A method to increase the yield of octadecin

Abstract

This invention provides a high-yield engineered strain of oviedomycin, ΔsabA / PGA-FGIH, from *Streptomyces ansochromogenes* 7100, with the accession number CGMCC No. 23634. This invention also provides a method for constructing a highly efficient oviedomycin expression strain. The method includes enhancing the transcription of the key enzyme-encoding gene ovmF-G-I-H, as well as the pyk2, gap1, and accA2 genes, using a strong promoter in a knockout strain (ΔsabA) of the *Streptomyces ansochromogenes* 7100 signal molecule synthase gene sabA. The method also includes screening for carbon sources in the culture medium.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic genetic engineering, specifically involving the construction of high-yield engineered strains of oviedomycin and the optimization and application of carbon sources in culture media. Background Technology

[0002] Streptomyces possess the ability to synthesize a wide range of secondary metabolites with diverse biological activities, including antifungal, antibacterial, antimalarial, antitumor, antiviral, and insecticidal properties, finding extensive applications in medicine, agriculture, and animal husbandry. Among the secondary metabolite biosynthetic gene clusters in Streptomyces, polyketide gene clusters constitute a high proportion, and the structural types of the secondary metabolites they synthesize are also diverse. Anthracycline drugs, such as daunorubicin (DNR), doxorubicin (ADM), and epirubicin (EPI), exhibit strong antitumor activity and are widely used in the clinical treatment of cancer. Oviedomycin, an anthracycline antibiotic with antitumor activity, can also inhibit Gram-positive bacteria such as Staphylococcus aureus, making it a drug with significant development potential. Previously, Méndez et al. isolated oviedomycin (Méndez C, Künzel E, Lipata F, Lombó F, Cotham W, Walla M, Bearden DW, Braña AF, Salas JA, and Rohr J. Oviedomycin, an unusual angucyclinone encoded by genes of the oleandomycin-producer Streptomyces antibioticus ATCC11891. J. Nat. Prod., 2002, 65: 779-782) from Streptomyces antibioticus ATCC11891 and identified its biosynthetic gene cluster (ovm) as having a full length of 27. The gene contains 25 complete open reading frames (ORFs). Furthermore, its biosynthetic pathway was determined through gene knockout and other methods: using one acetyl-CoA and nine malonyl-CoA as precursors, it undergoes polyketide synthesis, cyclization, and redox reactions to ultimately form the oviedomycin final product. A series of derivatives were obtained using knockout strains of different redox enzyme genes, with yields typically below 12.5 mg / L. The construction of high-yielding oviedomycin strains has not yet been reported.

[0003] Genome sequencing has revealed numerous gene clusters for the biosynthesis of secondary metabolites in Streptomyces, which can produce a variety of compounds and represent an important resource for obtaining novel drugs. However, these gene clusters remain largely recessive, or their products are unknown. Therefore, various strategies for activating recessive gene clusters have been developed to mine these hidden natural products (Liu G, Chater KF, Chandra G, Niu G, and Tan H. Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol. Mol. Biol. Rev., 2013, 77: 112-143).

[0004] Quorum sensing (QS) signaling systems, composed of signaling molecules and corresponding receptors, are widely distributed in Streptomyces. They play important roles in regulating the biosynthesis of secondary metabolites, morphological differentiation, and interspecies communication in Streptomyces. QS typically employs a cascaded regulatory mechanism accompanied by signal amplification, thus exhibiting high sensitivity and often functioning at concentrations as low as nM, suggesting a unique advantage in regulating secondary metabolite biosynthesis pathways (Niu G, Chater KF, Tian Y, Zhang J, and Tan H. Specialised metabolites regulating antibiotic biosynthesis in Streptomyces spp. FEMS Microbiol. Rev., 2016, 40: 554-573). Butenolate signaling systems are also widely distributed, found not only in terrestrial Streptomyces but also in marine microorganisms. The applicant isolated the butenolate signaling molecule SAB from Streptomyces cyclohexane 7100. Its receptor is SabR1. It can inhibit the biosynthesis of nicotinic acid through CprC, but the inhibition of nicotinic acid biosynthesis can be relieved when the signaling molecule SAB is present. At the same time, it was also found that SabR1 has multiple target genes, suggesting that SAB plays a regulatory role in the biosynthesis of a variety of secondary metabolites.

