Engineered bacteria producing tetramicin and construction and application thereof

Abstract

This invention discloses an engineered bacterium producing tetracrine, its construction, and its application. The invention utilizes a strong promoter to drive the high expression of regulatory genes ovmZn and ovmWn in *Streptomyces neyagawa*, constructing a recombinant engineered strain Sne / pZWn; alternatively, it utilizes a strong promoter to drive the high expression of homologous genes of both, including the regulatory genes ovmZ and ovmW from *Streptomyces chromogenicus*, in *Streptomyces neyagawa*, constructing a recombinant engineered strain Sne / pZW. These engineered strains can express previously silenced gene clusters, activating tetracrine biosynthesis and laying the foundation for the development and application of tetracrine.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial pharmaceuticals, and more specifically, to an engineered bacterium that produces tetracycline and its construction and application. Background Technology

[0002] Streptomyces are a group of Gram-positive bacteria widely found in soil, belonging to the kingdom Prokaryotes, phylum Firmicutes, class Actinomycetes, order Actinomycetes, and genus Streptomyces. Besides exhibiting a complex morphological development and differentiation cycle, Streptomyces can produce a wide variety of secondary metabolites. These secondary metabolites are important sources of antibiotics, anticancer drugs, immunosuppressants, enzyme inhibitors, pigments, and other bioactive substances, and have significant applications in medicine, industry, and agriculture.

[0003] Streptomyces have linearly arranged chromosomes, high GC content, and a large genome (8-10 Mb). In addition to genes related to primary metabolism, the Streptomyces genome also contains a large number of genes related to the biosynthesis of secondary metabolites, which are often clustered together. Under laboratory culture conditions, most of these secondary metabolite biosynthesis gene clusters in Streptomyces are silent. Activating these silent gene clusters is an important pathway for discovering new structural and bioactive natural products.

[0004] The expression of secondary metabolite gene clusters in Streptomyces is often tightly regulated. Overexpression of positive regulatory genes or suppression of negative regulatory genes can effectively activate certain silenced secondary metabolite biosynthetic gene clusters. For example, in *Streptomyces ansochromogenes* 7100, the positive regulatory genes *ovmZ* and *ovmW* in the *oviedomycin* biosynthesis gene cluster are transcriptionally repressed and cannot initiate *oviedomycin* synthesis. By overexpressing ovmZ and ovmW, the silenced oviedomycin biosynthetic gene cluster can be activated, thereby obtaining oviedomycin with inhibitory activity against cancer cells (see reference Xu, J., Zhang, J., Zhuo, J., Li, Y, Tian, ​​Y., Tan H. Activation and molecular mechanism of a cryptic oviedomycin biosynthetic gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes. Journal of Biological Chemistry, 2017, 292: 19708-19720).

[0005] Tetrangomycin is an antibiotic produced by Streptomyces and belongs to the benzo[a]anthraquinone class of antibiotics. Tetrangomycin not only inhibits Staphylococcus aureus but also exhibits inhibitory activity against various cancer cells, making it an important drug lead compound (see Ribeiro, L., Fumagalli, F., Mello, RB, Froes, TQ, da Silva, M., Villamizar Gómez, SM, Barros, TF, Emery, FS, & Castilho, MS: Structure-activity relations and mechanism of action of tetrangomycin derivatives as inhibitors of Staphylococcus aureus staphyloxanthin biosynthesis. Microbialpathogenesis, 2020, 144:104-127). Currently, there are few reports on the construction of recombinant engineered bacteria that can activate or increase tetrangomycin production. Summary of the Invention

[0006] The purpose of this invention is to provide an engineered bacterium that produces tetracycline, its construction, and its application.

[0007] The concept of this invention is as follows: Sequence analysis of the ovmZn and ovmWn regulatory genes within the silenced tetrazin biosynthesis gene cluster in *Streptomyces neyagawa* suggests they may be pathway-specific regulatory genes affecting tetrazin gene cluster expression. By introducing a strong promoter to drive high expression of ovmZn and ovmWn, tetrazin production was detected in the fermentation broth of the recombinant engineered strain, thus activating the tetrazin biosynthesis gene cluster in *Streptomyces neyagawa*. Furthermore, introducing ovmZ and ovmW from *Streptomyces chromogenicus* driven by a strong promoter into *Streptomyces neyagawa* can also activate the tetrazin biosynthesis gene cluster.

[0008] To achieve the objective of this invention, in a first aspect, this invention provides a method for constructing a tetrazolium-producing engineered bacterium, which enhances the genes ovmZn and ovmWn in Streptomyces neyagawaensis, and the resulting gene-enhanced strain is a tetrazolium-producing engineered bacterium.

[0009] In this invention, the enhancement method can be selected from the following 1) to 5), or an optional combination thereof:

[0010] 1) Enhancement is achieved by introducing a plasmid containing the gene;

[0011] 2) Enhanced by increasing the copy number of the aforementioned genes on the chromosome;

[0012] 3) Enhancement is achieved by altering the promoter sequence of the genes described on the chromosome;

[0013] 4) Enhancement is achieved by operatively linking a strong promoter to the gene;

[0014] 5) Enhancement through the introduction of enhancers.

