaL, and isaP, were significantly downregulated in the isaF-blocked strain, while transcription levels in the revertant strains were upregulated to varying degrees compared to the original strain.

[0154] 3.2 Research results of fermentation products

[0155] The original strain, knockout strain (CPCC 204095 / FKO) and complemented strain (CPCC 204095 / FKO / pL-isaF) were inoculated on ISP2 solid medium and fermented for 48 h. The culture was extracted with ethyl acetate, and the fermentation extract was treated and analyzed for changes in compound production by HPLC.

[0156] The results showed ( FIG10 ) that when isaF was blocked, the production of isatropolone C was significantly reduced, and isatropolone A was completely undetectable. When isaF was replenished, the production of isatropolone C and isatropolone A was also restored.

[0157] The above results further indicate that isaF is a positive regulatory gene for isatropolones biosynthesis.

[0158] Example 3: IsaJ regulates the hydroxylation of isatropolone C

[0159] 1. Bioinformatics analysis of IsaJ

[0160] The isaJ gene is located within the isa gene cluster (nt 6,465,704–6,466,495), is 792 base pairs long, and encodes the 263-amino acid IsaJ protein. Its gene sequence is shown in SEQ ID NO. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 4. Based on the alignment results, IsaJ is preliminarily identified as belonging to the Streptomyces SARP regulatory protein family.

[0161] Analysis of the IsaJ amino acid sequence using the Xtalpred-RF tool revealed that the N-terminus contained a DNA binding domain (DBD) with a wHTH structure typical of the SARP protein family, and the C-terminus contained a bacterial transcriptional activator domain (BTAD) composed of seven α-helical structures typical of the SARP family (Figure 11).

[0162] 2. Overexpression analysis of isaJ

[0163] 2.1 Construction of isaJ overexpression plasmid

[0164] This experiment adopted the same strategy as Example 1. The plasmid pICLSet containing the phage ΦC31 attP integration site was used as a vector. After inserting the isaJ gene, it was introduced into the wild-type strain Streptomyces sp. CPCC 204095 to achieve overexpression of the isaJ gene. Using the Streptomyces sp. CPCC 204095 genome as a template, primers isaJ-F and isaJ-R amplified the 792bp gene coding region of isaJ. The recombinant plasmid was identified by enzyme digestion using KpnI and XhoI. The expected band sizes were 4.4kb, 1.6kb, and 0.6kb, and the enzyme digestion band sizes were consistent with expectations. The plasmid that met the expectations was sequenced and verified. The recombinant plasmid with correct sequencing was named pL-isaJ.

[0165] 2.2 Verification of isaJ overexpression strains

[0166] Plasmid pL-isaJ was transformed into E. coli ET12567 / pUZ8002 and then introduced into Streptomyces sp. CPCC 204095 by conjugation. The genome of the conjugate was extracted and verified by PCR using primers pSET152 and attB_Strep. A 1.6 kb band was amplified in strains with correct integration, while no band was amplified in strains without integration (Figure 12). This strain was designated the overexpression strain CPCC 204095 / pL-isaJ.

[0167] 2.3 Analysis of fermentation products of isaJ overexpressing strains

[0168] The overexpression strain CPCC 204095 / pL-isaJ and the control strain CPCC 204095 / pSET152 were inoculated onto 9-cm-diameter plates containing 12 ml of ISP2 solid medium. Fermentation was continued for 48 hours, followed by extraction with 30 ml of ethyl acetate. The fermentation extract was processed and analyzed for compound yield changes using LC-MS. The structures of isatropolones A and C are shown in Figure 1.

[0169] The results showed ( FIG13 ) that, compared with the control strain, the total amount of isatropolone C and isatropolone A in the fermentation product of the isaJ overexpression strain did not change significantly, but the ratio of isatropolone C to isatropolone A changed significantly. The main product in the fermentation extract was isatropolone C, and isatropolone A was almost undetectable.

[0170] The above results suggested that IsaJ could affect the formation of hydroxyl groups of isatropolone C. The isatropolone C directed high-yield strain CPCC 204095 / pL-isaJ was constructed by overexpressing the isaJ gene.

[0171] 2.4 Transcriptional analysis of isaJ overexpression strains

[0172] To further analyze the function of isaJ, we examined the relative expression levels of individual genes in the overexpression strain. Using hrdB as an internal reference gene and the control strain CPCC 204095 / pSET152, where the transcription level of each gene is set to 1, we examined the transcription levels of potential regulatory genes, core biosynthesis genes, and genes potentially responsible for hydroxylation of isa in the overexpression strain CPCC 204095 / pL-isaJ.