[0005] In our previous work, knocking out the global regulatory gene adpA or overexpressing the regulatory gene ovmZ-W in Streptomyces ansochromogenes 7100 activated the transcription of the recessive biosynthetic gene cluster oviedomycin (ovm) (Xu J, Zhang J, Zhuo J, Li Y, Tian Y, and Tan H. Activation and mechanism of a crypticoviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes. J Biol Chem, 2017, 292: 19708-19720). This gene cluster can be heterologously expressed in Streptomyces azure, but the engineered strains obtained by these methods have reduced sporulation capacity or even fail to produce sporulation, which poses significant challenges to the subculturing, preservation, and large-scale fermentation of the strains. We previously investigated the regulatory mechanism of the SAB / SabR1 signaling system (Wang W, Zhang J, Liu X, Li D, Li Y, Tian Y, and Tan H. Identification of a butenolide signaling system that regulates nikkomycin biosynthesis in Streptomyces. J Biol Chem, 2018, 293: 20029-20040), and found that SAB-deficient strains cultured in SP medium with glucose as the carbon source could synthesize oviedomycin, and the strains maintained good growth and sporulation capabilities. This provides a crucial prerequisite for further strain modification and yield improvement. Based on this, this application establishes a novel, highly efficient oviedomycin expression and biosynthesis system to meet the needs of in-depth research and development, and also provides new insights for the discovery of recessive secondary metabolite biosynthesis gene clusters in Streptomyces and the construction of high-yielding antibiotic strains. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide a new system and method for increasing the yield of *Streptomyces oviedomycin*, creating conditions for the large-scale preparation and in-depth development of this compound. This invention utilizes the synergistic effect of the *Streptomyces* signal molecule knockout strain ΔsabA and glucose to activate the production of *oviedomycin*, and constructs strains with high expression of key biosynthetic genes. Furthermore, by optimizing the carbon source, the yield of *oviedomycin* is increased by 59 times. The main technical steps involved in this invention are as follows:

[0007] 1. To identify the rate-limiting step in oviedomycin synthesis, we performed real-time quantitative PCR analysis on the transcriptional levels of each transcription unit in its biosynthetic gene cluster (ovm), which includes 25 genes. The ovmOI-ovmOIII transcription unit contains the major structural genes, and its transcriptional level is significantly increased in the sabA knockout strain (ΔsabA). The transcriptional level of another transcription unit, ovmFGIH, is low, which may be the rate-limiting step in oviedomycin biosynthesis. Its transcriptional level needs to be increased to achieve balance with the expression of other genes.

[0008] 2. The inventors first constructed the ovmFGIH high expression plasmid pSET152::P hrdB After introducing the plasmid ::ovmFGIH (pFGIH) into ΔsabA, the oviedomycin yield was 3.8 times that of ΔsabA without the plasmid.

[0009] 3. pyk2, gap1, and accA2 are key enzyme genes for the synthesis of acetyl-CoA and malonyl-CoA, scattered across different locations in the genome. The inventors predicted and analyzed their gene sequences and the sequences of their ribosome binding sites, sequentially splicing these three genes together and assembling them into a gene module (PGA module) driven by a strong promoter. A high-expression plasmid, pIJ10500::P, was constructed using a Streptomyces integrative plasmid (e.g., the pIJ10500 vector). hrdB The plasmid ::pyk2::gap1::accA2 (pPGA) was inserted into the △sabA genome, and the resulting engineered strain produced 3.2 times more oviedomycin than the △sabA strain without the plasmid.

[0010] 4. Subsequently, the high expression plasmids of ovmFGIH and the PGA module (i.e., the pFGIH plasmid obtained in step 1 and the pPGA plasmid obtained in step 2) were co-transformed into ΔsabA. The resulting engineered strain (i.e., ΔsabA / PGA-FGIH) showed an oviedomycin yield that was 10.9 times that of ΔsabA. The inventors have deposited the ΔsabA / PGA-FGIH strain with the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 23634, on October 20, 2021.

[0011] 5. After screening different carbon sources, it was found that the yield of oviedomycin in ΔsabA / PGA-FGIH could be further increased under certain carbon source conditions, with the yield under galactose medium conditions being 59 times that of ΔsabA under glucose medium conditions.

[0012] The recombinant strains and recombinant plasmids involved in this invention are all within the scope of protection claimed by this invention. Those skilled in the art should understand that mutating or modifying one or more amino acids of the protein shown in the sequence listing—for example, replacing, adding, or deleting some amino acids—while still retaining the function of regulating oviedomycin, is still applicable to this invention. The gene encoding this polypeptide or protein can also be used in this invention to construct recombinant bacteria that can increase oviedomycin production. The promoter used in this invention is not limited to P. hrdB Promoters, including other promoters commonly used in Streptomyces genetic manipulation such as P ermE* Promoter (Bibb MJ, White J, Ward JM, and Janssen G R. The mRNA for the 23SrRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea istranslated in the absence of a conventional ribosome-binding site. Mol.Microbiol., 1994, 14: 533-545), P kasO*Promoter (Wang W, Li The pFGIH and pPGA high-expression plasmids in this invention are not limited to pSET152 (Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, and Schoner B E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichiacoli to Streptomyces spp. Gene, 1992, 116: 43-49) or pIJ10500 (Gregory MA, Till R, and Smith M C. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J. Bacteriol., 2003, 185:5320-5323), but can also be free high-copy plasmids such as pKC1139 (Kieser T, Bibb MJ, Buttner MJ, Chater KF, and Hopwood D A. Practical Streptomyces Genetics). Norwich, United Kingdom: John Innes Foundation, 2000, etc. In this invention, the aforementioned plasmids can be integrated into the Streptomyces genome using techniques such as conjugation transfer, protoplast fusion, electroporation, and chemical transformation. Conjugation transfer is preferred, but other techniques are also effective. The recombination technique used in this invention can be any suitable method used in the art, such as seamlessly knocking the target gene into any location in the strain genome, introducing a recombinant plasmid containing the target gene into the strain (e.g., free high-copy plasmid pKC1139), or integrating a recombinant plasmid containing the target gene into the bacterial genome.The Streptomyces ansochromogenes 7100 used in the embodiments herein is Streptomyces ansochromogenes 7100. However, those skilled in the art should understand that any suitable strain that can produce oviedomycin and the application of the methods in this specification to achieve the effect of increasing oviedomycin yield are still subject to the limitations of this invention.

