A genetically engineered bacterial strain for directional production of po tems compounds and application thereof

Abstract

The application discloses a genetically engineered strain for producing PoTeMs compounds and application thereof. The genetically engineered strain 306A is characterized in that the genetically engineered strain 306A is obtained by deleting an interfering gene fragment in a mutant strain ABC306 of Streptomyces sp. ZJ306 through a CRISPR-Cas9 gene editing method, and the interfering gene fragment is located between ikaA and orf(-1). The application further knocks out the gene fragment (about 11.1 kb, containing the ikaD gene) located between ikaA and Orf(-1) in the previously constructed engineered strain ABC306 (Chinese invention patent ZL202010086255.X) through a CRISPR-Cas9 method, and only the ikaA for producing a common polyene precursor 1 is reserved, so that a new chassis host 306A is obtained, and the compound polyene precursor compound polyene tetramate precursor 1 can be efficiently prepared from fermentation culture of the chassis host 306A.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of industrial microorganisms, and particularly relates to a preparation method of a genetically engineered strain for directional production of polycyclic tetramate macrolactam (PoTeM) compounds and application thereof. BACKGROUND

[0002] Polycyclic tetramate macrolactams (PoTeMs) have a unique chemical structure, in which a pyrrolidine-2,4-dione (tetramate) and a fused polycyclic carbon ring structure unit are fused in the macrolactam ring. According to the type of polycyclic carbon ring, PoTeMs are mainly divided into 5 / 6 / 5 ring type, 5 / 5 / 6 ring type and 5 / 5 ring type, wherein the 5 / 5 / 6 ring and 5 / 5 ring type polycyclic carbon rings are reported to have various cyclization and stereochemical types. Pharmacological studies have shown that PoTeMs have antibacterial, antifungal, antiviral, antitumor and antiprotozoal activities, and have great potential for application in the fields of agriculture and medicine. Among them, 5 / 5 / 6 type HSAF (dihydromaltophilin) has been developed as a new biological pesticide, which can inhibit plant pathogenic fungi by specifically interfering with the synthesis of pathogenic sphingolipids, has no side effects on mammals and plants, and is expected to become a new generation of fungicides or antifungal drugs; 5 / 5 type pactamide C and aburabubolactam E, 5 / 5 / 6 type pactamide A and maltophilins, and 5 / 6 / 5 type ikarugamycin all show tumor cell toxicity activity and selectivity similar to that of positive drug cisplatin; ikarugamycin also has significant antiprotozoal and methicillin-resistant Staphylococcus aureus (MRSA) inhibitory activity. Due to the complex chiral centers and functional groups of PoTeMs, especially the complex stereochemical polycyclic carbon ring, the chemical total synthesis of PoTeMs has many steps and low yield; at the same time, due to the problems of silencing of the PoTeMs biosynthesis gene cluster and production of only one PoTeM polycyclic carbon ring skeleton by a single strain in the fermentation production of wild-type strains, the high yield and directional production of PoTeM-type drug source compounds are limited.

[0003] The biosynthesis of PoTeMs compounds is characterized by the following: it begins with the formation of the same polyene precursor catalyzed by PKS / NRPS, followed by the formation of 5-, 5 / 5-, and 5 / 6-ring systems from the linear polyene precursor via 1-3 flavin cyclases, and further catalyzed by an alcohol dehydrogenase to form 5 / 5 / 6 and 5 / 6 / 5-ring systems from the internal 6- or 5-membered ring. Based on this concise and conserved biosynthetic characteristic, different types of PoTeMs can be custom-produced through combinations of PoTeMs biosynthetic gene clusters with different multi-scaffolds. However, currently, there is a lack of efficient and clean host systems for the heterologous expression of PoTeMs gene clusters. Previously, we used ikarugamycin knockout as a production host. Streptomyces cyclase gene in sp. ZJ306 ikaB and ikaC A chassis host, ΔBC306, was constructed, and downstream gene combinations demonstrated that the biosynthesis of different cyclic PoTeMs originates from the same polyene precursor. However, the ΔBC306 chassis host retained cytochrome P450 enzyme genes. ikaD This will cause unnecessary oxidative modification of the PoTeMs biosynthetic intermediates. Summary of the Invention

[0004] The first objective of this invention is to provide a genetically engineered strain 306A for the targeted production of polyene precursors for the biosynthesis of PoTeMs, which is derived by deleting Streptomyces via a CRISPR-Cas9 method. Streptomyces Genetically engineered strain 306A was obtained from the interfering gene fragment of the mutant strain ΔBC306 of sp. ZJ306, wherein the interfering gene fragment is located at... ikaA and orf(-1) Between, size 11.158kb, as attached Figure 2 As shown.