[0015] Among them, the gene ovmZn is a gene encoding either protein (A) or (B) as follows:

[0016] (A) A protein consisting of the amino acid sequence shown in SEQ ID NO:4;

[0017] (B) A protein derived from (A) with the sequence shown in SEQ ID NO:4 replaced, deleted or added with one or more amino acids and having the same function.

[0018] The gene ovm Wn is a gene that encodes either (a) or (b) the following protein:

[0019] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:5;

[0020] (b) Proteins derived from (a) with the sequence shown in SEQ ID NO:5 substituted, deleted or added with one or more amino acids and having the same function.

[0021] Secondly, the present invention provides a method for constructing a tetrazolium-producing engineered bacterium, wherein the homologous genes ovmZn and ovmWn, including the genes ovmZ and ovmW from Streptomyces ansochromogenes, are highly expressed in Streptomyces neyagawaensis, and the recombinant bacteria obtained are the tetrazolium-producing engineered bacterium.

[0022] Among them, the gene ovmZ is a gene encoding either (A′) or (B′) the following protein:

[0023] (A′) A protein consisting of the amino acid sequence shown in SEQ ID NO:8;

[0024] (B′) A protein derived from (A′) with the sequence shown in SEQ ID NO:8 substituted, deleted or added with one or more amino acids and having the same function.

[0025] The gene ovm W is a gene that encodes either (a′) or (b′) the following protein:

[0026] (a′) A protein consisting of the amino acid sequence shown in SEQ ID NO:9;

[0027] (b′) A protein derived from (a′) with the sequence shown in SEQ ID NO:9 replaced, deleted or added with one or more amino acids and having the same function.

[0028] Furthermore, the genes ovmZn and ovmWn, driven by strong promoters, or their homologous genes, are introduced into *Streptomyces nemakawa* via plasmids or integrated into the *Streptomyces nemakawa* chromosome through genetic engineering. The homologous genes may include ovmZ and ovmW.

[0029] Preferably, the genes ovmZ and ovmW, driven by strong promoters, are introduced into *Streptomyces neyagawa* via plasmids or integrated into the *Streptomyces neyagawa* chromosome through genetic engineering.

[0030] In this invention, the methods for introducing genes include, but are not limited to, the traceless knock-in method, the method of replicating different copy numbers of recombinant plasmids, and the homologous recombination method.

[0031] In this invention, the strong promoter can be selected from P hrdBpromoter, P ermE* promoter or P kasO* Promoters, etc. (see Wang, W., Li, X., Wang, J., Xiang, S., Feng, X., and Yang, K. An engineered strong promoter for streptomycetes. Appl Environ Microbiol., 2013, 79: 4484-4492), preferably P hrdB The promoter, more preferably P from Streptomyces coelicolor M145, is preferred. hrdB Promoter (sequence shown in SEQ ID NO:1).

[0032] In this invention, the plasmid can be selected from pSET152, pKC1139 or pIJ10500, etc., with pKC1139 being preferred.

[0033] Furthermore, the method includes: using pKC1139 as the starting plasmid, constructing a plasmid containing P... hrdB Recombinant plasmids expressing the ovmZn and ovmWn genes, driven by the promoter, were then introduced into *Streptomyces neyagawa* to obtain recombinant bacteria with high expression of the ovmZn and ovmWn genes, which are the tetracycline-producing engineered bacteria.

[0034] Furthermore, the method includes: using pKC1139 as the starting plasmid, constructing a plasmid containing P... hrdB Recombinant plasmids expressing the ovmZ and ovmW genes, driven by the promoter, were then introduced into *Streptomyces neiyagawa* to obtain recombinant bacteria with high expression of the ovmZ and ovmW genes.

[0035] Thirdly, the present invention provides a tetrazole-producing engineered bacterium constructed according to the above method.

[0036] Preferably, the engineered bacteria is constructed using *Streptomyces neyagawa* with accession number CGMCC 4.1990 (provided by the China General Microbiological Culture Collection Center) as the starting strain.

[0037] Fourthly, the present invention provides the application of the engineered bacteria in the production of tetracycline.

[0038] Fifthly, the present invention provides a method for producing tetracycline, the method comprising the following steps:

[0039] i) Cultivate the engineered bacteria to obtain a culture of the microorganism;

[0040] ii) Collect the resulting tetrazolium from the culture obtained in step i).

[0041] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0042] This invention provides a high-yield tetrazole-producing engineered strain and its construction method. This invention utilizes strong promoters (such as the promoter of the Streptomyces housekeeping gene hrdB) to drive the high expression of regulatory genes ovmZn and ovmWn or their homologs in *Streptomyces neyagawa*, constructing recombinant engineered strains Sne / pZWn and Sne / pZW. These engineered strains can express previously silenced gene clusters, activating the biosynthesis of tetrazole, laying the foundation for the development and application of tetrazole. Attached Figure Description

[0043] Figure 1 This document describes the construction and verification of ovmZn and ovmWn high-expression strains in a preferred embodiment of the present invention. A: Schematic diagram of the construction of ovmZn and ovmWn high-expression plasmids. B: Agarose gel electrophoresis image of the ovmZn and ovmWn high-expression plasmid pZWn. C: PCR verification electrophoresis image of the ovmZn and ovmWn high-expression plasmid pZWn. Using pZWn as a template, PCR amplification was performed using primers OvmZn-F / 1139-Wn-R to obtain a DNA fragment of approximately 1 kb, consistent with the expected result of 940 bp. D: PCR verification electrophoresis image of the recombinant engineered strain Sne / pZWn. The genomic DNA of the engineered strain Sne / pZWn was amplified using primers 1139-hB-F / 1139-Wn-R, yielding a DNA fragment (1.4 kb) consistent with the expected size. M: 1Kb DNA ladder (purchased from Biomed); N: pKC1139 control plasmid; 1: ovmZn and ovmWn high expression plasmid pZWn or PCR verification fragment.