[0013] More specifically, the present invention provides the following:

[0014] 1. An engineered strain of Streptomyces cyclohexensis 7100, which is a sabA gene knockout strain (△sabA) of Streptomyces cyclohexensis 7100, and has enhanced ovmF-GIH gene expression and enhanced pyk2, gap1 and accA2 gene expression.

[0015] 2. The engineered strain according to Project 1, wherein the ovmF-GIH gene is operatively linked to a strong promoter, and the pyk2, gap1 and accA2 genes are operatively linked to a strong promoter;

[0016] Optionally, the strong promoter is a strong constitutive promoter, such as P hrdB promoter, P ermE* promoter or P kasO* Promoter, preferably P hrdB Promoter.

[0017] 3. The engineered strain described in Project 1 or 2 has the accession number CGMCC No. 23634.

[0018] 4. The use of any of the engineered strains described in Items 1-3 in the production of oviedomycin.

[0019] 5. A method for constructing a high-oviedomycin-producing engineered strain of Streptomyces circumvallate 7100, the method comprising introducing a plasmid overexpressing the ovmF-GIH gene and a plasmid overexpressing the pyk2, gap1 and accA2 genes into a sabA gene knockout strain (△sabA) of Streptomyces circumvallate 7100.

[0020] 6. The method according to Project 5, wherein the ovmF-GIH gene is operatively linked to a strong promoter, and the pyk2, gap1 and accA2 genes are operatively linked to a strong promoter;

[0021] Optionally, the strong promoter is a strong constitutive promoter, such as P hrdB promoter, P ermE* promoter or P kasO*Promoter, preferably P hrdB Promoter.

[0022] 7. A method for the biosynthesis of oviedomycin, comprising culturing any of the engineered strains described in items 1-3 under conditions sufficient to produce oviedomycin.

[0023] 8. The method according to Project 7, further comprising isolating the oviedomycin from the culture medium.

[0024] 9. The method according to item 7, wherein the conditions sufficient to produce oviedomycin include using any one of glucose, sucrose, fructose and galactose as a carbon source, preferably using galactose as a carbon source.

[0025] 10. The method according to Item 7, wherein the nucleotide sequence of ovmF is SEQ ID No. 3 and the amino acid sequence is SEQ ID No. 4; the nucleotide sequence of ovmG ​​is SEQ ID No. 5 and the amino acid sequence is SEQ ID No. 6; the nucleotide sequence of ovmI is SEQ ID No. 7 and the amino acid sequence is SEQ ID No. 8; and the nucleotide sequence of ovmH is SEQ ID No. 9 and the amino acid sequence is SEQ ID No. 10;

[0026] Optionally, the nucleotide sequence of pyk2 is SEQ ID No. 11 and the amino acid sequence is SEQ ID No. 12; the nucleotide sequence of gap1 is SEQ ID No. 13 and the amino acid sequence is SEQ ID No. 14; and the nucleotide sequence of accA2 is SEQ ID No. 15 and the amino acid sequence is SEQ ID No. 16.

[0027] Technical effect

[0028] Previous studies have shown that knocking out the globally regulatory gene adpA or the positive regulatory gene within the constitutively highly expressed oviedomycin gene cluster of *Streptomyces zonalensis* 7100 can activate the expression of this gene cluster. However, the sporulation capacity and growth of the strain are severely inhibited, which is detrimental to the large-scale preparation of the compound and further development of the strain. In our study of ΔsabA, we found that the addition of glucose can synthesize oviedomycin, and the strain maintains good growth and differentiation. This provides a necessary prerequisite for constructing high-yield engineered strains. Based on this, this invention constructs a highly efficient expression system for oviedomycin. Its technical effects are as follows:

[0029] 1) First, transcriptional analysis was used to identify genes with low transcriptional levels within the oviedomycin gene cluster in order to discover potential rate-limiting steps. Results showed that the transcriptional levels of genes in the structural gene ovmOI-OIII transcription units were 86-146 times higher than wild-type, while the transcriptional level of genes in ovmFGIH was only 7.2 times higher, far lower than other structural genes, and therefore may constitute a bottleneck in oviedomycin biosynthesis. To ensure balanced transcription of these genes, this invention overexpressed ovmFGIH, resulting in an increase in oviedomycin yield of 3.8 times that of ΔsabA.

[0030] 2) Pyk2, gap1, and accA2 are located outside the oviedomycin secondary metabolic gene cluster and are scattered in different regions of the genome. They are key genes responsible for the synthesis of acetyl-CoA and malonyl-CoA in the primary metabolic pathway of Streptomyces. These two compounds are precursors of oviedomycin. Therefore, high expression of pyk2-gap1-accA2 was performed to increase the supply of precursors. The results showed that the oviedomycin yield was increased by 3.2 times in strains with high expression of this gene module.

