A method for increasing the yield of carbamoyltobramycin and a production strain

Abstract

This invention provides a method and a production strain for increasing the yield of carbamoyl tobramycin, belonging to the field of genetic engineering technology. By knocking out the tobR gene, which encodes an Lrp / AsnC family transcriptional regulator, within the tobramycin synthesis gene cluster region of the *Streptoalloteichus tenebrarius* genome, and by overexpressing the tobO gene, which encodes a TauD / TfdA family oxidase directly acted upon by TobR, using a constitutive strong promoter, a hybrid mutant strain was obtained, exhibiting a significantly increased fermentation yield of carbamoyl tobramycin. The shake-flask fermentation level of carbamoyl tobramycin by this hybrid mutant strain reached 3.76 g / L, a 42.5% increase compared to the 2.64 g / L of the starting strain. The advantages of this invention are: by inactivating the tobR gene and simultaneously overexpressing the tobO gene in *S. tenebrarius*, a high-yielding carbamoyl tobramycin strain was constructed. This strain can increase the fermentation yield of carbamoyl tobramycin without increasing the cost of additional culture medium, thus reducing production costs and showing significant industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for constructing and applying a method to increase the fermentation yield of carbamoyl tobramycin. Background Technology

[0002] Streptoalloteichus tenebrarius (formerly known as Streptomyces tenebrarius, Streptomyces darkense) (Int J Syst Evol Microbiol 2008, 58, 3, 688-691) is an important Streptomyces species that can produce carbamoyl tobramycin, carbamoyl kanamycin, and apramycin through fermentation. Industrially, tobramycin is often produced by alkaline hydrolysis and purification of its fermentation products (J Antibiot 1973, 26, 12, 745-751). To reduce the cost of purification in industrial production, the synthesis of apramycin can be blocked by knocking out genes (e.g., aprJ) in the biosynthetic pathway of the major byproduct apramycin (Xiao Jianping. Research on Directed Biosynthesis of Apramycin [D]. Fuzhou University, 2014.).

[0003] Tobramycin, also known as tobramycin, belongs to the second class of aminoglycoside antibiotics. Tobramycin binds to the aminoacyl-tRNA recognition site on the 30S subunit of bacterial ribosomes, preventing the formation of the 70S complex, hindering translation, affecting peptide chain elongation, causing codon misreading, producing abnormal proteins, and thus killing bacteria (Nature Communications 2015, 6). Tobramycin is a broad-spectrum and highly effective antibiotic. In animal experiments, tobramycin has lower ototoxicity and nephrotoxicity than gentamicin. Clinically, it is mainly used to treat some serious infections, such as burn infections, sepsis, respiratory infections, urinary tract infections, gallbladder and biliary tract infections, and severe soft tissue infections caused by Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae (Nat Prod Rep 2013, 30, 1, 11-20).

[0004] Aminoglycoside antibiotics are typically produced by Streptomyces and Micromonas, and like most other antibiotics, they are secondary metabolites produced by actinomycetes. The metabolic synthesis of antibiotics in actinomycetes is usually regulated by pathway-specific or global regulatory proteins. By studying and modifying these regulatory proteins, the secondary metabolic processes of actinomycetes can be precisely controlled, thereby affecting antibiotic yield. The purpose of this invention is to obtain high-yielding tobramycin-producing strains through targeted gene modification via genetic engineering for the industrial production of tobramycin.

[0005] Lrp / AsnC family transcription factors are widely distributed in bacteria and archaea. Lrp / AsnC family regulatory proteins have broad functions, with most involved in regulating amino acid metabolism and small molecule transport (Mol Microbiol 2003, 48, 2, 287-294). Based on gene sequence inference, the tobR gene belongs to the Lrp / AsnC family. Laboratory studies have revealed that TobR in *S. tenebrarius* may be a negative regulator involved in the biosynthesis of carbamoyl tobramycin, and EMSA experiments have demonstrated that the TobR protein can directly bind to the promoter region DNA of its neighboring gene, tobO.

[0006] Based on the sequence alignment analysis of the tobO gene in S. tenebrarius, it is speculated that TobO belongs to the TauD / TfdA family of proteins and is an α-ketoglutarate-dependent oxidase. It may affect the secondary metabolism of bacteria by participating in the catabolism of amino acids. Laboratory studies have shown that overexpression of the tobO gene in S. tenebrarius can promote the fermentation production of carbamoyltobramycin.