[0044] Figure 2 The above are the HPLC analysis results of the fermentation broth of the recombinant engineered strain *Streptomyces nekogawa* in a preferred embodiment of the present invention.

[0045] Figure 3 The mass spectrometry analysis (A) and chemical structure diagram (B) of tetracycline are shown in the preferred embodiment of the present invention.

[0046] Figure 4 In a preferred embodiment of the present invention, tetracycline 13 C nuclear magnetic resonance (C 13 C NMR analysis results.

[0047] Figure 5 In a preferred embodiment of the present invention, tetracycline 1H nuclear magnetic resonance (H nuclear magnetic resonance) 1 Results of H NMR analysis. Detailed Implementation

[0048] The present invention provides a method for constructing a tetrazolium-producing Streptomyces neiyagawa, the method comprising introducing a strongly promoter-driven ovmZn and ovmWn gene or its homologous genes ovmZ and ovmW into Streptomyces neiyagawa.

[0049] The strong promoter is selected from P. hrdB promoter, P ermE* promoter or P kasO* Promoter, preferably P hrdB The promoter, whose sequence is shown in SEQ ID NO:1 (from Streptomyces azureus).

[0050] Among them, the gene sequences of ovmZn and ovmWn from Streptomyces neyagawa are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences of the proteins encoded by ovmZn and ovmWn are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0051] The gene sequences of ovmZ and ovmW from *Streptomyces circumvallate* are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively, and the amino acid sequences of the proteins encoded by ovmZ and ovmW are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0052] Among them, the strong promoter and ovmZ-ovmW gene, ovmZn and ovmWn gene are introduced by methods including traceless knock-in, recombinant plasmid copy number replication, and recombinant plasmid integration.

[0053] The plasmid is selected from pSET152, pKC1139 or pIJ10500, etc., with pKC1139 being preferred.

[0054] Furthermore, the recombinant plasmid used to activate tetrazolemycin production contains a strong promoter sequence P in its plasmid backbone. hrdB Expression cassettes of the ovmZn and ovmWn genes.

[0055] The present invention also provides *Streptomyces neiyakawa* expressing tetrazolemycin (recombinant engineered strains Sne / pZWn and Sne / pZW) constructed according to the above method.

[0056] Specifically, the recombinant engineered strain Sne / pZWn is constructed as follows: the promoter sequences of the ovmZn and ovmWn genes in *Streptomyces nekogawa* are replaced with strong promoter sequences, preferably P. hrdB The promoter sequence is shown in SEQ ID NO:1, the gene sequences of ovmZn and ovmWn are shown in SEQ ID NO:2 and 3, respectively, and the amino acid sequences of the proteins encoded by ovmZn and ovmWn are shown in SEQ ID NO:4 and 5; the method includes transforming the recombinant plasmid into Streptomyces nemyogae, wherein the recombinant plasmid contains a strong promoter P. hrdB A gene expression cassette composed of ovmZn and ovmWn. The ovmZn and ovmWn expression cassettes are co-expressed.

[0057] Specifically, the recombinant engineered strain Sne / pZW is constructed as follows: the promoter sequences of the ovmZ and ovmW genes from *Streptomyces chromogenicus* are replaced with strong promoter sequences, preferably P. hrdB The promoter sequence is shown in SEQ ID NO:1, the gene sequences of ovmZ and ovmW are shown in SEQ ID NO:6 and 7 respectively, and the amino acid sequences of the proteins encoded by ovmZn and ovmWn are shown in SEQ ID NO:8 and 9; the method includes transforming the recombinant plasmid into Streptomyces nekogawa.

[0058] The present invention also provides the application of the engineered bacteria in the production of tetracycline.

[0059] In this invention, the recombinant engineered strains *Streptomyces neiyakawa* Sne / pZWn and *Streptomyces neiyakawa* Sne / pZW that produce tetrakeratin are non-type strains. However, those skilled in the art should understand that other strains capable of producing tetrakeratin, as well as strains obtained using the method of this invention that can produce tetrakeratin effects, are all within the scope of protection of this invention.

[0060] The gene sequences encoding the regulatory genes ovmZn and ovmWn involved in this invention are shown in SEQ ID NO:2 and 3, respectively, and the amino acid sequences of the regulatory proteins they encode are shown in SEQ ID NO:4 and 5, respectively.

[0061] Those skilled in the art will understand that proteins shown in SEQ ID NO:4 and 5 that undergo mutations or modifications of one or more amino acids, such as substitution, addition, or deletion of some amino acids, but still retain the function of positively regulating tetraxylacetin or even enhance protein activity, are still applicable to the present invention. Furthermore, the gene encoding this polypeptide or protein can also be used in the present invention to construct recombinant engineered bacteria that can activate tetraxylacetin production or increase tetraxylacetin yield.