[0173] The results are as follows (Figure 14). In the overexpression strain CPCC 204095 / pL-isaJ, the transcription level of the regulatory gene isaJ itself was significantly increased, approximately 7 times that of the original strain, further proving that the overexpression strain was successfully constructed. Within the isa gene cluster, the relative expression levels of the three type II pks core genes isaG, isaH, and isaI, the cyclase gene isaP, and the oxidase gene isaE did not change significantly. IsaG, IsaH, IsaI, and IsaP are speculated to be responsible for the assembly of the tropone core skeleton, and the function of IsaE is currently unknown. It is worth noting that the relative expression level of the cytochrome P450 enzyme isaS was upregulated, approximately 17 times that of the control strain. The expression levels of the transcriptional regulatory genes isaF, isaA, and isaB did not change significantly.

[0174] The results showed that the transcription levels of the core genes responsible for isatropolones biosynthesis did not change, which is consistent with the presence of isatropolone C in the fermentation products of the overexpression strain CPCC 204095 / pL-isaJ. The upregulation of cytochrome P450 isaS transcription levels suggested that IsaJ may affect the expression level of the isaS gene, thereby promoting the formation of the hydroxyl group at the C-3 position of isatropolone C.

[0175] 3. Gene transcription analysis and fermentation product analysis of isaJ-blocked and revertant strains

[0176] To further confirm the function of IsaJ, this experiment used the in-frame deletion method to construct the isaJ-blocking strain CPCC 204095 / JKO and the complementing strain CPCC 204095 / JKO / pL-isaJ, and analyzed the changes in the transcription levels of various biosynthetic genes and the production of isatropolones homologs in the blocking and complementing strains.

[0177] 3.1 Transcriptional level analysis

[0178] The results (Figure 15) showed that compared to the original strain, undetectable levels of isaJ mRNA were observed in the blocked strain, confirming that the isaJ gene was blocked. Introduction of the pL-isaJ plasmid into the isaJ-blocked strain restored isaJ gene expression. After blocking isaJ, the transcription level of the cytochrome P450 gene isaS was significantly downregulated. However, after complementation of isaJ, the transcription level of the isaS gene was upregulated, reaching approximately 10-fold that of the original strain.

[0179] 3.2 Fermentation product analysis

[0180] The original strain, the knockout strain CPCC 204095 / JKO, and the complemented strain CPCC 204095 / JKO / pL-isaJ were inoculated on ISP2 solid medium and fermented for 48 hours. The fermentation extract was then extracted with ethyl acetate, and the fermentation extract was analyzed by HPLC for changes in compound production. The results (Figure 16) showed that when isaJ was blocked, isatropolone C production was almost undetectable. The secondary metabolites of the blocked strain were primarily isotropolone A, which lacks a hydroxyl group on its side chain, and its isomer isotropolone B. In contrast, fermentation products from the complemented strain CPCC 204095 / JKO / pL-isaJ restored isotropolone C production.

[0181] The above results further indicated that IsaJ was involved in the hydroxyl formation of isatropolone C, and we constructed an isatropolone A-directed high-producing strain CPCC 204095 / JKO by blocking the isaJ gene.

[0182] Example 4: IsaS affects the hydroxylation of isatropolone C

[0183] Isatropolone A and isatropolone C differ only in the hydroxyl group at the C-3 position, and isatropolone A has better inhibitory activity against Leishmania donovani than isatropolone C. [1] In the isaJ-blocked strain, the fermentation products were mainly isotropolone A and isotropolone B, which do not contain a hydroxyl group at the C-3 position. The production of isotropolone C, which contains a hydroxyl group at the C-3 position, decreased significantly and almost ceased to be produced.

[0184] Within the isa gene cluster, blocking isaJ decreased the transcription level of isaS, while overexpressing isaJ also increased the transcription level of isaS, suggesting that IsaS may be responsible for the hydroxylation of isatropolone C C-3.

[0185] 1. Bioinformatics analysis of IsaS

[0186] The isaS gene is located within the isa gene cluster (nt 6,455,391 to nt 6,456,620), is 1230 bp long, and encodes the 409-amino acid IsaS protein. Its gene sequence is shown in SEQ ID NO. 5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 6.