[0007] To further improve the fermentation level of carbamotobramycin, an industrialized method is needed to increase the fermentation yield of carbamotobramycin and reduce production costs by utilizing the tobR and tobO genes of S. tenebrarius. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for increasing the yield of carbamotobramycin.

[0009] The present invention solves the above-mentioned technical problems by adopting the following technical solutions.

[0010] In a first aspect, the present invention provides a method for increasing the yield of carbamoyl tobramycin: by inactivating the tobR gene in S. tenebrarius through genetic engineering technology, and / or by overexpressing the encoding gene of the TauD / TfdA family oxidase in S. tenebrarius, a high-yielding carbamoyl tobramycin strain is obtained, and the strain is used for the fermentation production of carbamoyl tobramycin; wherein the nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2; the S. tenebrarius is S. tenebrarius with the aprJ gene knocked out.

[0011] According to an embodiment of the present invention, the method for inactivating the tobR gene in S. tenebrarius is as follows: knocking out the tobR gene in S. tenebrarius.

[0012] According to an embodiment of the present invention, the method for inactivating the tobR gene in S. tenebrarius is as follows:

[0013] (a) Using the S. tenebrarius genome as a template, primer pairs were designed to amplify the upstream and downstream homologous arm fragments of the tobR gene;

[0014] (b) Fuse the upstream and downstream homologous arm fragments of the tobR gene;

[0015] (c) The fusion product of the upstream and downstream homologous arm fragments of the tobR gene was recombined with the knockout plasmid vector fragment to obtain the knockout plasmid of the tobR gene.

[0016] (d) The tobR gene knockout plasmid was introduced into Escherichia coli ET12567 (pUZ8002), and then introduced into S. tenebrarius by conjugation transfer. After two rounds of homologous recombination exchange were completed, mutant strains lacking the tobR gene were screened.

[0017] According to an embodiment of the present invention, the encoding gene of the TauD / TfdA family oxidase is the tobO gene. The nucleotide sequence of the tobO gene is as shown in SEQ ID NO.3, or the nucleotide sequence of SEQ ID NO.3 is obtained by substituting one or more bases to obtain the nucleotide sequence encoding the same functional protein. The amino acid sequence of the protein encoded by the nucleotide sequence of the tobO gene is as shown in SEQ ID NO.4, or the amino acid sequence of SEQ ID NO.4 is obtained by substituting one or more amino acid bases to obtain the same functional amino acid sequence.

[0018] According to an embodiment of the present invention, the *S. tenebrarius* uses a *Streptomyces* strong promoter to overexpress the tobO gene.

[0019] According to an embodiment of the present invention, the method for overexpressing the tobO gene is as follows:

[0020] (a) Using the S. tenebrarius genome as a template, the tobO gene fragment was amplified;

[0021] (b) The gene fragment of tobO was recombined with an integrative overexpression vector containing a strong streptomyces promoter to obtain an overexpression plasmid of tobO;

[0022] (c) The tobO overexpression plasmid was first introduced into Escherichia coli ET12567 (pUZ8002), and then introduced into S. tenebrarius by conjugation transfer to obtain mutant strains that integrate the tobO gene.

[0023] In step (c), S. tenebrarius is preferably a mutant strain of S. tenebrarius lacking the tobR gene, and the mutant strain finally obtained in step (c) is preferably a mutant strain of S. tenebrarius lacking the tobR gene but overexpressing the tobO gene.

[0024] Secondly, the present invention provides a high-yield tobramycin-producing strain, which is constructed by the following method: inactivating the tobR gene in S. tenebrarius using genetic engineering technology, and / or overexpressing the encoding gene of the TauD / TfdA family oxidase in S. tenebrarius to obtain a high-yield tobramycin-producing strain; wherein the nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2.

[0025] Preferably, the encoding gene of the TauD / TfdA family oxidase is the tobO gene, and the nucleotide sequence of the tobO gene is as shown in SEQ ID NO.3 or the nucleotide sequence of SEQ ID NO.3 obtained by substituting one or more bases to obtain the nucleotide sequence encoding the same functional protein. The amino acid sequence of the protein encoded by the nucleotide sequence of the tobO gene is as shown in SEQ ID NO.4 or the amino acid sequence of SEQ ID NO.4 obtained by substituting one or more amino acid bases to obtain the same functional amino acid sequence.

[0026] Thirdly, the present invention also provides the application of the high-yield strain of carbamotobramycin in the production of carbamotobramycin.