[0062] The promoters used in this invention are not limited to P hrdB Promoters, including other promoters commonly used in Streptomyces genetic manipulation such as P ermE* promoter, P kasO* Promoters, etc.

[0063] The plasmids used for high expression of ovmZn and ovmWn in this invention are not limited to the free high-copy plasmid pKC1139 (see Bierman, M., Logan, R., O'Brien, K., Seno, ET, Rao, RN, and Schoner, BEPlasmid cloning vectors for the conjugal transfer of DNA from Escherichiacoli to Streptomyces spp. Gene, 1992, 116:43-49.), but can also be integrative plasmids such as pSET152 (see Kieser, T., Bibb, M., Buttner, M., et al. Practical Streptomyces genetics [M]. Norwich, United Kingdom: John Innes Foundation, 2000), or other integrative plasmids such as pIJ10500 (see Pullan, S., Chandra G., Bibb, M., & Merrick, M., Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes. BMC Genomics, 2011,12:175.) etc.

[0064] 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 in this invention, but other techniques are also applicable.

[0065] The recombination technology used in this invention can be any suitable method used in the art, such as knocking the target gene into any position in the strain genome without leaving a trace, introducing a recombinant plasmid containing the target gene into the strain, or integrating a recombinant plasmid containing the target gene into the strain genome.

[0066] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0067] The main experimental materials involved in the following examples (Tables 1 to 3):

[0068] Table 1. Plasmids used in this invention

[0069]

[0070] Table 2. Strains used in this invention

[0071]

[0072] Table 3 Primers used in this invention

[0073]

[0074] Example 1: Sequence Analysis and Functional Identification of ovmZn and ovmWn

[0075] ovmZ and ovmW are a novel pair of actinomycete genes encoding proteins that regulate secondary metabolic biosynthesis. OvmZ consists of 248 amino acids, and bioinformatics analysis indicates that it lacks any known conserved domains; almost all of its homologous proteins have unknown functions. OvmW consists of 63 amino acids, and database comparisons revealed that the function of this type of protein has never been studied, only suggesting the possible presence of a helix-turn-helix DNA-binding domain. OvmZ and OvmW synergistically regulate the biosynthesis of oviedomycin in *Streptomyces cyclohexim.*

[0076] ovmZn and ovmZn are located within a type II PKS biosynthetic gene cluster. Based on antiSMASH software analysis and gene sequence alignment, this type II PKS biosynthetic gene cluster encodes a horn-anthracene compound, presumably tetracycline. However, tetracycline and any horn-anthracene compounds were not detected during fermentation of *Streptomyces neyagawa*. This indicates that under the current laboratory conditions, this gene cluster is in a silenced state.

[0077] The silenced tetrazolium biosynthesis gene cluster in the *Streptomyces neyagawa* genome includes two genes, ovmZn and ovmWn, which are homologous to ovmZ and ovmW in *Streptomyces chromogenicus*, respectively. OvmZn is a 213-amino acid protein, and sequence alignment revealed a 43% sequence identity with OvmZ in *Streptomyces chromogenicus*. OvmWn is a 78-amino acid protein, with an 81% sequence identity with OvmW in *Streptomyces chromogenicus*. These results suggest that OvmZn-OvmWn in *Streptomyces neyagawa* may function similarly to OvmZ-OvmW in *Streptomyces chromogenicus*, potentially positively regulating the biosynthesis of related secondary metabolites.

[0078] Example 2: Construction of Tetracornemycin-Generating Engineered Strains

[0079] 1. Construction of recombinant plasmids for high expression of ovmZn and ovmWn

[0080] Using the genome of *Streptomyces neyagawa* (Table 2) as a template, PCR amplification was performed using primers ovmZn-F and 1139-Wn-R to obtain DNA fragment 1 (SEQ ID NO: 10) containing the ovmZn and ovmWn genes; using the genome of *Streptomyces coelicolor* M145 as a template, PCR amplification was performed using primers 1139-hB-F and hB-Zn-R to obtain DNA fragment 1 containing P... hrdB The promoter region DNA fragment (SEQ ID NO:1). The DNA fragment obtained above was combined with the pKC1139 plasmid fragment digested with XbaI / EcoRI and assembled using Gibson to obtain the recombinant plasmid pZWn (Table 1), as shown. Figure 1 As shown in Figure A, the recombinant plasmid pZWn was extracted and subjected to agarose gel electrophoresis. The plasmid size was as expected, 7.9 kb. Figure 1 B). PCR and sequencing confirmed that pZWn contains the correct ovmZn and ovmWn gene fragments. Figure 1 C).