[0005] Preferably, using endonuclease Bam HI and Hin CRISPR-Cas9 knockout plasmid pCSG8006 was digested with dIII to obtain two fragments, pCas9F1 and pCas9F2. The fragments sgRNA, UHA, DHA, pCas9F1, and pCas9F2 were then ligated end-to-end to obtain a clean chassis host 306A CRISPR-Cas9 knockout plasmid pCSG7021. Plasmid pCSG7021 was transformed into *E. coli*. E. coli Obtained from ET12567 / pUZ8002 E. coli ET12567 / pUZ8002 / pCSG7021, serving as the donor bacteria for conjugation transfer, ikaB and ikaC Clean chassis host of double gene deletion mutant StreptomycesThe sp. ΔBC306 is used as a receptor strain for conjugation transfer, and a genetically engineered strain 306A is obtained by conjugation transfer, wherein the nucleotide sequences of the sgRNA, UHA and DHA are shown in SEQ ID NO. 1, 2 and 3.

[0006] The second object of the present application is to provide an application of the genetically engineered strain 306A in preparing a PoTeM compound, polyene tetramate precursor 1.

[0007] The structure of the PoTeM compound, polyene tetramate precursor 1 is shown in formula (1).

[0008] (1)

[0010] The third object of the present application is to provide a production strain 306A. ptmB1B2C The gene fragment inserted into the genetically engineered strain 306A is ptmB1B2C The production strain 306A is obtained. ptmB1B2C The nucleotide sequence of the gene fragment ptmB1B2C is shown in SEQ ID NO. 4.

[0011] Preferably, the gene fragment ptmB1B2C and the promoter gene fragment erm E*p are connected to the vector pSET152 linearized by restriction endonuclease Xba I / Bcu I to obtain plasmid pCSG7022. E. coli The plasmid pCSG7022 is transformed into E. coli E. coli ET12567 / pUZ8002 to obtain ptmB1B2C ET12567 / pUZ8002 / pCSG7022, which is used as a donor strain for conjugation transfer, and the genetically engineered strain 306A is used as a receptor strain for conjugation transfer to obtain the production strain 306A. erm The nucleotide sequence of the promoter gene fragment E*p is shown in SEQ ID NO. 8.

[0012] The fourth object of the present application is to provide a method and application of the production strain 306A. ptmB1B2C in preparing 5-membered ring, 5 / 5 ring and 5 / 5 / 6 ring PoTeM compounds of the pactamides type.

[0013] The structural formula of the 5-membered ring, 5 / 5 ring and 5 / 5 / 6 ring pataciamide PoTeM compound is shown as compounds 2-4 in formula (2);

[0014] (2)

[0016] A fifth object of the present application is to provide a production strain 306A: flaB1B2CD , which is a genetically engineered strain 306A inserted with a gene fragment flaB1B2CD to obtain the production strain 306A: flaB1B2CD , wherein the nucleotide sequence of the gene fragment flaB1B2CD is shown as SEQ ID NO. 9;

[0017] The genetically engineered strain 306A is obtained by using endonuclease Bam HI and Hin dIII to cut the CRISPR-Cas9 knockout plasmid pCSG8006 into two fragments pCas9F1 and pCas9F2, connecting the fragments sgRNA, UHA, DHA, pCas9F1 and pCas9F2 end to end to obtain the CRISPR-Cas9 knockout plasmid pCSG7021 of the clean chassis host 306A, transforming the plasmid pCSG7021 into Escherichia coli E. coli ET12567 / pUZ8002 to obtain E. coli ET12567 / pUZ8002 / pCSG7021, which is used as a donor strain for conjugation transfer, and the genetically engineered strain 306A is used as a receptor strain for conjugation transfer to obtain the production strain 306A, wherein the nucleotide sequences of sgRNA, UHA and DHA are shown as SEQ ID NO. 1, 2 and 3. ikaB ikaC The double gene deletion mutant strain clean chassis host Streptomyces sp.△BC306 is used as a receptor strain for conjugation transfer, and the genetically engineered strain 306A is used as a receptor strain for conjugation transfer to obtain the production strain 306A, wherein the nucleotide sequences of sgRNA, UHA and DHA are shown as SEQ ID NO. 1, 2 and 3.

[0018] Preferably, the gene fragment flaB1B2CD and the promoter gene fragment erm E*p are connected to the vector pSET152 linearized by restriction endonuclease Nde I / Bcu I to obtain the plasmid pCSG7026, and the plasmid pCSG7026 is transformed into Escherichia coli E. coli ET12567 / pUZ8002 to obtain E. coli ET12567 / pUZ8002 / pCSG7026, which is used as a donor strain for conjugation transfer, and the genetically engineered strain 306A is used as a receptor strain for conjugation transfer to obtain the production strain 306A.​flaB1B2CD , the promoter gene fragment erm The nucleotide sequence of E*p is shown as SEQ ID NO. 8.