[0027] The advantages of this invention compared to the prior art are:

[0028] In this study, the gene tobR, which negatively regulates the biosynthesis of carbamoyl tobramycin, was screened. Non-denaturing gel retardation electrophoresis confirmed that the transcriptional regulator TobR directly binds to the promoter region DNA of its neighboring gene tobO. In S. tenebrarius, by knocking out the tobR gene and overexpressing the tobO gene, a high-yielding carbamoyl tobramycin strain could be obtained, providing technical support for increasing the fermentation yield of carbamoyl tobramycin in industrial production.

[0029] Specifically, knocking out the tobR gene in S. tenebrarius (Tb-ΔaprJ in the specific embodiment) increased the production of carbamotobramycin by 22.38%; while overexpressing the tobR gene in Tb-ΔaprJ decreased the production of carbamotobramycin by 10.32%; thus, it is indicated that TobR is a negative regulator involved in the biosynthesis of carbamotobramycin.

[0030] Specifically, when the tobO gene was overexpressed in Tb-ΔaprJ, the yield of carbamotobramycin increased by 36.25%; while when the tobO gene was overexpressed in the S. tenebrarius mutant strain lacking the tobR gene (Tb-ΔaprJ-ΔtobR in the specific embodiment), the yield of carbamotobramycin increased by 16.40% compared with Tb-ΔaprJ-ΔtobR and by 42.42% compared with Tb-ΔaprJ; thus, it is shown that overexpression of tobO promotes the biosynthesis of carbamotobramycin. Attached Figure Description

[0031] Figure 1 It is the location of the tobR gene.

[0032] Figure 2 This is an SDS-PAGE gel image of TobR protein expression and purification (in the image, M: Protein Ladder; 1. Whole cell; 2. Supernatant; 3. Precipitate; 4. Crude protein (supernatant); 5. Purified TobR protein).

[0033] Figure 3 It is TobR protein and P tobO A schematic diagram of the non-denaturing gel retardation electrophoresis results of the probe.

[0034] Figure 4 This is a schematic diagram illustrating the construction process of the tobR gene inactivation mutant Tb-△aprJ-△tobR and the tobR gene overexpression mutant Tb-△aprJ / ermE*p-tobR;

[0035] Figure 5 This is a PCR verification diagram of the tobR inactivated mutant strain (a 428bp nucleotide fragment was deleted from the tobR gene (492bp). Validation primers were designed on both sides of the tobR gene. The mutant strain ΔtobR obtained a fragment length of 1035bp through PCR, while the original strain obtained a band of 1463bp. In the diagram, M: 5000bp DNA Marker, 1: original strain, 2: mutant strain).

[0036] Figure 6This is a chromatogram of the yield analysis of carbamoyl tobramycin shake-flask fermentation of mutant strains (specifically, HPLC analysis of the products from the starting strain Tb-△aprJ, the tobR-inactivated mutant strain Tb-△aprJ-△tobR, the tobR-overexpressing strain Tb-△aprJ / ermE*p-tobR, the empty vector-overexpressing control strain Tb-△aprJ / pSpc8660, the tobR-inactivated mutant strain Tb-△aprJ-△tobR / ermE*p-tobO, and the tobR-inactivated mutant strain Tb-△aprJ-△tobR / pSpc8660, which were fermented in shake-flasks at 37°C for 144 h in fermentation medium for 144 h).

[0037] Figure 7 This is a graph showing the results of shake-flask fermentation biomass detection (specifically, the analysis of the wet weight of the cells after 144 h of fermentation culture using the starting strain Tb-△aprJ and the inactivated tobR mutant superimposed with the overexpression of tobO strain Tb-△aprJ-△tobR / ermE*p-tobO). Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below. These embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0039] The strains and plasmids used in the following examples are shown in Table 1. *Escherichia coli* was cultured in liquid LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) or on LB solid plates supplemented with 2.0% agar at 37°C. The carbamoyl tobramycin-producing strain Tb-ΔaprJ was cultured in YEME liquid medium (0.3% yeast extract, 0.3% malt extract, 2.5% sucrose, 0.5% peptone and 1% glucose), MS solid plates (2% soybean flour, 2% mannose and 2% agar powder), seed medium (2% soybean flour, 0.3% casein, 0.5% glucose, 1% dextrin, 0.1% potassium chloride, 0.025% calcium chloride, potassium dihydrogen phosphate and 0.5% magnesium sulfate), and fermentation medium (5% soybean flour, 1% corn flour, 1.5% glucose, 0.6% silkworm pupa powder, 1% fish meal, 0.8% ammonium chloride, 0.025% light calcium carbonate and 3% soybean oil) at 37°C and 220 rpm.