[0081] 2. Construction of Tetracornemycin-producing engineered strains

[0082] The verified recombinant plasmid pZWn was transformed into *Escherichia coli* ET12567 / pUZ8002 (see Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, and Hopwood, DAP Practical Streptomyces Genetics, 2000, John Innes Foundation, Norwich, United Kingdom), and then transferred into *Streptomyces neyagawa* via conjugation. The target engineered strain Sne / pZWn, resistant to apramycin, was screened on a medium containing naphthylpyridinic acid and apramycin (Table 2). The recombinant strain Sne / pZWn was transferred to TSB liquid medium, and after 48 h, the plasmid was extracted as a template and amplified using primers 1139-hB-F and 1139-Wn-R, yielding a fragment of approximately 1.4 kb, consistent with the expected result. Figure 1 D) This demonstrates the correct construction of the recombinant engineered strain Sne / pZWn. Simultaneously, the recombinant plasmid pZW, which highly expresses ovmZ and ovmW from *Streptomyces chromogenicus*, was transformed into ET12567 / pUZ8002 and transferred to *Streptomyces neyagawa* via conjugation. The target engineered strain Sne / pZW, resistant to apramycin, was screened on a medium containing nalidixic acid and apramycin (Table 2). Furthermore, the empty vector pKC1139 was transformed into ET12567 / pUZ8002 and transferred to *Streptomyces neyagawa* via conjugation. The target engineered strain Sne / pKC1139, resistant to apramycin, was screened on a medium containing nalidixic acid and apramycin and used as a control strain (Table 2).

[0083] Example 3: Fermentation of the Tetracornemycin-producing engineered strain and HPLC analysis of tetracornemycin.

[0084] 1. Culture conditions for producing tetracycline-producing engineered strains

[0085] Spores of recombinant Streptomyces neyagawa strains Sne / pZWn and Sne / pZW were inoculated into MS or TSB medium and cultured at 28°C and 220 rpm for 48 h. 1 mL of the culture was transferred to 100 mL of MS medium and cultured at 28°C and 220 rpm for 6 days. The fermentation broth was then collected and extracted with an equal volume of chloroform for 1-2 h. The upper organic phase was concentrated and dissolved in 1 mL of methanol.

[0086] MS medium: Weigh 20g soybean flour, add 500-800mL of cold water and mix well. Heat until the soybean flour boils. Filter through two layers of gauze to remove the residue. After cooling, add 20g mannitol and distilled water to a final volume of 1L. Sterilize at 115℃ for 30min.

[0087] TSB medium: Weigh 16g Difco Bacto Tryptone, 10g yeast extract and 5g sodium chloride, add distilled water to a final volume of 1L, and sterilize at 121℃ for 30min.

[0088] 2. HPLC analysis of tetracycline

[0089] Instrument: Agilent 1260 HPLC; Analytical column: Agilent Technologies An SB-C18 column (4.6 × 250 mm, 5 μm) was used, pre-connected to an Agilent SB-C18 pre-column (4.6 × 12.5 mm, 5 μm). The UV wavelength for detecting tetracycline was set to 280 nm. Mobile phase A was ddH2O containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 1 mL / min, and the elution conditions are shown in Table 4.

[0090] Table 4 HPLC elution conditions

[0091]

[0092] The products of *Streptomyces nekogawa* Sne, Sne / pZWn, Sne / pZW, and Sne / pKC1139 fermented for 6 days were analyzed by HPLC. The HPLC analysis results showed that ( Figure 2 Compared with wild-type Streptomyces neiyakawa Sne and strain Sne / pKC1139 containing an empty vector, the fermentation broths of engineered strains Sne / pZWn and Sne / pZW showed a difference peak at a retention time of 23 min. Compounds exhibiting this difference peak were selected for separation and purification.

[0093] Example 4: Chemical Structure Analysis of Tetracornemycin

[0094] Tetracycline derived from the fermentation broths of engineered strains Sne / pZWn and Sne / pZW in Example 3 was enriched and purified by HPLC and analyzed by liquid chromatography-mass spectrometry (LC-MS). The instrument used was an Agilent Triple Quadrupole LC / MS system 1260 / 6460. The analysis mode was negative ion mode. Mass spectrometry analysis showed that the mass-to-charge ratio of this compound was 322 (…). Figure 3 The structural formula is presumably C. 19 H 15O5, consistent with the molecular weight and formula of tetragonaline (see reference Kuntsmann, M., Mitscher, LA The structural characterization of tetragonaline and tetrangulol. Journal of Organic Chemistry. 1966, 31: 2920-2925). The above-isolated and purified tetragonaline sample was dissolved in deuterated DMSO as solvent for [further processing]. 1 H NMR, 13 C NMR, 1 H- 1 H COSY、 1 H- 13 C HMBC and 1 H- 13 Nuclear magnetic resonance (NMR) analysis, including C HSQC, was performed using a Bruker AVANCE III 500M instrument. The attribution of relevant proton and carbon spectral data is shown in Table 5. Tetrakeratin... 13 C NMR, 1 The H NMR analysis results are shown in the figures below. Figure 4 , Figure 5 Based on the nuclear magnetic resonance results, it can be determined that the structure of this compound is consistent with that of tetrakeratin. Figure 3 This demonstrates that high expression of ovmZn and ovmWn, ovmZ and ovmW activates the expression of the silent tetracycline biosynthesis gene cluster in *Streptomyces neyagawa*.

[0095] Table 5. Summary of NMR analysis results for tetracycline (carbon and proton spectra).