[0019] A sixth object of the present application is to provide a production strain 306A: flaB1B2CD The method and application in preparing 5 / 5 and 5 / 5 / 6 ring type PoTeM compounds with oxidative modification at C14 position;

[0020] The structural formula of the 5 / 5 and 5 / 5 / 6 ring type PoTeM compounds with oxidative modification at C14 position is shown as compound 5-9 in formula (3);

[0021] (3)

[0023] The original strain of the present application Streptomyces sp. Streptomyces Streptomyces sp. ZJ306 is a marine Streptomyces from the sediment of Zhujiang River Estuary, which contains ikarugamycin biosynthesis gene cluster (Genbank accession number: KF954540) and can efficiently produce 5 / 6 / 5 ring type ikarugamycin PoTeM compounds. The ikarugamycin biosynthesis gene cluster has been granted patent ZL20141020626.6. The biosynthesis principle of PoTeM compounds such as ikarugamycin shows that PoTeM compounds of different types of polycyclic rings are derived from the same polyene precursor 1 formed by PKS / NRPS catalyzed biosynthesis (reference: Zhang, G.; Zhang, W.; Zhang, Q.; Shi, T.; Ma, L.; Zhu, Y.; Li, S.; Zhang, H.; Zhao, Y. L.; Shi, R.; Zhang, C. Mechanistic insights into polycycle formation by reductive cyclization in ikarugamycin biosynthesis. Angew Chem Int Ed Engl 2014, 53 , 4840-4844. Jin, H.; Zhang, W.; Zhang, G.; Zhang, L.; Liu, W.; Zhang, C. Engineered Biosynthesis of 5 / 5 / 6 Type Polycyclic Tetramate Macrolactams in an Ikarugamycin (5 / 6 / 5 Type)-Producing Chassis.Org Lett 2020, 22 , 1731-1735.), via 1-3 flavin cyclase catalyzed linear polyene precursor 1 to form 5-type, 5 / 5-type and 5 / 6-type ring systems, 1 ethanol dehydrogenase further catalyzes the formation of 5 / 5 / 6-type and 5 / 6 / 5-type ring systems. This simple, conservative biosynthetic logic is the theoretical basis of the present application.

[0024] The present application further knocks out the gene fragment (about 11.1 kb, containing ikaD gene) between ikaA and Orf(-1) in the previously constructed engineering strain ΔBC306 (Chinese invention patent ZL202010086255.X) by CRISPR-Cas9 method, only retaining ikaA producing common polyene precursor 1, to obtain a new chassis host 306A, from which the above-mentioned compound polyene tetramate precursor (1) Figure 3 can be efficiently prepared. The cytochrome P450 enzyme gene ikaD left in the original ΔBC306 chassis host will cause unnecessary oxidative modification of the PoTeMs synthesized by gene combination in the next step (reference: Jiang, P.; Jin, H. B.; Zhang, G. T.; Zhang, W. J.; Liu, W.; Zhu, Y. G.; Zhang, C. S.; Zhang, L. P. A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates. Angew Chem Int Ed 2023, 62 . e202310728) interferes with the directed biosynthesis of PoTeM compounds. The new chassis host 306A solves this problem by deleting the gene fragment where ikaD is located. By sequentially assembling flavin cyclase, ethanol dehydrogenase and cytochrome P450 enzyme from different types of PoTeM biosynthetic gene clusters into the genetically engineered strain 306A for heterologous expression, different types of PoTeM compounds can be produced. In the application cases of the genetically engineered strain 306A listed in the present application, the flavin cyclase, ethanol dehydrogenase and cytochrome P450 enzyme from different types of PoTeM biosynthetic gene clusters are sequentially introduced. S. pactumSCSIO 02999 ptm The first flavin cyclase gene in the gene cluster ptmB2, Second flavin cyclase gene ptmB1 Ethanol dehydrogenase gene ptmC 5-membered ring, 5 / 5 ring and 5 / 5 / 6 ring pactamide PoTeM compounds can be obtained respectively. Figure 4 ); sequentially introduce actinomycetes from the South China Sea Streptomyces flavogriseus PoTeMs gene cluster in SCSIO 40032 fla The first flavin cyclase gene in (Genbank accession number: PP539903) flaB2, Second flavin cyclase gene flaB1 Ethanol dehydrogenase gene flaC and P450 enzyme gene flaD , can respectively obtain pactamide E, 5 / 5 ring type and 5 / 5 / 6 ring type PoTeM compounds with oxidation modification at C14 position 5-9 ( Figure 5 The structure of the compounds was determined based on HPLC comparison with standards and characteristic UV absorbance values, combined with our previous research experience on this series of compounds in patents and papers.

[0025] This invention also provides the use of Streptomyces flavogriseus SCSIO 40032 originates from the PoTeMs biosynthetic gene cluster fla Application of cytochrome P450 oxidase FlaD (Genbank accession number: PP539903) in the targeted production of 5 / 5-cyclic and 5 / 5 / 6-cyclic PoTeM compounds with oxidative modification at C14.

[0026] The nucleotide sequence or at least a portion thereof provided by this invention can be modified or mutated in vivo or in vitro, including insertion, substitution or deletion, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutation, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical reagents, etc.