[0040] All biological reagents used in the following examples were purchased from reagent companies. General procedures for handling *E. coli* and *Streptomyces* were performed according to standard operating procedures. Primer synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd., and DNA sequencing was performed by Zhejiang Youkang Biotechnology Co., Ltd.

[0041] Table 1. Strains and plasmids used in the experiments of this invention.

[0042]

[0043]

[0044] Example 1

[0045] Information related to the tobR gene:

[0046] According to genome annotation, the tobR gene is 492 bp in length and has a protein monomer size of 18.1 kDa. The nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and its encoding amino acid sequence is shown in SEQ ID NO.2.

[0047] Amino acid sequence alignment revealed that TobR is an Lrp / AsnC family transcription regulator. Lrp family transcription regulators exhibit relatively low sequence conservation, with average amino acid sequence homology between 20% and 30%, but their structure is highly conserved. Lrp-like protein monomers are typically around 15 kDa and contain two domains: an N-terminal DNA domain with a common helical-to-helical fold (HTH motif) and a C-terminal ligand-binding domain with a typical αβ-sandwich fold. This latter terminal domain, also known as the RAM (Regulation of amino acid metabolism) domain, is typically involved in amino acid metabolism. Lrp / AsnC family transcription regulator genes are usually located near target genes; it is speculated that the Lrp / AsnC family transcription regulator TobR likely also directly regulates its neighboring gene tobO (see [link to relevant documentation]). Figure 2 ).

[0048] Example 2

[0049] Heterologous expression and purification of TobR:

[0050] (1) To overexpress TobR protein in Escherichia coli BL21(DE3), PCR primers for tobR gene amplification, namely tobR-F and tobR-R (as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively), were designed. The tobR gene fragment was amplified from S. tenebrarius;

[0051] (2) The amplified and recovered tobR gene fragment was recombined with the pET28a vector to construct the expression plasmid pET28a-tobR, and a His fusion tag was added to the N-terminus of the tobR gene.

[0052] (3) The constructed pET28a-tobR plasmid was introduced into *E. coli* BL21(DE3) and induced for expression for 16 h using 0.05 mM IPTG at 28 °C and 220 rpm. His6-labeled TobR protein was expressed using Ni... 2+ Extraction and purification were performed using an NTA spin column (Sangon Biotech). The quality of the purified protein was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see [link]). Figure 3 Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Shanghai).

[0053] Example 3

[0054] Electrophoretic mobility assays (EMSAs):

[0055] The electrophoretic mobility assay was performed according to the standard EMSA procedure. DNA molecular probe P was obtained by PCR amplification using primer pairs ptobO-F and ptobO-R (as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively). tobO The DNA probe was incubated with a certain concentration of purified TobR protein in a binding buffer (10 mL Tris-HCl (pH 7.5), 5 mM MgCl2, 60 mM KCl, 10 mM DTT, 50 mM EDTA, and 10% glycerol) at 30°C for 20 min, with a reaction volume of 20 μL. After incubation, the sample was electrophoresed in a 6% non-denaturing gel at 100 V on ice for 120 min using 0.5×TBE buffer. After electrophoresis, the gel block was removed, and the DNA was stained in 0.5×TBE containing green-blue nucleic acid dye. The results were observed under a gel imaging system (see [link to gel imaging]). Figure 4 ).

[0056] Example 4

[0057] Construction of the tobR gene deletion mutant strain Tb-△aprJ-△tobR:

[0058] (1) In order to knock out the tobR gene in S. tenebrarius, the S. tenebrarius genome was used as a template and tobR-F1, tobR-R1, tobR-F2 and tobR-R2 (nucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively) were used as primers to amplify the upstream and downstream homologous arm fragments of the tobR gene, respectively;

[0059] (2) Using the amplified and recovered homologous arm fragments of the tobR gene as templates, and tobR-F1 and tobR-R2 as primers, fusion PCR was performed.

[0060] (3) The fusion fragments of the upstream and downstream homologous arms of the amplified and recovered tobR gene were recombined with the pSpc260 vector to obtain the pSpc260-ΔtobR plasmid (see Figure 5 );

[0061] (4) The constructed pSpc260-△tobR plasmid was first introduced into Escherichia coli ET12567 (pUZ8002), and then introduced into Tb-△aprJ by conjugation transfer.