[0031] 3) In strains that co-express the two gene modules ovmFGIH and PGA (△sabA / PGA-FGIH) both within and outside the cluster, oviedomycin was 10.9 times higher than that of △sabA, indicating that the two have a synergistic effect.

[0032] 4) Glucose is typically used as an energy source, primarily affecting the growth and development of bacterial strains. However, in this invention, transcriptional analysis revealed that glucose can significantly enhance the transcription of secondary metabolic gene clusters when the SAB signaling molecule is absent (ΔsabA). This synergistic regulation between glucose and the signaling system, leading to the promotion of secondary metabolic gene cluster expression, highlights the importance of carbon sources. Therefore, carbon source screening revealed that in fructose, sucrose, or galactose media, the oviedomycin yield of ΔsabA was 15, 18, and 44 times higher than that of glucose, respectively, while the yield of ΔsabA / PGA-FGIH in galactose increased dramatically to 59 times. This demonstrates that optimizing the oviedomycin biosynthesis process through multiple pathways, including signal regulation systems, combined high expression of key intra- and extra-cluster biosynthetic genes, and carbon source modification, produces a significant synergistic effect. Furthermore, this system does not affect the normal growth and differentiation of bacterial strains. Therefore, the constructed high-efficiency expression system can be used for the large-scale preparation and further development of oviedomycin.

[0033] Preservation of biological materials

[0034] The engineered strain △sabA / PGA-FGIH of *Streptomyces ansochromogenes* 7100 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 20, 2021, with accession number CGMCC No. 23634. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0035] To make the objectives, technical solutions, and product effects of this invention clearer, the present invention provides the following figures and tables, and provides a detailed description.

[0036] Figure 1 To analyze the production of oviedomycin and the biomass of Streptomyces circumvallate 7100 and its derivative strains. Figure 1 A represents the HPLC analysis and bioactivity detection of fermentation products of WT, △sabA, and △sabAc in SP medium with glucose as the carbon source, using Staphylococcus aureus as the indicator bacterium. WT refers to wild-type Streptomyces 7100; △sabA is the sabA knockout strain; and △sabAc is the sabA replacement strain. Figure 1 B represents the chemical structure of oviedomycin. Figure 1 C represents the oviedomycin production curve of ΔsabA in glucose-SP medium. Figure 1 D represents the biomass change curve of the strain.

[0037] Figure 2 Transcriptional analysis of the oviedomycin biosynthesis gene cluster and related genes. Figure 2 A represents the oviedomycin biosynthetic gene cluster. Figure 2 B represents the transcriptional analysis of genes related to the biosynthesis of oviedomycin.

[0038] Figure 3 The construction and validation of ovmFGIH high expression plasmids and strains, and their impact on oviedomycin yield. Figure 3 A is pSET152::P hrdB Schematic diagram of ::ovmFGIH plasmid construction. Figure 3 B is the electrophoresis image verified by double plasmid digestion (XbaI / EcoRI). Figure 3 C is the PCR verification electrophoresis image of the high-yielding strain △sabA / FGIH (using primer 152 seq F / R). M: 1 kbplus ladder. Figure 3D represents the oviedomycin yield of △sabA and △sabA / FGIH. Figure 3 E represents the bioactivity assay, with Staphylococcus aureus as the indicator bacterium.

[0039] Figure 4 Construction and validation of pyk2-gap1-accA2 (module PGA) high expression plasmid and strain. Figure 4 A is pIJ10500::P hrdB Schematic diagram of plasmid construction::pyk2::gap1::accA2. Figure 4 B is an electrophoresis image verifying plasmid double digestion (XbaI / XhoI). Figure 4 C is the PCR verification electrophoresis image of the △sabA / PGA strain (using primers gap1-accA2 F / accA2-10500 R). M: 1 kb plus ladder. Figure 4 D represents the oviedomycin yield of △sabA and △sabA / PGA. Figure 4 E represents the bioactivity assay, with Staphylococcus aureus as the indicator bacterium.

[0040] Figure 5 The effect of high expression of the ovmFGIH and pyk2-gap1-accA2 combination on oviedomycin yield. Figure 5 A is an electrophoresis image of the PCR verification of the △sabA / PGA-FGIH strain. The primers used are 152-Ph F / PhrdBR, 10500-PhF / PhrdBR (left side) and gap1-accA2 F / accA2-10500 R, 152 seq F / R (right side). Figure 5 B represents the oviedomycin yields of △sabA and △sabA / PGA-FGIH. Figure 5 C represents the bioactivity assay, with Staphylococcus aureus as the indicator bacterium.

[0041] Figure 6 The effects of different carbon sources on WT and ΔsabA fermentation products. Figure 6 A and B represent the HPLC analysis of WT and ΔsabA fermentation products in SP medium with different carbon sources. Figure 6 C represents the detection of antimicrobial bioactivity of WT and ΔsabA fermentation products in SP medium with different carbon sources. The indicator strains were Candida albicans and Staphylococcus aureus, respectively. The carbon sources were 1: mannitol; 2: glucose; 3: sucrose; 4: maltose; 5: galactose; 6: lactose; 7: sorbitol; 8: glycerol; and 9: fructose.