[0096]

[0097] The recombinant Streptomyces nemophila and its establishment method provided by this invention can be widely used in fields involving tetracycline, such as scientific research, medicine, industry and agriculture, and has important application value.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Tetracycline-producing engineered bacteria, their construction and application <130> KHP221113509.4 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 483 <212> DNA <213> Streptomyces coelicolor <400> 1 ccagtgccaa gcttgggctg caggtcgact cgagcactga ccgccttccg ccggaacggc 60 ggggtccggg cacgccaaac ccctcctgtg gctgtggccg gccaccgccg tcaccttcgg 120 accccgtgga gccgctcccg gttccacggg gtccgaaggt gtgatgagca ggctgcgcct 180 tcctcgcgcg gccgcaaggt acgagttgat gaccttgttt atccgcatct gaccaatttt 240 gatcgcttac ggggtgtgac tcgggccacg cggattgggc gtaacgctct tgggaacaac 300 acgatgacct aagaggtgac agccgcggag ggaatacgga cgccgttcac ggcgctgtgc 360 atctccccgg cccgcccgca ccgtcggccc attcccaagc cggtggtcgg cccctgtccg 420 ccgtggacgg ggccggaagc cgtttttcaa cgttccgaga ggttgttcgt gccctcatcc 480 agg 483 <210> 2 <211> 642 <212> DNA <213> Streptomyces neyagawaensis <400> 2 gtgccctcat ccaggaaatt acctccaact cttcacatat ccctcaacac atctctcgaa 60 agcgtccatg cgccaagctg cctccgagca gagggcctgc gcgtcgctct gcgcaaagtc 120 gtgaatttcc accacgagag cgagcacgaa ctgacgtcgt ccgcgcacgg gctgcgggag 180 cgtgtcagcg gcagcaggcc gatgggtatc accctcgacg agtccgtgct cgccctgcgg 240 tcgcggatca ccgagctgct cacctcgtgg gcccggctgg tcatcgagga gcgcggagtc 300 gccgcgcccc ggaaccgcag cgttccgggc ctggccgaat tcctcggccg acacctctcc 360 tggctgacgg agcatccggc gggaccggac ttcgaccggg aggtcaccgc gctgatgcgc 420 tcctgccact cagtgcgcga agagaccagg tcggcacgcc gggagttggg cggctgcccg 480 gagccaggct gctccggaac gctgtacgca gtgctgcgtc cgaccgaaag cgccccggga 540 cggcctggtt caggcgtggt gtgcgaccag gggcatgcct tcgcgccgca cgactggctg 600 ctgctcgccg gtccacggca gtctgccggg atccggccgt ga 642 <210> 3 <211> 237 <212> DNA <213> Streptomyces neyagawaensis <400> 3 gtgaccaccg gggaaggcaa gcacaccgta ccgaccgaac tcgcggccct ggccatgggg 60 gtctcgtcgg cgaccatccg gaaatgggcc agtcgaggaa agatcacccg tcttggcacc 120 gcacagcgcg cggagtacga cctcgacgag ttgtacaggt tgcgacagac ccgacaccac 180 gaagccccga gccgggccag gaccgtcgat gacgcagccg actccggccc ggattga 237 <210> 4 <211> 213 <212> PRT <213> Streptomyces neyagawaensis <400> 4 Val Pro Ser Ser Arg Lys Leu Pro Pro Thr Leu His Ile Ser Leu Asn 1 5 10 15 Thr Ser Leu Glu Ser Val His Ala Pro Ser Cys Leu Arg Ala Glu Gly 20 25 30 Leu Arg Val Ala Leu Arg Lys Val Val Asn Phe His His Glu Ser Glu 35 40 45 His Glu Leu Thr Ser Ser Ala His Gly Leu Arg Glu Arg Val Ser Gly 50 55 60 Ser Arg Pro Met Gly Ile Thr Leu Asp Glu Ser Val Leu Ala Leu Arg 65 70 75 80 Ser Arg Ile Thr Glu Leu Leu Thr Ser Trp Ala Arg Leu Val Ile Glu 85 90 95 Glu Arg Gly Val Ala Ala Pro Arg Asn Arg Ser Val Pro Gly Leu Ala 100 105 110 Glu Phe Leu Gly Arg His Leu Ser Trp Leu Thr Glu His Pro Ala Gly 115 120 125 Pro Asp Phe Asp Arg Glu Val Thr Ala Leu Met Arg Ser Cys His Ser 130 135 140 Val Arg Glu Glu Thr Arg Ser Ala Arg Arg Glu Leu Gly Gly Cys Pro 145 150 155 160 Glu Pro Gly Cys Ser Gly Thr Leu Tyr Ala Val Leu Arg Pro Thr Glu 165 170 175 Ser Ala Pro Gly Arg Pro Gly Ser Gly Val Val Cys Asp Gln Gly His 180 185 190 Ala Phe Ala Pro His Asp Trp Leu Leu Leu Ala Gly Pro Arg Gln Ser 195 200 205 Ala Gly Ile Arg Pro 210 <210> 5 <211> 78 <212> PRT <213> Streptomyces neyagawaensis <400> 5 Val Thr Thr Gly Glu Gly Lys His Thr Val Pro Thr Glu Leu Ala Ala 1 5 10 15 Leu Ala Met Gly Val Ser Ser Ala Thr Ile Arg Lys Trp Ala Ser Arg 20 25 30 Gly Lys Ile Thr Arg Leu Gly Thr Ala Gln Arg Ala Glu Tyr Asp Leu 35 40 45 Asp Glu Leu Tyr Arg Leu Arg Gln Thr Arg His His Glu Ala Pro Ser 50 55 60 Arg Ala Arg Thr Val Asp Asp Ala Ala Asp Ser Gly Pro Asp 65 70 75 <210> 6 <211> 747 <212> DNA <213> Streptomyces ansochromogenes <400> 6 atggccggcc agccacgacc