[0062] The zygotes were transferred to MS solid plates containing 100 μg / mL spectinomycin for concentrated enrichment, and spores were obtained by incubation at 37°C for 5–7 days. A small amount of spores were then incubated in YEME liquid medium for 24 h, and the cultured solution was diluted 10⁻⁶. 5 Take 100 μL of the culture and spread it onto a non-antibiotic MS plate. Incubate at 37°C for 5–7 days to complete the relaxation culture for homologous recombination single exchange. When single colonies grow on the plate, pick single colonies and perform replica screening on both non-antibiotic MS plates and MS plates containing 100 μg / mL spectinomycin. Strains that grow on antibiotic-free MS plates but not on spectinomycin plates will be validated by PCR.

[0063] Using di-tobR-F and di-tobR-R (as shown in SEQ ID NO.15 and SEQ ID NO.16) as identification primers, and the control group being the Tb-△aprJ genome, the tobR gene deletion mutant strain Tb-△aprJ-△tobR (see [link to PCR product]) was obtained based on the DNA fragment size of the PCR product. Figure 6 ).

[0064] Example 5

[0065] The tobR gene is overexpressed in the originating strain Tb-ΔaprJ:

[0066] (1) To overexpress the tobR gene in Tb-ΔaprJ, primers for PCR amplification of the tobR gene, namely ermE-tobR-F and ermE-tobR-R (as shown in SEQ ID NO.17 and SEQ ID NO.18), were designed. The tobR gene fragment was amplified from S. tenebrarius;

[0067] (2) The amplified and recovered tobR gene fragment was recombined with the pSpc8660 vector to construct the expression plasmid pSpc8660-tobR (see Figure 3 );

[0068] (3) pSpc8660-tobR was introduced into Tb-△aprJ via conjugation transfer transformation. Preliminary screening was performed using spectinomycin, and PCR identification was conducted using the spectinomycin resistance gene (spc) as the target. Spc-F and Spc-R were used as identification primers (as shown in SEQ ID NO.19 and SEQ ID NO.20). The tobR overexpressing strain was named Tb-△aprJ / ermE*p-tobR.

[0069] Example 6

[0070] Construction of the control strain Tb-ΔaprJ / pSpc8660 overexpressing the empty vector:

[0071] pSpc8660 was introduced into Tb-△aprJ via conjugation transfer transformation. Preliminary screening was performed using spectinomycin, and PCR identification was conducted using the spectinomycin resistance gene (spc) as the target. Spc-F and Spc-R were used as identification primers (as shown in SEQ ID NO. 19 and SEQ ID NO. 20). The control strain overexpressing the empty vector was named Tb-△aprJ / pSpc8660.

[0072] Example 7

[0073] The tobO gene is overexpressed in the originating strain Tb-ΔaprJ:

[0074] (1) To overexpress the tobO gene in Tb-ΔaprJ, primers for PCR amplification of the tobO gene, namely ermE-tobO-F and ermE-tobO-R (as shown in SEQ ID NO.9 and SEQ ID NO.10), were designed. The tobO gene fragment was amplified from the S. tenebrarius genome;

[0075] (2) The amplified and recovered tobO gene fragment was recombined with the pSpc8660 vector to construct the expression plasmid pSpc8660-tobO;

[0076] (3) pSpc8660-tobR was introduced into Tb-△aprJ via conjugation transfer transformation. Preliminary screening was performed using spectinomycin, and PCR identification was conducted using the spectinomycin resistance gene (spc) as the target. Spc-F and Spc-R were used as identification primers (as shown in SEQ ID NO.19 and SEQ ID NO.20). The tobO overexpressing strain was named Tb-△aprJ / ermE*p-tobO.

[0077] Example 8

[0078] The tobO gene is overexpressed in the tobR gene deletion mutant Tb-△aprJ-△tobR:

[0079] pSpc8660-tobO was introduced into Tb-△aprJ-△tobR via conjugation transfer transformation. Preliminary screening was performed using spectinomycin, and PCR identification was conducted using the spectinomycin resistance gene (spc) as the target. Spc-F and Spc-R were used as identification primers (as shown in SEQ ID NO.19 and SEQ ID NO.20). The resulting mutant strain with the tobR gene deletion and tobO gene overexpression was named Tb-△aprJ-△tobR / ermE*p-tobO.