[0187] The amino acid sequence of IsaS was analyzed, and the three-dimensional structure of IsaS was simulated using the SWISS-MODEL tool, which contained a cavity structure that could bind heme. It was preliminarily determined that IsaS was a cytochrome P450 enzyme protein.

[0188] 2. Overexpression analysis of IsaS

[0189] We used the same method as in Examples 2 and 3 to achieve overexpression of the isaS gene.

[0190] Plasmid construction: Using the genome of Streptomyces sp. CPCC 204095 as a template, primers isaS-F and isaS-R were used to amplify the 1332-bp coding region of the isaS gene. This was then ligated into pICLSet digested with NdeI and BamHI. The recombinant plasmid was double-digested with KpnI and NcoI, yielding four expected bands of 4.5 kb, 1.5 kb, 0.7 kb, and 0.6 kb, respectively. The band sizes were consistent with expectations. The plasmid identified as correctly digested was then sequenced to obtain the recombinant plasmid, named pL-isaS.

[0191] The constructed pL-isaS plasmid was introduced into Streptomyces sp. CPCC 204095 by conjugation. The correctly integrated strain amplified a 1.6 kb band, and the target band size was consistent with the expected size ( Figure 17 ). The recombinant strain was successfully constructed and named CPCC 204095 / pL-isaS.

[0192] The control strain CPCC 204095 / pSET152 and the overexpression strain CPCC 204095 / pL-isaS were fermented for 48 hours, and the metabolite production changes were analyzed by LC-MS. The results showed that the metabolite profile changed significantly after overexpression of isaS. The main product in the fermentation extract was isatropolone C, and isatropolone A was almost undetectable (Figure 18). The extracted isatropolone A / B molecular ion peak was m / z 457 [M+H] + Compared with the control strain, the production of isatropolone A and isatropolone B was significantly reduced (Figure 18), suggesting that IsaS catalyzes the formation of the hydroxyl group of isatropolone C, and we constructed the isatropolone C-directed high-yielding strain CPCC 204095 / pL-isaS by overexpressing the isaS gene.

[0193] 3. Cross-complementation of IsaS to isaJ-blocked strains

[0194] To further analyze the relationship between IsaS and IsaJ and explore the function of IsaS, we cross-complemented IsaS with the isaJ-blocking strain and examined the changes in the products.

[0195] The constructed pL-isaS plasmid was introduced into CPCC 204095 / JKO by conjugation. The correctly integrated strain amplified a 1.6 kb band, and the target band size was consistent with the expectation. The recombinant strain was successfully constructed and named CPCC 204095 / JKO / pL-isaS.

[0196] Solid-state fermentation results (Figure 19) show that IsaS can restore the function of the isaJ-blocked strain. In the restored strain, isatropolone C is primarily produced, while the yield of isatropolone A is extremely low. This further demonstrates that IsaS is responsible for catalyzing the hydroxylation of isatropolones at the C-3 position, and IsaJ influences the ratio of these microbial secondary metabolites by regulating IsaS.

[0197] 4. IsaS blocking analysis

[0198] The isaS gene is transcribed in the opposite direction to its upstream and downstream genes. Modification of this gene is unlikely to induce polarity effects and affect the transcription of other genes. Therefore, this experiment employed a double-crossover strategy using homologous recombination to replace the CDS sequence of the thiostrepton resistance gene (tsr), thereby blocking gene function and generating the blocked strain CPCC 204095 / SKO. Both the wild-type strain CPCC 204095 and the blocked strain were fermented, and metabolite changes were analyzed.

[0199] The results showed ( FIG. 20 ) that when isaS was blocked, the isatropolone C peak disappeared, and the production of isatropolone A and B was significantly increased compared with the original strain.

[0200] The results of this example suggest that IsaS is responsible for catalyzing the formation of the C-3 hydroxyl group in isatropolone C. Blocking isaS results in the accumulation of isatropolone A or isatropolone B. We constructed the isatropolone A-directed high-yielding strain CPCC 204095 / SKO by blocking the isaS gene.