caacgacggg tccgacaaac agcaattcca gttcaaaaaa 60 cggggaaacg gaatgcgcca agctctgcca ttgtcagaaa cgtcggagga acaacccagc 120 gcggacgcgc tgatcccaac tctgcgtatc ctcgcggagc tttacgaaga atgcgggcga 180 atgttagcgc ataccccgta cagcctgcgc cagcgtgtca ccggaagccg cgccaccgga 240 atttctctca acgagaaagc catggcgata cggacggaa cgcagaacac gctcggctcc tgggcgcgcc tggtggtgga cgagtgcggc acggccggcc cgtcccagga ggggatccct 360 cccctcgtct ccttcctcat ccgccattcc ggatggctgg ccgcgcatcc ggcggccggt 420 gacttcgccg aggagatcgc cgcgctcgcg aggtccgccc gccggatcgc gggccccggc 480 cccgcccacc gcgtcgacct ggggcgctgc ctgcggtccg gctgcgcggg cacgctgcgc 540 gccgtggtgc acgacgggga cgacgcgacg ccgggacagg tgtcgtgcga cgccgggcac 600 gcgctgccgc cgcagcagtg gctgctggtc gcgcaccgca tgcggcggac cgcctcgcgc 660 ggcgagcgcg tcacccggcg ccacgacggc gaccggggcg gtgtccaggc cgaccggggcc 720 ggtgtccgcg aggaggccgc ccgatga 747 <210> 7 <211> 192 <212> DNA <213> Ansochromogenes (Streptomyces ansochromogenes) <400> 7 atgagccgta cgccacggcg gcgcttcgtg cccacggac tcgccgcgct cgcggccggg 60 gtgacggagg ccaccatccg caagtgggcc agccgcggca agatcacccg ctacggcagc 120 ccccggcgcg cccagtacga cctggacgaa ctcatcgcca tcgtcgccga ccggtccgag 180 gccctcggct ga 192 <210> 8 <211> 248 <212> PRT <213> Streptomyces ansochromogenes <400> 8 Met Ala Gly Gln Pro Arg Pro Asn Asp Gly Ser Asp Lys Gln Gln Phe 1 5 10 15 Gln Phe Lys Lys Arg Gly Asn Gly Met Arg Gln Ala Leu Pro Leu Ser 20 25 30 Glu Thr Ser Glu Glu Gln Pro Ser Ala Asp Ala Leu Ile Pro Thr Leu 35 40 45 Arg Ile Leu Ala Glu Leu Tyr Glu Glu Cys Gly Arg Met Leu Ala His 50 55 60 Thr Pro Tyr Ser Leu Arg Gln Arg Val Thr Gly Ser Arg Ala Thr Gly 65 70 75 80 Ile Ser Leu Asn Glu Lys Ala Met Ala Ile Arg Thr Glu Thr Gln Asn 85 90 95 Thr Leu Gly Ser Trp Ala Arg Leu Val Val Asp Glu Cys Gly Thr Ala 100 105 110 Gly Pro Ser Gln Glu Gly Ile Pro Pro Leu Val Ser Phe Leu Ile Arg 115 120 125 His Ser Gly Trp Leu Ala Ala His Pro Ala Ala Gly Asp Phe Ala Glu 130 135 140 Glu Ile Ala Ala Leu Ala Arg Ser Ala Arg Arg Ile Ala Gly Pro Gly 145 150 155 160 Pro Ala His Arg Val Asp Leu Gly Arg Cys Leu Arg Ser Gly Cys Ala 165 170 175 Gly Thr Leu Arg Ala Val Val His Asp Gly Asp Asp Ala Thr Pro Gly 180 185 190 Gln Val Ser Cys Asp Ala Gly His Ala Leu Pro Pro Gln Gln Trp Leu 195 200 205 Leu Val Ala His Arg Met Arg Arg Thr Ala Ser Arg Gly Glu Arg Val 210 215 220 Thr Arg Arg His Asp Gly Asp Arg Ala Gly Val Gln Ala Asp Arg Ala 225 230 235 240 Gly Val Arg Glu Glu Ala Ala Arg 245 <210> 9 <211> 63 <212> PRT <213> Streptomyces ansochromogenes <400> 9 Met Ser Arg Thr Pro Arg Arg Arg Phe Val Pro Thr Glu Leu Ala Ala 1 5 10 15 Leu Ala Ala Gly Val Thr Glu Ala Thr Ile Arg Lys Trp Ala Ser Arg 20 25 30 Gly Lys Ile Thr Arg Tyr Gly Ser Pro Arg Arg Ala Gln Tyr Asp Leu 35 40 45 Asp Glu Leu Ile Ala Ile Val Ala Asp Arg Ser Glu Ala Leu Gly 50 55 60 <210> 10 <211> 875 <212> DNA <213> Artificial Sequence <400> 10 gtgccctcat ccaggaaatt acctccaact cttcacatat ccctcaacac atctctcgaa 60 agcgtccatg cgccaagctg cctccgagca gagggcctgc gcgtcgctct gcgcaaagtc 120 gtgaatttcc accacgagag cgagcacgaa ctgacgtcgt ccgcgcacgg gctgcgggag 180 cgtgtcagcg gcagcaggcc gatgggtatc accctcgacg agtccgtgct cgccctgcgg 240 tcgcggatca ccgagctgct cacctcgtgg gcccggctgg tcatcgagga gcgcggagtc 300 gccgcgcccc ggaaccgcag cgttccgggc ctggccgaat tcctcggccg acacctctcc 360 tggctgacgg agcatccggc gggaccggac ttcgaccggg aggtcaccgc gctgatgcgc 420 tcctgccact cagtgcgcga agagaccagg tcggcacgcc gggagttggg cggctgcccg 480 gagccaggct gctccggaac gctgtacgca gtgctgcgtc cgaccgaaag cgccccggga 540 cggcctggtt caggcgtggt gtgcgaccag gggcatgcct tcgcgccgca cgactggctg 600 ctgctcgccg gtccacggca gtctgccggg atccggccgt gaccaccggg gaaggcaagc 660 acaccgtacc gaccgaactc gcggccctgg ccatgggggt ctcgtcggcg accatccgga 720 aatgggccag tcgaggaaag atcacccgtc ttggcaccgc acagcgcgcg gagtacgacc 780 tcgacgagtt gtacaggttg cgacagaccc gacaccacga agccccgagc cgggccagga 840 ccgtcgatga cgcagccgac tccggcccgg attga 875