[0027] Cloned genes containing the nucleotide sequences or at least a portion thereof provided by this invention can be expressed in exogenous hosts using suitable expression systems to obtain the corresponding enzymes or other substances with higher bioactivity or yield. These exogenous hosts include Escherichia coli, Streptomyces, Micromonospora, Pseudomonas, Bacillus, yeast, plants, and animals.

[0028] The amino acid sequence provided by this invention can be used to isolate the desired protein and can be used for antibody preparation.

[0029] The DNA fragment or gene can be used to construct a mutant strain for directed production of PoTeMs or derivatives thereof, and the present application provides a pathway for directed use in genetically engineered microorganisms.

[0030] The starting strain of the present application is Streptomyces sp. Streptomyces sp. ZJ306 was preserved in China Center for Type Culture Collection (CCTCC) on March 13, 2014, the address of which is Wuhan University, Wuhan, China, and the preservation number thereof is CCTCC NO. M2014081. The strain is disclosed in the patent with the patent number CN201410120626.6 and the title of a biosynthetic gene cluster of pseurotin and its application. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a schematic diagram of plasmid pCSG7021.

[0032] Figure 2 is a clean chassis host Streptomyces is a schematic diagram of construction and verification of Streptomyces sp. 306A. (A) is a schematic diagram of the genome of 306A derived from △BC306; (B) is a schematic diagram of gene knockout of △BC306; (C) is a verification of the genotype of 306A using primers 306A-UHA-F and 306A-DHA-R, and a fragment with a size of 2040 bp represents the sizes of the homologous arms UHA and DHA. The fragment is detected in 306A, which represents that the gene fragment located between ikaA and Orf (-1) is successfully knocked out; (D) is a verification of the genotype of 306A using primers ikaD-28a-F / R. The fragment has a size of 1262 bp, and the fragment is not detected in 306A, which represents that ikaD is successfully knocked out.

[0033] Figure 3 is a clean chassis host Streptomyces is a high performance liquid chromatogram detection result of a fermentation extract sample of 306A of Streptomyces sp. 306A.

[0034] Figure 4 is a high performance liquid chromatogram detection result of a fermentation extract sample of a pactamide A production strain.

[0035] Figure 5 is a high performance liquid chromatogram detection result of a fermentation extract sample of a 10- epi -FI-2 production strain. DETAILED DESCRIPTION

[0036] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0037] Example 1: Construction of a clean chassis host StreptomycesS. sp. 306A (i.e. ΔBC306::pCSG7021, hereinafter referred to as 306A)

[0038] 1. Construction of conjugative donor strain

[0039] Three nucleic acid fragments sgRNA, UHA and DHA (see SEQ ID NO. 1, 2, 3 of the sequence listing) were amplified from the genome of S. sp. ZJ306 by PCR method using three pairs of primers 306A-sgRNA-F / R, 306A-UHA-F / R and 306A-DHA-F / R (primer sequences are shown in Table 1). Streptomyces Bam HI and Hin dIII to cut our published CRISPR-Cas9 knockout plasmid pCSG8006 (reference: Tan, B.; Zhang, Q.; Zhu, Y.; Jin, H.; Zhang, L.; Chen, S.; Zhang, C., Deciphering Biosynthetic Enzymes Leading to 4-Chloro-6-Methyl-5,7-Dihydroxyphenylglycine, a Non-Proteinogenic Amino Acid in Totopotensamides. ACS Chem. Biol. 2020, 15 , 766-773.) to get two fragments pCas9F1 (191 bp in size) and pCas9F2 (12.5 kb in size). Subsequently, the fragments sgRNA, UHA, DHA, pCas9F1 and pCas9F2 were ligated end to end to get the CRISPR-Cas9 knockout plasmid pCSG7021 for the clean chassis host 306A (plasmid map is shown in Figure 1 E. coli ET12567 / pUZ8002 to get E. coli ET12567 / pUZ8002 / pCSG7021, i.e. as the conjugative donor strain.

[0040] Table 1. Primers used in the present application

[0041]

[0042] 2. Obtaining clean chassis host through conjugation Streptomyces S. sp. 306A ​​