[0201] Example 5: Construction of an Isatropolone A High-yielding Strain

[0202] 1. Construction of a second generation high-yielding strain based on the first generation high-yielding isatropolone A strains (JKO, SKO)

[0203] Examples 2-4 have demonstrated that the cytochrome P450 enzyme IsaS can convert isatropolone A to isatropolone C. Therefore, blocking the isaS gene can accumulate isatropolone A. Furthermore, blocking the regulatory gene isaJ or overexpressing the regulatory gene isaF can increase the yield of isatropolone A. Therefore, the above results suggest that overexpressing the isaF gene in the first-generation strain constructed in Example 4 (the JKO strain is the strain CPCC 204095 / JKO constructed in Example 3 with the isaJ gene blocked; the SKO strain is the strain CPCC 204095 / SKO constructed in Example 4 with the isaS gene blocked) that already produces high levels of isatropolone A may further increase isatropolone A yield. This approach was used to construct a second-generation, targeted high-yield strain.

[0204] (1) Introduction of isaF overexpression plasmid into JKO and SKO strains

[0205] Using the genome of Streptomyces sp. CPCC 204095 as a template, the gene coding region of isaF was amplified, and the PCR product was ligated to a T vector and sequenced. The correct fragment was double-digested with SpeI and NotI, and recovered and purified by agarose gel electrophoresis. Using the pICLSet plasmid as a template, the strong ermE*p promoter was amplified, and the PCR product was ligated to a T vector and sequenced. The correct fragment was double-digested with XbaI and SpeI, and recovered and purified by agarose gel electrophoresis. The pSET152 plasmid was digested with XbaI and NotI, and recovered and purified by agarose gel electrophoresis. The recovered PCR product and linearized vector were ligated with T4 ligase in an appropriate ratio, and the ligation product was transformed into E. coli DH5α. The plasmid was extracted and verified by enzyme digestion. The expected plasmid was named pSET-eF. The plasmid pSET-eF was transformed into E. coli ET12567 / pUZ8002 and then introduced into the JKO and SKO strains constructed earlier in the laboratory through conjugation. The genomes of the conjugates were extracted and verified by PCR. The correctly verified strains were named JKO / eF and SKO / eF, respectively, which were the second-generation directional high-yield strains to be investigated.

[0206] (2) Analysis of fermentation product yield of recombinant strains

[0207] The original strain, high-yield first-generation strains (JKO, SKO), and directed high-yield second-generation strains (JKO / eF, SKO / eF) were inoculated on 9-cm-diameter plates containing 12 ml of M1(ISP2) solid medium, with three biological replicates each. The cells were fermented for 46 h and extracted with 24 ml of ethyl acetate. The fermentation extracts were treated and analyzed for changes in isatropolones production by HPLC-MS.

[0208] The results showed that both JKO and SKO significantly increased isotropolone A production compared to the original strain, reaching 7.3-fold and 6.8-fold, respectively, compared to the original strain under the same batch conditions. JKO / eF and SKO / eF also significantly increased isotropolone A production, increasing it by 5.1-fold and 6.3-fold, respectively, compared to the first-generation high-yield strains JKO and SKO, and approximately 40-fold that of the original strain (Figure 21).

[0209] Since a small amount of isatropolone C could still be detected in JKO / eF, while no isatropolone C was produced in SKO / eF ( FIG. 21 ), SKO / eF was a relatively more suitable second-generation strain for directed high-yield isatropolone A.

[0210] 2. Construction of a third-generation directional high-yield strain based on the second-generation directional high-yield isatropolone A strain (SKO / eF)

[0211] In addition to regulatory genes, the regulatory elements of microbial gene expression also include RNA polymerase, promoters, ribosome binding sites and terminators. The strength of the promoter and ribosome binding site is directly related to the expression level of downstream genes, which in turn affects the biosynthesis efficiency of the target metabolites. The modification and optimization of the promoter and ribosome binding site is one of the important means to achieve fine regulation of target gene expression. In the second-generation directional high-yield isatropolone A strain SKO / eF, the strong promoter that controls the expression of isaF is ermE*p in the plasmid. Replacing different promoters may increase the expression level of isaF and further increase the yield of isatropolone A. Based on this idea, the third-generation directional high-yield strain was constructed. The selected promoters include the strong promoter (containing ribosome binding site) Pgapdh reported in the literature (EL) 、PrpsL (XC) , kasO*p, Psco5768 and the promoter regions of two RNA polymerase sigma factors, Pisa2027 and Pisa4727, in this bacterium (Table 3).