[0042] Figure 7The effect of different carbon sources on the yield of oviedomycin. Figure 7 A represents the oviedomycin yield of ΔsabA and ΔsabA / PGA-FGIH in SP medium with different carbon sources. Figure 7 B represents the antimicrobial bioactivity assay, with Staphylococcus aureus as the indicator strain. The assay results are as follows: 1: Glucose-SP fermentation ΔsabA; 2: Fructose-SP fermentation ΔsabA; 3: Sucrose-SP fermentation ΔsabA; 4: Galactose-SP fermentation ΔsabA; 5: Galactose-SP fermentation ΔsabA / PGA-FGIH. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0044] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0045] Example 1: Analysis of oviedomycin yield by △sabA strain

[0046] Previously, we constructed a SAB signal molecule deletion strain △sabA from Streptomyces 7100 (Wang W, Zhang J, Liu X, Li D, Li Y, Tian Y, and Tan H. Identification of a butenoli designaling system that regulates nikkomycin biosynthesis in Streptomyces. JBiol Chem, 2018, 293: 20029-20040), and fermented and cultured it to detect ovieodmycin production. The specific fermentation and culture methods and the ovieodmycin detection procedure are as follows:

[0047] The formulation of seed culture medium and fermentation medium (SP medium): mannitol 30 g, potato starch 10 g, neutral soybean peptone 5 g, yeast extract 8 g, add distilled water to 1000 mL, adjust pH to 6.0, 121 o Sterilize at C for 30 min. SP fermentation media with different carbon sources are obtained by replacing mannitol with other carbon sources, such as glucose-SP when glucose is used instead of mannitol.

[0048] The HPLC analysis conditions for Oviedomycin were as follows: Reagent A was water (containing 0.1% trifluoroacetic acid); Reagent B was acetonitrile (containing 0.1% trifluoroacetic acid); gradient elution conditions are shown in the table below, with a flow rate of 1 mL / min and a detection wavelength of 280 nm. The HPLC analyzer used was an Agilent 1260 HPLC; the analytical column was an Agilent ZORBAX® SB-C18 column (4.6 × 250 mm, 3.5 μm), pre-columnned with an Agilent SB-C18 pre-column (4.6 × 12.5 mm, 5 μm).

[0049]

[0050] SP medium with mannitol as the carbon source was selected as the seed culture medium. Spores of wild-type Streptomyces chromogenicus 7100 and the SAB signal molecule deletion strain △sabA were inoculated into the seed culture medium and cultured at 28°C. o C. Incubate at 220 rpm for 24 h with shaking; then transfer the seed culture at a 1% inoculum to SP medium with mannitol and glucose as carbon sources, respectively, and incubate for 28 h. o After culturing on a shaker at 220 rpm for 5 days, the supernatant of the fermentation broth was collected by centrifugation and filtered through a 0.22 μm microporous membrane. This supernatant could then be used as a sample for activity testing or for HPLC analysis. HPLC analysis showed that the wild-type strain could not produce oviedomycin in either of the two carbon sources, and ΔsabA could not produce oviedomycin in a medium containing mannitol. Only ΔsabA, in a medium containing glucose, began to produce oviedomycin at 24 h, reaching its peak at 36 h, and then remained stable. The biological activity appeared after 36 h, which is because the compound needs to accumulate to a certain level to exhibit its antibacterial activity. Figure 1 ).

[0051] Example 2: Effect of high expression of the gene ovmFGIH within the ovm cluster on oviedomycin yield

[0052] To determine the rate-limiting steps that may exist in the synthesis of oviedomycin, we examined its biosynthetic gene cluster (ovm). Figure 2A) Representative genes from each transcription unit were analyzed by real-time quantitative PCR. The results showed that, with mannitol as the carbon source, ovmZ (positive regulatory gene), ovmOI (structural gene), and ovmS (structural gene) were almost not transcribed in the wild-type and △sabA knockout strains of *Streptomyces chromogenicus* 7100. In the presence of glucose, the transcription levels of ovmZ, ovmOI, and ovmS in △sabA increased significantly at 24 h, being 9.5-fold, 86-fold, and 146-fold higher than in the wild-type, respectively. Furthermore, the transcription of these genes partially or completely recovered to wild-type levels in the complement strain (△sabAc). Figure 2 B), but the transcriptional level of ovmF in the knockout strain was only 7.2 times that of the wild type at 24 h. Figure 2 B). Sequence alignment revealed that ovmF (gene sequence SEQ ID No. 3, protein sequence SEQ ID No. 4) in the oviedomycin biosynthesis gene cluster (ovm) is a PPTase enzyme encoding a polyketide biosynthesis pathway. This enzyme modifies acyl carrier proteins, transforming them from an inactive state to an active state. In the ovm gene cluster, ovmS is an acyl carrier protein encoding gene located in the transcription units ovmOI-OIII. The low transcriptional level of ovmF may not be sufficient to modify OvmS, therefore, it is necessary to increase OvmF to obtain sufficiently active OvmS. Furthermore, ovmG ​​(gene sequence SEQ ID No. 5, protein sequence SEQ ID No. 6) encodes the β subunit of acetyl-CoA carboxylase, responsible for carboxyl transfer; ovmI (gene sequence SEQ ID No. 7, protein sequence SEQ ID No. 8) is the gene encoding the ε subunit; and ovmH (gene sequence SEQ ID No. 9, protein sequence SEQ ID No. 10) encodes the α subunit responsible for biotin carboxylation. The inventors infer that, under the premise that the transcription level of the structural genes ovmOI-OIII in the ovm gene cluster of ΔsabA is significantly increased in SP medium with glucose as the carbon source, ( Figure 2 B) Increasing the transcriptional level of ovmFGIH will help increase the yield of oviedomycin.