[0043] Using our previously constructed ikaB and ikaC The double-gene deletion mutant strain ABC306 was used as the recipient strain for conjugation (Reference: Jin, H.; Zhang, W.; Zhang, G.; Zhang, L.; Liu, W.; Zhang, C., Engineered biosynthesis of 5 / 5 / 6 type Polycyclic Tetramate Macrolactams in an Ikarugamycin (5 / 6 / 5 Type)-Producing chassis. Org. let. 2020, 22 , 1731-1735.). The conjugation process was described as follows: ikaB and ikaC The double-gene deletion mutant strain ABC306 was streaked on SFM plates (Formula: Soybean flour 15 g, Mannitol 15 g, Sodium chloride 2 g, Calcium carbonate 5 g, add water to 1 L, pH 7.4, preparation: dissolve each component of the medium in water, stir to dissolve, sterilize, and then obtain) for 5-7 days. The spores that grew were collected in TSB medium (Formula: Take 100 g of purchased TSB medium powder, add water to 1 L, pH 7.4, preparation: dissolve the TSB medium powder in water, stir to dissolve, sterilize, and then obtain) with a sterile cotton swab. The spores were dispersed by vortexing. The mycelium and spores were separated by filtration. The spores were suspended in 5 mL of TSB medium, heat shocked at 50°C for 10 min, and then germinated at 28°C for 2 h as the recipient strain for conjugation. The donor strain E. coli ET12567 / pUZ8002 / pCSG7021 was grown in 50 mL of LB liquid medium containing 50 pg / mL apramycin at 37°C to OD 600about 0.8. The above-mentioned 400 μL of acceptor bacteria and 100 μL of donor bacteria were mixed uniformly and spread on ISP4 solid medium without any antibiotic (medium formula: soluble starch 10 g, bacteriological peptone 1 g, yeast extract powder 0.5 g, potassium phosphate dibasic 1 g, magnesium sulfate 1 g, ammonium sulfate 2 g, sodium chloride 1 g, sea salt 30 g, calcium carbonate 2 g, agar powder 18 g, add water to 1 L, pH 7.4, preparation: dissolve each component of the medium in water, stir to dissolve, sterilize, and then obtain). After blowing dry, the plate was cultured at 28°C for 18-20 h. Then the plate was taken out and covered with water containing antibiotics, and the final concentration of the antibiotics was 50 μg / mL apramycin, 100 μg / mL trimethoprim, and 25 μg / mL thiostrepton. After blowing dry, the plate was placed in a 28°C incubator and cultured for 5-7 days, and then observed. When colonies grew on the conjugation transfer plate, they were transferred to SFM plates containing 15 μg / mL 5-fluorocytosine and 15 μg / mL 5-fluorocytosine at the same time to detect whether the pCSG7021 plasmid was lost. After being cultured at 28°C for 3 days, single colonies that grew only on the SFM plate containing 15 μg / mL 5-fluorocytosine were selected as candidate strains for genotype verification. Genomic DNA of each candidate strain was extracted, and positive clones were obtained by PCR detection using primers 306A-UHA-F and 306A-DHA-R (Table 1) Figure 2 C, with a fragment size of 2040 bp), and the positive clone also met the condition that no ikaD gene fragment band (D, with a fragment size of 1262 bp) could be detected using primers ikaD-28a-F / R, that is, a clean chassis host Figure 2 sp. 306A was obtained. Streptomyces

[0044] 3. Fermentation detection of clean chassis host Streptomyces sp. 306A

[0045] The strain of Streptomyces sp. 306A that grew on the SFM solid plate was selected and cultured in 50 ml of A1 medium (soluble starch 10 g, yeast extract 4 g, bacterial peptone 2 g, sea salt 10 g, add water to 1 L, pH 7.0-7.4. Preparation: dissolve each component of the medium in water, stir to dissolve, sterilize, and then obtain) at 28°C under a shaking condition of 200 rpm for 5 days to obtain a fermentation broth. 5 ml of the fermentation broth was ultrasonically extracted with 5 ml of butanone, extracted, and centrifuged (3500 r•min -1 ​, 8 min), 1.6 ml of the upper organic solvent phase was taken and evaporated to dryness to obtain the fermentation extract of 306A, and 120 μΐ of methanol was added to dissolve the fermentation extract of 306A to obtain the fermentation extract sample of 306A. The fermentation extract sample of 306A was detected by high performance liquid chromatography. The high performance liquid chromatography (HPLC) conditions were as follows: the chromatographic column was phenomex 150 x 4.6 mm (SphereClone SAX), the mobile phase included phase A and phase B, the mobile phase A phase: 10% (volume fraction) of acetonitrile + 0.08% (volume fraction) of formic acid, the solvent was water, the mobile phase B phase: 90% (volume fraction) of acetonitrile, the solvent was water; the injection program: 0-18 min, the mobile phase ratio A phase / B phase (volume ratio): 95:5-0:100, 18-23 min, the mobile phase ratio A phase / B phase (volume ratio): 0:100, 23-25 min, the mobile phase ratio A phase / B phase (volume ratio): 0:100-95:5, 25-30 min, the mobile phase ratio A phase / B phase (volume ratio): 95:5, the detection wavelength was 360 nm, and the flow rate was 1 mL / min. The high performance liquid chromatogram of the fermentation extract sample of 306A is shown in FIG. 2. Figure 3

[0046] Example 2: Construction of the active metabolite pactamide A production strain (306A::pSET152- ermE p*- ptmB1B2C )