Claims

1. A method for constructing tetracycline-producing engineered bacteria, characterized in that, Strengthening the room's Streptomyces ( Streptomycesneyagawaensis genes in ) ovmZn and ovmWn The resulting gene-enhanced strain is the tetrazolium-producing engineered bacterium; or, High expression in Streptomyces reyokawa ovmZn and ovmWn The homologous gene comes from Streptomyces cyclohexenum ( Streptomyces ansochromogenes ) genes ovmZ and ovmW The recombinant bacteria obtained are the tetrazole-producing engineered bacteria; Among them, genes ovmZn The gene encoding a protein with the amino acid sequence shown in SEQ ID NO:4; Gene ovmWn a gene encoding a protein as set forth in SEQ ID NO: 5; Gene ovmZ a gene encoding a protein as set forth in SEQ ID NO: 8; Gene ovmW A gene encoding a protein as set forth in SEQ ID NO:

9.

2. The method of claim 1, wherein, The enhancement method is selected from the following 1) to 5), or an optional combination thereof: 1) Enhancement is achieved by introducing a plasmid containing the gene; 2) Enhanced by increasing the copy number of the aforementioned genes on the chromosome; 3) Enhancement is achieved by altering the promoter sequence of the aforementioned genes on the chromosome; 4) Enhancement is achieved by operatively linking a strong promoter to the gene; 5) Enhancement through the introduction of enhancers.

3. The method according to claim 1 or 2, characterized in that, Genes driven by strong promoters ovmZn and ovmWn Alternatively, its homologous gene may be introduced into *Streptomyces nemyoga* via plasmid or integrated into the chromosome of *Streptomyces nemyoga* through genetic engineering; the homologous gene is... ovmZ and ovmW .

4. The method according to claim 3, characterized in that, Methods for gene introduction include traceless knock-in, replication of different copy numbers of recombinant plasmids, and homologous recombination.

5. The method of claim 3, wherein, The strong promoter is selected from P. hrdB promoter, promoter or Promoter.

6. The method of claim 5, wherein, The strong promoter is the P Streptomyces coelicolor ) M145. The strong promoter is the P hrdB promoter.

7. The method of claim 3, wherein, The plasmids are selected from pSET152, pKC1139 or pIJ10500.

8. The method of claim 7, wherein, The plasmid is pKC1139.

9. The method of claim 3, wherein, The method includes: using pKC1139 as the starting plasmid, constructing a plasmid containing P... hrdB Promoter-driven genes ovmZn and ovmWn The recombinant plasmid of the expression cassette was then introduced into *Streptomyces neyagawa* to obtain the gene. ovmZn and ovmWn High expression of recombinant bacteria is what we call tetracycline-producing engineered bacteria; or, Using pKC1139 as the starting plasmid, a system containing P... hrdB Promoter-driven genes ovmZ and ovmW The recombinant plasmid of the expression cassette was then introduced into *Streptomyces neyagawa* to obtain the gene. ovmZ and ovmW High expression of recombinant bacteria.

10. Tetracycline-producing engineered bacteria constructed according to any one of claims 1-9.

11. The use of the engineered bacteria of claim 10 in the production of tetracycline.

12. A method for producing tetracycline, characterized in that, The method includes the following steps: i) Cultivate the engineered bacteria of claim 10 to obtain a culture of the engineered bacteria; ii) Collect the resulting tetrazolium from the culture obtained in step i).

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