[0212] Table 3 Promoter sequences

[0213] (1) Construction of promoter-replaced isaF overexpression plasmid and introduction into SKO strain

[0214] Amplification of Pgapdh (EL) 、PrpsL (XC) Six promoters, kasO*p, Psco5768, Pisa2027 and Pisa4727, were selected. The PCR products were ligated to T vectors and sequenced. The correct fragments were double-digested with XbaI and SpeI and recovered and purified by agarose gel electrophoresis. The pSET-eF plasmid was digested with XbaI and SpeI and recovered and purified by agarose gel electrophoresis. The recovered PCR products and the linearized vector were ligated with T4 ligase in an appropriate ratio. The ligation products were transformed into E. coli DH5α, and the plasmids were extracted and verified by enzyme digestion. The expected plasmids were named pSET-gF, pSET-rF, pSET-kF, pSET-5768F, pSET-2027F and pSET-4727F, respectively. The above plasmids were transformed into E. coli ET12567 / pUZ8002 and introduced into the SKO strain constructed earlier in the laboratory through conjugation transfer. The genome of the conjugants was extracted and PCR verification was performed. The correctly verified strains were named SKO / gF, SKO / rF, SKO / kF, SKO / 5768F, SKO / 2027F and SKO / 4727F, respectively, which were the third generation of directional high-yield strains to be investigated.

[0215] (2) Analysis of fermentation product yield of recombinant strains

[0216] The second-generation directional high-yield strain (SKO / eF) and six directional high-yield strains to be investigated in the third generation were inoculated on 9-cm diameter plates containing 12 ml of M1 (ISP2) solid medium, with three biological replicates each. The plates were fermented for 46 h and extracted with 24 ml of ethyl acetate. The fermentation extracts were treated and the changes in isatropolones production were analyzed by HPLC-MS.

[0217] The results showed that SKO / rF(PrpsL (XC) The SKO / kF (kasO*p promoter) and SKO / rF (kasO*p promoter) strains further increased isatropolone A production by 1.34-fold and 1.39-fold, respectively, compared to the second-generation targeted high-yield strain SKO / eF ( Figure 22 ). Therefore, SKO / rF and SKO / kF are relatively more suitable third-generation strains for targeted high-yield isatropolone A production.

[0218] Example 6: Optimization of Isatropolone A High-yield Culture Conditions

[0219] To further increase the yield of isatropolone A, we optimized the fermentation conditions of the constructed second and third generation directional high-yield strains.

[0220] (1) Screening of high-yield culture medium

[0221] Six culture media (M1, M2, M3, M4, M5, and M6) were selected, mainly derived from the fermentation medium of strains producing isatropolones reported in the literature. The second-generation directional high-yielding strain (SKO / eF) and the third-generation directional high-yielding strain (SKO / rF and SKO / kF) were inoculated on 15-cm-diameter plates containing 35 ml of the above six solid culture media, with an inoculum size of 10 9 The spores were grown on a plate for 36 h, 42 h, and 48 h, and the culture was extracted with 2 volumes of ethyl acetate. The fermentation extract was treated and analyzed for changes in isatropolone A production using HPLC-MS.

[0222] LC-MS analysis was not performed on M3 and M4 media due to their extremely low isotropolone A yields (no obvious yellow product was produced until 48 hours of fermentation). The results of the other four cultures are shown in Figure 23 . The SKO / eF, SKO / rF, and SKO / kF strains all achieved the highest isotropolone A yields when fermented in M5 medium. In another batch fermentation experiment, additional biological replicates (two zygotes each) were added, and the high-yield fermentation results of the three strains in M5 medium were reproducible (Figure 24). The strain with the highest isotropolone A production was SKO / kF, with an average yield of 946.3 mg / L. Therefore, M5 is the optimal high-yield fermentation medium.

[0223] (2) Investigation of inoculation amount and fermentation time

[0224] The effect of increasing the inoculum size on the yield of isatropolone A was then investigated. The third generation high-yield strain SKO / kF was inoculated with an inoculum size of 10 9 Spores / plate and 10 10 The spores / plate were inoculated on 15 cm diameter plates containing 35 ml of M5 solid medium and fermented for 24 to 45 h. The plates were extracted with 2 volumes of ethyl acetate, and the fermentation extracts were treated and analyzed for changes in isatropolone A production using HPLC-MS.

[0225] The results showed that the inoculum size of SKO / kF increased to 10 10 Spores / plate can advance the peak time of isatropolone A production (Figure 25), compared with 10 9The spore / plate is prepared 6 hours in advance while maintaining a high yield, with the highest average reaching 980.8 mg / L.