[0047] 1. Construction of the conjugation transfer donor strain

[0048] A pair of primers 152-ptmB1B2C-F / R (the primer sequences are shown in Table 1) were used to amplify the gene fragment (SEQ ID NO. 4) from the genome of SCSIO 02999 (disclosed in CN106434702, a patent for the biosynthetic gene cluster of pactamides and its application) by PCR method. Streptomyces pactum The gene fragment (SEQ ID NO. 4) contains the sequences of three genes (SEQ ID NO. 5, 6, 7), see SEQ ID NO. 5, 6, 7 of the sequence listing), and a pair of primers ErmF / R were used to amplify the vector pPWW50 (reference: M. Doumith, P. Weingarten, U. F. Wehmeier, K. Salah-Bey, B. Benhamou, C. Capdevila, J. M. Michel, W. Piepersberg, M. C. Raynal, ptmB1B2C ptmB1B2C ptmB1, ptmB2 ptmC Mol. Gen. Genet. ​​​​​Promoter gene fragment was amplified in the method described in the reference (Chen, Y.; Zhang, Y.; Zhang, X.; Liu, Z. J. Biol. Chem. 2000, 264, 477-485.) erm E*p (see SEQ ID NO. 8 of the sequence listing). Subsequently, the fragment ptmB1B2C and erm E*p was ligated to the vector pSET152 (reference: Bierman, M.; Logan, R.; O'Brien, K.; Seno, E. T.; Rao, R. N.; Schoner, B. E., Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Xba I / Bcu I linearized by restriction enzyme Gene 1992, 116, 43-9.) to obtain plasmid pCSG7022 (pSET152- ermE p*- ptmB1B2C ). The plasmid pCSG7022 was transformed into Escherichia coli E. coli ET12567 / pUZ8002 to obtain E. coli ET12567 / pUZ8002 / pCSG7022, i.e. as the donor strain for conjugal transfer.

[0049] 2, Obtain the pactamide A producing strain (306A::pSET152- ermE p*- ptmB1B2C )

[0050] 306A as the recipient strain for conjugal transfer. The conjugal transfer process is described as follows: 306A was streaked on SFM medium (medium formula: soybean flour 15 g, mannitol 15 g, sodium chloride 2 g, calcium carbonate 5 g, add water to 1 L, pH 7.4, preparation: dissolve each component of the medium in water, stir to dissolve, sterilize, and obtain) for 5-7 days, and the spores grown were collected in TSB medium (formula: take purchased TSB medium powder 100 g, add water to 1 L, pH 7.4, preparation: dissolve TSB medium powder in water, stir to dissolve, sterilize, and obtain) with a sterile cotton swab, vortexed to disperse the spores. Filtration separated the mycelium and spores, and the spores were suspended in 5 mL of TSB medium, heat shocked at 50°C for 10 min, and then germinated at 28°C for 2 h as the recipient strain for conjugal transfer. The donor strain E. coliET12567 / pUZ8002 / pCSG7022 was grown to OD in 50 mL of LB liquid medium containing 50 µg / mL apopramycin at 37°C. 600 The value was approximately 0.8. 400 µL of the recipient bacteria and 100 µL of the donor bacteria were mixed thoroughly and spread onto ISP4 solid medium (medium formulation: 10 g soluble starch, 1 g bacteriological peptone, 0.5 g yeast extract, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 2 g ammonium sulfate, 1 g sodium chloride, 30 g sea salt, 2 g calcium carbonate, 18 g agar powder, diluted to 1 L with water, pH 7.4; preparation: dissolve all components in water, stir to dissolve, sterilize) without antibiotics. After drying, the medium was incubated at 28°C for 18-20 h. Then, the plate was removed and covered with water containing antibiotics (final antibiotic concentration: 50 µg / mL apopramycin, 100 µg / mL trimethoprim). After drying, the plate was placed in a 28°C incubator and incubated for 5-7 days before observation. After colonies grew on the conjugation transfer plate, they were simultaneously transferred to an SFM plate containing 50 µg / mL apopramine using a sterile toothpick to check whether the pCSG7022 plasmid had been introduced. After incubation at 28°C for 3 days, single colonies growing on SFM plates containing 50 µg / mL apopramine were selected as candidate strains for genotyping. Genomic DNA was extracted from each candidate strain, and positive clones were obtained by PCR detection using primers ErmF and 152-ptmB1B2C-R (Table 1), thus obtaining the pactamide A producing strain (306A::pSET152-). ermE p*- ptmB1B2C ).