[0053] Therefore, we first constructed the high expression plasmid pFGIH of ovmFGIH ( Figure 3A) The specific operation steps are as follows: The plasmid pSET152 (Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, and Schoner B E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43-49) was double-digested with NotI and EcoRV to obtain linear plasmid DNA fragments; using the genomic DNA of Streptomyces m1146 (Wang W, Zhang J, Liu X, Li D, Li Y, Tian Y, and Tan H. Identification of a butenolide signaling system that regulates nikkomycin biosynthesis in Streptomyces. J Biol Chem, 2018, 293:20029-20040) as a template, 152-Ph F / PhrdB R (SEQ ID No. 17, SEQ ID No. 18) was used as a primer to amplify the promoter sequence P. hrdB (SEQ ID No. 1); Using the genomic DNA of *Streptomyces chromogenicus* 7100 as a template, and Ph-ovmF F / ovmH-152 R (SEQ ID No. 19, SEQ ID No. 20) as primers, ovmFGIH (gene sequence SEQ ID No. 2) was amplified. The three DNA fragments (i.e., the linear plasmid DNA fragment, the promoter sequence P) were then assembled using Gibson assembly. hrdB The ligation products (SEQ ID No. 1 and ovmFGIH SEQ ID No. 2) were ligated, transformed into E. coli JM109 (Invitrogen), and verified by plasmid extraction, enzyme digestion analysis, and sequencing. Figure 3 B) Obtain the plasmid with the correct sequence and name it pSET152::P hrdB::ovmFGIH (pFGIH). pFGIH was first transformed into *E. coli* ET12567 / pUZ8002 (for details, see Flett F, Mersinias V, and Smith CP. High efficiency intergeneric conjugal transfer of plasmid DNA from *Escherichia coli* to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett., 1997, 155: 223-229), then transferred via conjugation into ΔsabA. The resulting strain was verified by PCR using primers 152 seq F / R (SEQ ID No. 28, SEQ ID No. 29). Finally, we obtained the desired strain with the hrdB promoter (P... hrdB ) driven by the ovmFGIH integrated high-expression strain △sabA / FGIH ( Figure 3 C).

[0054] The PCR reaction system used above consisted of: 50 μL of 2×PCR Buffer KOD FX buffer, 20 μL of 2 mM dNTPs, 2 μL of KOD FX (1 U / μL) (all reagents purchased from TOYOBO, Japan), 3 μL each of 10 μM primers, 5 μL (1-50 ng) of genomic template, 17 μL of ddH2O, and a total reaction volume of 100 μL. PCR cycling conditions: Pre-denaturation: 94 °C o C, 3 min. Denaturation: 94 o C, 30 sec; Annealing: 60 o C, 30 sec. Extension: 68 o C, 1 kb / min, 30 cycles; 68 o C, 5 min, 4 o C, Save.

[0055] The bonding transfer method used above is a conventional technique in this field, and the specific method is as follows:

[0056] The plasmid to be transferred was first transformed into *E. coli* ET12567 / pUZ8002 [E. coli ET12567 / pUZ8002 strain information see (Flett F, Mersinias V, and Smith CP. Highefficiency intergeneric conjugal transfer of plasmid DNA from *Escherichiacoli* to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett., 1997, 155: 223-229)]. Single colonies of ET12567 / pUZ8002 containing the recombinant plasmid were selected and inoculated into 3 mL of liquid LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl sodium chloride, distilled water added to 1000 mL, 121...). o Sterilize at 37°C for 30 minutes. o Incubate overnight at 220 rpm with shaking. Transfer the overnight culture at a 1% inoculum to 10 mL of liquid LB containing the appropriate antibiotic and 0.1% glucose, and incubate at 37°C. o C, incubate on a shaker at 220 rpm until OD. 600 Approximately 0.4-0.6; collect bacterial cells by centrifugation at 5000 rpm, wash the cells twice with an equal volume of liquid LB medium, and suspend the cells in 500 μL of LB medium for later use; collect Streptomyces spores in LB medium, centrifuge at 4500 rpm for 3 min, wash twice with LB medium, and suspend in 50 μL of LB medium for later use. o Heat shock in a C water bath for 10 min, then cool to room temperature; mix equal amounts of E. coli cells and Streptomyces spores evenly, and spread on MS solid medium plates containing 10 mM MgCl2 (mannitol 2%, soybean meal 2%, agar 2%, pH natural, 115). o C, sterilize for 30 minutes), 28 o After culturing at C for 16-20 h, apply nalidixic acid to a final concentration of 25 μg / mL and other appropriate antibiotics to their corresponding concentrations. Specifically, for Streptomyces chromogenicus 7100, the concentration of apramycin used was 10 μg / mL, and the concentration of hygromycin used was 25 μg / mL. o C was cultured for another 3-5 days, and the correct conjugates were obtained after screening and PCR verification.