[0226] Example 7: Construction of a high-yielding strain of Isatropolone C

[0227] The above examples show that overexpression of isaF, isaJ and isaS can increase the yield of isatropolone C. We co-expressed the above three genes in pairs or in combination to construct the second generation of isatropolone C directed high-yield strains.

[0228] (1) Construction of multi-gene overexpression plasmids with shared promoters or individual promoters and introduction into the original strain

[0229] Construction of multi-gene overexpression plasmids with a shared promoter: The genome of Streptomyces sp. CPCC 204095 was used as a template to amplify the gene coding regions of isaF, isaJ and isaS. The PCR products were ligated to a T vector and sequenced. The correct fragments were double-digested with SpeI+NotI, NotI+EcoRV, and EcoRV+EcoRI, respectively, and recovered and purified by agarose gel electrophoresis. The ermE*p strong promoter was amplified using the pICLSet plasmid as a template. The PCR products were ligated to a T vector and sequenced. The correct fragments were double-digested with XbaI and SpeI, and recovered and purified by agarose gel electrophoresis. The above fragments were ligated into the pSET152 plasmid with T4 ligase in pairs or triplets of gene combinations and the ermE*p strong promoter fragment. The ligation products were transformed into E. coli DH5α, the plasmids were extracted, and the enzyme digestion was verified. The expected plasmids were named pLeFJ, pLeFS, pLeJS and pLeFJS, respectively.

[0230] Construction of multi-gene overexpression plasmids with separate promoters: The genome of Streptomyces sp. CPCC 204095 was used as a template to amplify the gene coding regions of isaF, isaJ and isaS. The PCR products were ligated to a T vector and sequenced. The correct fragments were double-digested with SpeI+NotI, NdeI+EcoRV and BglII+EcoRI, respectively, and recovered and purified by agarose gel electrophoresis. Using the pICLSet plasmid as a template, three ermE*p strong promoter fragments containing different restriction sites were amplified. The PCR products were ligated to a T vector and sequenced. The correct fragments were double-digested with XbaI+SpeI, NotI+NdeI and EcoRV+BglII, respectively, and recovered and purified by agarose gel electrophoresis. The above fragments were ligated to the pSET152 plasmid in pairs or triplets with the ermE*p strong promoter fragment using T4 ligase, and the ligation products were transformed into E. coli DH5α was used to extract the plasmids, and the enzyme digestion was performed to verify the plasmids. The expected plasmids were named pLeFeJ, pLeFeS, pLeJeS, and pLeFeJeS, respectively.

[0231] The above eight plasmids were transformed into E. coli ET12567 / pUZ8002 and introduced into the original strain by conjugation transfer to obtain multi-gene overexpression strains with a common promoter (623 / pLeFJ, 623 / pLeFS, 623 / pLeJS, 623 / pLeFJS) and multi-gene overexpression strains with separate promoters (623 / pLeFeJ, 623 / pLeFeS, 623 / pLeJeS, 623 / pLeFeJeS), which are the second-generation directional high-yielding strains of isatropolone C to be investigated.

[0232] (2) Analysis of fermentation product yield of recombinant strains

[0233] The control strain (control) transformed with the empty plasmid pSET152 and the above eight recombinant strains (623 / pLeFJ, 623 / pLeFS, 623 / pLeJS, 623 / pLeFJS, 623 / pLeFeJ, 623 / pLeFeS, 623 / pLeJeS, and 623 / pLeFeJeS) were inoculated on 9-cm-diameter plates containing 12 ml of M1(ISP2) solid medium, with three biological replicates each. The cells were fermented for 46 h and extracted with 24 ml of ethyl acetate. The fermentation extracts were treated and the changes in isatropolones production were analyzed by HPLC-MS.

[0234] The results (Figure 26) show that under identical fermentation conditions, the six strains overexpressing the isaS gene significantly reduced isatropolone A production (by 85% to 100%) compared to the control strain, making them ideal strains for directed isatropolone C production. The strains co-overexpressing all three genes (623 / pLeFJS and 623 / pLeFeJeS) showed the greatest increases in isatropolone C production, reaching 2.6-fold and 2.2-fold, respectively, compared to the control strain. These results indicate that 623 / pLeFJS and 623 / pLeFeJeS are relatively suitable strains for directed high-yield isatropolone C production, with little difference in the effects of using a shared promoter versus a single promoter.