[0051] 3. Pactamide A producing strain (306A::pSET152- ermE p*- ptmB1B2C Fermentation detection

[0052] Select pactamide A producing strains (306A::pSET152-) grown on SFM solid plates containing 50 µg / mL apopramine. ermE p*- ptmB1B2C The fermentation broth was prepared by dissolving the components of the culture medium in 50 ml of A1 medium (10 g soluble starch, 4 g yeast extract, 2 g bacterial peptone, 10 g sea salt, diluted to 1 L with water, pH 7.0-7.4. Preparation: Dissolve all components of the culture medium in water, stir to dissolve, sterilize, and then culture at 28℃ and 200 rpm for 5 days). 5 ml of the fermentation broth was then extracted with 5 ml of butanone by sonication and centrifugation (3500 r•min). -1The fermentation extract of the pactamide A-producing strain was obtained by rotary evaporation of 1.6 ml of the upper organic solvent phase (8 min) and dissolved in 120 µl of methanol. The fermentation extract sample was then analyzed by high performance liquid chromatography. High-performance liquid chromatography (HPLC) conditions: chromatographic column: Phenomix 150×4.6mm (SphereClone SAX); mobile phases: Phase A: 10% (v / v) acetonitrile + 0.08% (v / v) formic acid, solvent: water; Phase B: 90% (v / v) acetonitrile, solvent: water; injection program: 0-18 min, mobile phase ratio A / B (v / v): 95:5-0:100; 18-23 min, mobile phase ratio A / B (v / v): 0:100; 23-25 ​​min, mobile phase ratio A / B (v / v): 0:100-95:5; 25-30 min, mobile phase ratio A / B (v / v): 95:5; detection wavelength: 360 nm; flow rate: 1 mL / min. The HPLC chromatogram of the fermentation extract sample from the pactamide A producing strain is shown below. Figure 4 As shown.

[0053] Example 3: Construction of active metabolite 10- epi -FI-2 production strain (306A::pSET152- ermE p*- flaB1B2CD )

[0054] 1. Construction of conjugation transfer donor strains

[0055] Using primers 152-flaB1B2-F and 152-flaB1B2CD-R (primer sequences are shown in Table 1), PCR was performed to extract... Streptomyces falvogriseus Gene fragments amplified from the SCSIO 40032 genome flaB1B2CD (SEQ ID NO. 9) (gene fragment) flaB1B2CD Contains four genes flaB1 , flaB2 , flaC and flaD sequence ， See SEQ ID NOs 10, 11, 12, and 13 in the sequence listing. The fragments were then cloned using a multi-fragment one-step cloning kit (manufactured by Nanjing Novizan Biotechnology Co., Ltd.). flaB1B2CD Linked with restriction endonuclease Nde I / Bcu I linearized, with erm The plasmid pCSG7026 (pSET152-) was obtained on the vector pSET152 containing the E*p promoter. ermE p*-flaB1B2CD ). Plasmid pCSG7026 was transformed into E. coli ET12567 / pUZ8002 E. coli ET12567 / pUZ8002 E. coli ET12567 / pUZ8002 / pCSG7026, i.e. as the donor strain for conjugation.

[0056] 2. Obtaining 10- epi Production strain of FI-2 (306A::pSET152- ermE p*- flaB1B2CD )

[0057] 306A as the recipient strain for conjugation. The conjugation process is described in detail as follows: 306A was streaked on SFM medium (medium formula: soybean flour 15 g, mannitol 15 g, sodium chloride 2 g, calcium carbonate 5 g, add water to 1 L, pH 7.4, preparation: dissolve each component of the medium in water, stir to dissolve, sterilize, and then obtain) for 5-7 days, and the spores that grew were collected in TSB medium (formula: take 100 g of purchased TSB medium powder, add water to 1 L, pH 7.4, preparation: dissolve the TSB medium powder in water, stir to dissolve, sterilize, and then obtain) with a sterile cotton swab, vortexed to disperse the spores. The mycelium and spores were separated by filtration, the spores were suspended in 5 mL of TSB medium, heat shocked at 50°C for 10 min, and then germinated at 28°C for 2 h as the recipient strain for conjugation. The donor strain E. coli ET12567 / pUZ8002 / pCSG7026 was grown in 50 mL of LB liquid medium containing 50 μg / mL apramycin at 37°C to OD 600The value was approximately 0.8. 400 µL of the recipient bacteria and 100 µL of the donor bacteria were mixed thoroughly and spread onto ISP4 solid medium (medium formulation: 10 g soluble starch, 1 g bacteriological peptone, 0.5 g yeast extract, 1 g dipotassium hydrogen phosphate, 1 g magnesium sulfate, 2 g ammonium sulfate, 1 g sodium chloride, 30 g sea salt, 2 g calcium carbonate, 18 g agar powder, diluted to 1 L with water, pH 7.4; preparation: dissolve all components in water, stir to dissolve, sterilize) without antibiotics. After drying, the medium was incubated at 28°C for 18-20 h. Then, the plate was removed and covered with water containing antibiotics (final antibiotic concentration: 50 µg / mL apopramycin, 100 µg / mL trimethoprim). After drying, the plate was placed in a 28°C incubator and incubated for 5-7 days before observation. After colonies grew on the conjugation transfer plate, they were simultaneously transferred to an SFM plate containing 50 µg / mL apopramine using a sterile toothpick to check for pCSG7022 plasmid introduction. After incubation at 28°C for 3 days, single colonies growing on SFM plates containing 50 µg / mL apopramine were selected as candidate strains for genotyping. Genomic DNA was extracted from each candidate strain, and positive clones were obtained by PCR detection using primers ErmF and 152-flaB1B2CD-R (Table 1), i.e., 10- epi -FI-2 production strain (306A::pSET152- ermE p*- flaB1B2CD ).