[0057] Fermentation of ΔsabA and engineered strain ΔsabA / FGIH in glucose-SP was performed. Antimicrobial activity and HPLC analysis of the fermentation products showed that the oviedomycin yield in ΔsabA / FGIH was 3.8 times that in ΔsabA. Figure 3 D, E), wherein the fermentation conditions, activity detection methods and HPLC detection methods are the same as in Example 1.

[0058] Example 3: Effect of combined high expression of pyk2, gap1, and accA2 on oviedomycin yield

[0059] Studies have shown that pyk2, gap1, and accA2 are key genes in the synthesis of acetyl-CoA and malonyl-CoA in Streptomyces coelicolor, and are regulated by the SCBs / ScbR signaling system (Li X, Wang J, Li S, Ji J, Wang W, and Yang K. ScbR- and ScbR2-mediated signal transduction networks coordinate complex physiological responses in Streptomyces coelicolor. Sci. Rep., 2015, 5: 14831). pyk2 encodes pyruvate kinase, gap1 encodes glyceraldehyde-3-phosphate dehydrogenase, and accA2 encodes the α subunit of acetyl-CoA carboxylase. These three enzymes are involved in the biosynthesis of acetyl-CoA and malonyl-CoA from glucose, and these two coenzymes are precursors to oviedomycin. Through sequence alignment, three genes corresponding to Streptomyces circumvallate 7100 were identified. Their sequences and the sequences of the ribosome binding site region were predicted and analyzed. Then, they were sequentially assembled and driven by a strong promoter to achieve high expression. The specific process is as follows:

[0060] The vector pIJ10500 (Gregory MA, Till R, and Smith MC. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J. Bacteriol., 2003, 185: 5320-5323) was double-digested with NdeI and XhoI to obtain its linear DNA fragment; using primers 10500-Ph F / PhrdB R (sequence listing SEQ ID No. 21 and SEQ ID No. 18), the promoter sequence P was amplified using the genomic DNA of Streptomyces cyanobacterium M1146 as a template. hrdB (Gene sequence SEQ ID No. 1); using primers Ph-pyk2 F / pyk2-gap1 R (sequence listing SEQ ID No. 22 and SEQ ID No. 24), pyk2-gap1 F / gap1-accA2 R (sequence listing SEQ ID No. 23 and SEQ ID No. 26), and gap1-accA2 F / accA2-10500 R (sequence listing SEQ ID No. 25 and SEQ ID No. 27), pyk2 (gene sequence SEQ ID No. 11, protein amino acid sequence see SEQ ID No. 12), gap1 (gene sequence SEQ ID No. 13, protein amino acid sequence see SEQ ID No. 14), and accA2 (gene sequence SEQ ID No. 15, protein amino acid sequence see SEQ ID No. 16) were amplified using the genomic DNA of *Streptomyces chromogenicus* 7100 as a template. The PCR products of gap1 and accA2 contained their respective ribosome binding sites. The P obtained from the above PCR amplification was assembled using Gibson assembly. hrdB DNA fragments containing the promoter, pyk2, gap1, and accA2 were ligated into a linearly digested vector to obtain plasmid pIJ10500::P hrdB ::pyk2::gap1::accA2 (pPGA) ( Figure 4 A) where pyk2 utilizes the strong promoter P hrdB The plasmid was then subjected to enzyme digestion and sequencing verification to identify the ribosome binding site. Figure 4B). pPGA was introduced into △sabA using a conjugation transfer method, and PCR verification was performed using primers gap1-accA2 F / accA2-10500 R (SEQ ID No. 25, SEQ ID No. 27). This ultimately yielded the desired engineered strain △sabA / PGA with high expression of pyk2, gap1, and accA2. Figure 4 C).

[0061] Fermentation of ΔsabA / PGA in glucose-SP medium was performed, and the fermentation products were analyzed for antibacterial activity and by HPLC. The results showed that the oviedomycin yield in ΔsabA / PGA increased to 3.2 times that of ΔsabA. Figure 4 (D, E). The fermentation conditions, activity detection methods, and HPLC detection methods are the same as in Example 1, and the conjugation transfer method is the same as in Example 2.

[0062] Example 4: Effect of combined high expression of ovmFGIH and pyk2-gap1-accA2 on oviedomycin yield

[0063] Since expressing either pFGIH or pPGA alone can increase the yield of oviedomycin in ΔsabA, to further increase the yield, two high-expression plasmids, pFGIH and pPGA, were sequentially introduced into ΔsabA via conjugation transfer. PCR validation was then performed using primer pairs 152-Ph F / PhrdBR (SEQ ID No. 17, SEQ ID No. 18), 10500-Ph F / PhrdBR (SEQ ID No. 21, SEQ ID No. 18), gap1-accA2 F / accA2-10500 R (SEQ ID No. 25, SEQ ID No. 27), and 152 seq F / R (SEQ ID No. 28, SEQ ID No. 29). Figure 5 A) The engineered strain △sabA / PGA-FGIH was obtained.