[0235] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

Claims

1. A biosynthetic gene of isatropolones, a tropone compound, wherein the isatropolones are isatropolone A and / or isatropolone C; the gene is: (1) isaF, 891 bp in length, encoding 296 amino acids of IsaF protein Its gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2; or (2) isaJ, 792 bp in length, encoding the 263 amino acid IsaJ protein Its gene sequence is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4; or (3) isaS, 1230 bp in length, encoding the 409 amino acid IsaS protein The gene sequence is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

6.

2. The isa gene cluster of Streptomyces sp. CGMCC No. 15540 comprising the biosynthetic gene according to claim 1, wherein the gene cluster comprises the isaF, isaJ and isaS genes; Preferably, the position of the isa gene cluster in the genome of Streptomyces sp. CPCC 204095 is: 6,151,891-6,159,911nt+6,451,081-6,476,781nt.

3. Use of the gene according to claim 1 or the gene cluster according to claim 2 in the biosynthesis of isatropolones, a tropolone compound; preferably, the isatropolones are isatropolone A and / or isatropolone C.

4. Use of the protein encoded by the gene according to claim 1 in the biosynthesis of isatropolones, a tropolone compound; preferably, the isatropolones are isatropolone A and / or isatropolone C.

5. Use of the biosynthetic gene and / or the protein encoded by the biosynthetic gene according to claim 1 in regulating the production of isatropolones in a host bacterium, wherein the regulation refers to: (1) overexpressing the isaF gene and / or its encoded protein to increase the biosynthesis of isatropolones; and / or (2) blocking the isaJ and / or isaS genes and / or their encoded proteins to increase the biosynthesis of isatropolone A; and / or (3) overexpressing the isaJ and / or isaS genes and / or their encoded proteins to increase the biosynthesis of isatropolone C; Preferably, the regulation refers to, (1) while blocking the isaJ gene or the isaS gene, overexpressing the isaF gene and / or its encoded protein to increase the biosynthesis of isotropolone A; more preferably, using PrpsL (XC) The promoter or kasO*p promoter drives the overexpression of isaF; The PrpsL (XC) The sequence of the promoter is shown in SEQ ID NO.9, and the sequence of the kasO*p promoter is shown in SEQ ID NO.10; or (2) overexpression: a) isaF, isaS genes and / or their encoded proteins, or b) isaJ, isaS genes and / or their encoded proteins, or c) isaF, isaJ, isaS genes and / or their encoded proteins; To increase the biosynthesis of isatropolone C.

6. The use according to claim 5, characterized in that: The host bacteria include, but are not limited to, Streptomyces and Escherichia coli; preferably Streptomyces sp. CGMCC No. 15540.

7. A genetically engineered strain for directional high production of isatropolone A, wherein the strain is: Streptomyces sp. CGMCC No. 15540 is used as a host, the host isaJ gene is blocked or the host isaS gene is blocked, and PrpsL is used (XC) The strains overexpressing isaF were driven by the promoter or the kasO*p promoter.

8. A method for producing isotropolone A by fermenting the strain directed to produce high isotropolone A according to claim 7, characterized in that: The host bacteria is cultured and fermented in M5 medium to obtain the isotropolone A; the formula of the M5 medium is: 2.5% malt extract, 0.4% glucose, 0.4% yeast extract, 0.6% soy powder, 2% agar, and deionized water. Preferably, when fermenting the directional high-yield isotropolone A strain, the inoculum size is 10 10 The spores were inoculated into 35 ml of M5 solid medium and fermented for 24 to 45 h.

9. A genetically engineered strain for directed high production of isatropolone C, the strain being: a strain overexpressing isaF and isaS genes, or a strain overexpressing isaJ and isaS genes, or a strain overexpressing isaF, isaJ and isaS genes, using Streptomyces sp. CGMCC No. 15540 as a host.

10. A biosynthetic gene of the tropone compound isatropolones, wherein the gene is: (1) isaF, 891 bp in length, encoding 296 amino acids of IsaF protein Its gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2; or (2) isaJ, 792 bp in length, encoding the 263 amino acid IsaJ protein Its gene sequence is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4; or (3) isaS, 1230 bp in length, encoding the 409 amino acid IsaS protein The gene sequence is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

6.

11. Use of the genetically engineered strain with directed high production of isatropolone C according to claim 9 in the preparation of a preparation for preventing and treating potato scab.

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