[0058] 3, 10- epi -FI-2 production strain (306A::pSET152- ermE p*- flaB1B2CD Fermentation detection

[0059] Select 10- ... epi The FI-2 production strain was cultured in 50 ml of A1 medium (10 g soluble starch, 4 g yeast extract, 2 g bacterial peptone, 10 g sea salt, diluted to 1 L with water, pH 7.0-7.4. Preparation: Dissolve all components of the medium in water, stir to dissolve, sterilize, and the solution is ready). The culture was then grown for 5 days at 28℃ and 200 rpm to obtain the fermentation broth. 5 ml of the fermentation broth was extracted with 5 ml of butanone by sonication and centrifugation (3500 r•min). -1 (8 min), take 1.6 ml of the upper organic solvent and evaporate to dryness to obtain 10- epiThe fermentation extract of the FI-2 production strain was dissolved with 120 μl of methanol to obtain a fermentation extract sample. The fermentation extract sample was subjected to high performance liquid chromatography detection. The high performance liquid chromatography (HPLC) conditions were as follows: the chromatographic column was phenomex 150×4.6 mm (SphereClone SAX), the mobile phase included phase A and phase B, the mobile phase A phase: 10% (volume fraction) of acetonitrile + 0.08% (volume fraction) of formic acid, the solvent was water, the mobile phase B phase: 90% (volume fraction) of acetonitrile, the solvent was water; the injection procedure was as follows: 0-18 min, the mobile phase ratio A phase / B phase (volume ratio): 95:5-0:100, 18-23 min, the mobile phase ratio A phase / B phase (volume ratio): 0:100, 23-25 min, the mobile phase ratio A phase / B phase (volume ratio): 0:100-95:5, 25-30 min, the mobile phase ratio A phase / B phase (volume ratio): 95:5, the detection wavelength was 360 nm, and the flow rate was 1 mL / min.10- epi The high performance liquid chromatogram of the fermentation extract sample of the FI-2 production strain is shown in FIG. 1. Figure 5

[0060] The above merely describes the preferred embodiments of the present application, and it should be noted that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be subject to the scope defined by the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.​

Claims

1. A production strain 306A:: flaB1B2CD Its characteristics are, A gene fragment was inserted into the genetically engineered strain 306A. flaB1B2CD Production strain 306A:: flaB1B2CD The aforementioned gene fragment flaB1B2CD The nucleotide sequence is shown in SEQ ID NO.9; The genetically engineered strain 306A is produced using endonuclease. Bam HI and Hin CRISPR-Cas9 knockout plasmid pCSG8006 was digested with dIII to obtain two fragments, pCas9F1 and pCas9F2. The fragments sgRNA, UHA, DHA, pCas9F1, and pCas9F2 were then ligated end-to-end to obtain a clean chassis host 306A CRISPR-Cas9 knockout plasmid pCSG7021. Plasmid pCSG7021 was transformed into *E. coli*. Escherichia coil ) ET12567 / pUZ8002 obtained E. coli ET12567 / pUZ8002 / pCSG7021, serving as the donor bacteria for conjugation transfer, ikaB and ikaC Double gene deletion mutant strain clean chassis host Streptomyces ( Streptomyces sp.) △BC306 was used as the recipient strain for conjugation transfer to obtain genetically engineered strain 306A. The nucleotide sequences of sgRNA, UHA and DHA are shown in SEQ ID NO.1, 2 and 3.

2. The production strain 306A according to claim 1:: flaB1B2CD Its characteristics are, gene fragments flaB1B2CD and promoter gene fragments erm E*p is ligated to a restriction endonuclease. Nde I / Bcu The plasmid pCSG7026 was obtained by linearizing the vector pSET152. The plasmid pCSG7026 was then transformed into Escherichia coli. E. coli Obtained from ET12567 / pUZ8002 E. coli ET12567 / pUZ8002 / pCSG7026, serving as the donor strain for conjugation transfer, was used with genetically engineered strain 306A as the recipient strain to obtain the production strain 306A:: flaB1B2CD The promoter gene fragment mentioned above erm The nucleotide sequence of E*p is shown in SEQ ID NO.

8.

3. The production strain 306A according to claim 1 or 2:: flaB1B2CD Methods and applications in the preparation of 5 / 5-cyclic and 5 / 5 / 6-cyclic PoTeM compounds with oxidation modification at C14 position; The structural formulas of the 5 / 5 ring and 5 / 5 / 6 ring PoTeM compounds with oxidation modification at C14 position are shown in compounds 5-9 of formula (3); (3)。

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