[0064] The results showed that the oviedomycin yield in ΔsabA / PGA-FGIH reached 10.9 times that of ΔsabA, far exceeding the yield when pFGIH or pPGA were expressed alone. Furthermore, antibacterial activity analysis also indicated a significant increase in oviedomycin yield. Therefore, high expression of the pFGIH and pPGA combination produced a significant synergistic effect, further increasing oviedomycin yield. Figure 5(B, C). The fermentation conditions, activity detection methods, and HPLC detection methods were the same as in Example 1, and the conjugation transfer method was the same as in Example 2.

[0065] Example 5: Effects of different carbon sources on the activation of the oviedomycin gene cluster

[0066] Considering that the addition of glucose can activate the recessive oviedomycin gene cluster in the SAB-deficient strain and significantly enhance the transcription of structural genes within the cluster, other carbon sources were screened, including mannitol, glucose, sucrose, maltose, galactose, lactose, sorbitol, glycerol, and fructose, which were then used to ferment ΔsabA. HPLC analysis and bioactivity assays revealed that, except for glucose, sucrose, galactose, and fructose, when used as carbon sources, could activate oviedomycin in ΔsabA. Figure 6 ).

[0067] Example 6 Effect of different carbon sources on ΔsabA / PGA-FGIH yield

[0068] Further quantitative analysis revealed that when fructose, sucrose, and galactose were used as carbon sources, the yield of oviedomycin was 15 times, 18 times, and 44 times that of glucose, respectively. Figure 7 Next, SP medium with galactose as the carbon source was used to ferment ΔsabA / PGA-FGIH. HPLC and bioactivity analysis of the product revealed that the yield of oviedomycin was further increased, reaching 59 times that of ΔsabA when glucose was used as the carbon source. Figure 7 (), up to 160 mg / L.

[0069] Table 1. Plasmids used in this invention and their descriptions

[0070]

[0071] Table 2. Strains used in this invention and their descriptions

[0072]

[0073] Table 3 Primer Sequences

[0074]

[0075] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered strain of Streptomyces 7100, which is a sabA gene knockout strain (△sabA) of Streptomyces 7100, and has enhanced ovmF-GIH gene expression and enhanced pyk2, gap1 and accA2 gene expression. in, The sequence of the ovmF-GIH gene is shown in SEQ ID NO:

2.

2. The engineered strain according to claim 1, wherein the ovmF-GIH gene is operatively linked to a strong promoter, and the pyk2, gap1, and accA2 genes are operatively linked to a strong promoter.

3. The engineered strain according to claim 2, wherein, The strong promoter is a strongly constitutive promoter.

4. The engineered strain according to claim 2, wherein the strong promoter is P hrdB promoter, P ermE* promoter or P kasO* Promoter.

5. The engineered strain according to claim 4, wherein, The strong promoter is P. hrdB Promoter.

6. The engineered strain according to any one of claims 1-5, with accession number CGMCC No. 23634.

7. Use of the engineered strain according to any one of claims 1-6 in the production of olividomycin.

8. A method for constructing a high-yield olividomycin-producing engineered strain of Streptomyces chromogenicus 7100, the method comprising introducing a plasmid overexpressing the ovmF-GIH gene and a plasmid overexpressing the pyk2, gap1 and accA2 genes into a sabA gene knockout strain (△sabA) of Streptomyces chromogenicus 7100; in, The sequence of the ovmF-GIH gene is shown in SEQ ID NO:

2.

9. The method of claim 8, wherein the ovmF-GIH gene is operatively linked to a strong promoter, and the pyk2, gap1, and accA2 genes are operatively linked to a strong promoter.

10. The method according to claim 9, wherein, The strong promoter is a strongly constitutive promoter.

11. The method according to claim 9, wherein, The strong promoter is P. hrdB promoter, P ermE* promoter or P kasO* Promoter.

12. The method according to claim 11, wherein, The strong promoter is P. hrdB Promoter.

13. A method for the biosynthesis of olividomycin, comprising culturing the engineered strain according to any one of claims 1-6 under conditions sufficient to produce olividomycin.

14. The method of claim 13, further comprising isolating the olividomycin from the culture medium.

15. The method of claim 13, wherein the conditions sufficient to produce olividomycin include using any one of glucose, sucrose, fructose, and galactose as a carbon source.

16. The method of claim 15, wherein galactose is used as the carbon source.

17. The method according to claim 13, wherein the nucleotide sequence of ovmF is SEQ ID No. 3 and the amino acid sequence is SEQ ID No. 4; the nucleotide sequence of ovmG ​​is SEQ ID No. 5 and the amino acid sequence is SEQ ID No. 6; the nucleotide sequence of ovmI is SEQ ID No. 7 and the amino acid sequence is SEQ ID No. 8; and the nucleotide sequence of ovmH is SEQ ID No. 9 and the amino acid sequence is SEQ ID No.

10.

18. The method according to claim 13, wherein the nucleotide sequence of pyk2 is SEQ ID No. 11 and the amino acid sequence is SEQ ID No. 12; the nucleotide sequence of gap1 is SEQ ID No. 13 and the amino acid sequence is SEQ ID No. 14; and the nucleotide sequence of accA2 is SEQ ID No. 15 and the amino acid sequence is SEQ ID No. 16.

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