[0080] Example 9

[0081] Construction of the control strain Tb-ΔaprJ-ΔtobR / pSpc8660 overexpressing the empty vector:

[0082] pSpc8660 was introduced into Tb-△aprJ-△tobR via conjugation transfer. Preliminary screening was performed using spectinomycin, and PCR identification was conducted using the spectinomycin resistance gene (spc) as the target. Spc-F and Spc-R were used as identification primers (as shown in SEQ ID NO.19 and SEQ ID NO.20). The resulting tobR gene deletion combination, tobO overexpression empty vector control mutant, was named Tb-△aprJ-△tobR / ermE*p-tobO.

[0083] Example 10

[0084] Information related to the tobO gene:

[0085] According to genome annotation, the tobO gene is 984 bp in length and its protein monomer size is 35.6 kDa. The nucleotide sequence of the tobO gene is shown in SEQ ID NO.3, and its encoded amino acid sequence is shown in SEQ ID NO.4.

[0086] Sequence alignment of the amino acid sequence corresponding to tobO in the NCBI database revealed that tobO is an oxidase belonging to the TauD / TfdA family.

[0087] Example 11

[0088] HPLC detection of fermentation products:

[0089] S. tenebrarius was inoculated into YEME medium and cultured at 37°C with shaking at 220 rpm for 24 h. The culture was then transferred to seed culture medium and cultured at 37°C with shaking at 220 rpm for 20 h. Finally, the culture was transferred to fermentation medium and cultured at 37°C with shaking at 220 rpm for 144 h. After fermentation, the supernatant obtained by centrifugation was diluted several times and derivatized with 2,4-dinitrofluorobenzene. The reaction solution was filtered through a 0.22 μm organic filter and analyzed by high-performance liquid chromatography (HPLC). Detection was performed at 365 nm using a C-18 column (Hypersil BDS 5 μm, 4.6 mm × 250 mm). The mobile phase was 0.01 mM ammonium acetate aqueous solution (pH adjusted to 4.0 with phosphoric acid) / acetonitrile (47:53, v / v) at a flow rate of 1 mL / min. All results were repeated three times and the average value was taken.

[0090] The yield results of carbamoyl tobramycin for Tb-△aprJ, deletion mutant Tb-△aprJ-△tobR, overexpression strains Tb-△aprJ / ermE*p-tobR, Tb-△aprJ / ermE*p-tobO, Tb-△aprJ-△tobR / ermE*p-tobR, and empty vector control strains Tb-△aprJ / pSpc8660 and Tb-△aprJ-△tobR / pSpc8660 are shown in the table below. Figure 7 .

[0091] Example 12

[0092] Biomass detection during shake-flask fermentation:

[0093] The same inoculum was used to simultaneously inoculate the starting strain Tb-△aprJ and the tobR gene deletion combination tobO gene overexpression mutant Tb-△aprJ-△tobR / ermE*p-tobO into the fermentation medium. Sterile water with the same inoculum volume was used as a blank control. After fermentation at 37℃ for 144 h, biomass was measured. 10 mL of fermentation broth was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the weight of the precipitate after centrifugation in the experimental group was subtracted from that in the control group. The weight difference was defined as the wet cell biomass. The biomass of the fermentation broths of the mutant strain and the starting strain were compared. The results are as follows: Figure 7As shown, the combined mutant strain showed a slight increase in wet cell mass compared to the starting strain, indicating that tobR may be involved in regulating mycelial growth during fermentation.

[0094] Example 13

[0095] Observation of the mycelial growth status and spore growth of S. tenebrarius:

[0096] To determine whether the knockout of the tobR gene affects the morphological differentiation of the bacteria, the deletion mutant and the starting strain were streaked on MS solid plates and cultured at 37°C for 120 h. The mycelial growth and spore growth of each strain were observed and compared, and no significant differences were found.

[0097] Example 14

[0098] This embodiment is used to analyze and summarize the experimental results of the above embodiments:

[0099] 1. Basic information about the tobR gene:

[0100] The location of the tobR gene in the tobramycin synthesis gene cluster of S. tenebrarius is shown in [reference]. Figure 1 The nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2.

[0101] 2. TobR binds to the promoter region DNA of the adjacent gene tobO:

[0102] TobR protein and P tobO See probe binding Figure 4 EMSA analysis revealed that TobR protein is related to P tobO The probe exhibited significant in vitro binding activity; in the control group without TobR, P... tobO The probe migration rate is relatively fast, as evidenced by P at point a. tobO Strips, some P after adding TobR to the system tobO When the probe binds to the TobR protein, the migration rate of the protein-DNA complex decreases, and a new band appears at position b in the gel image, which represents P. tobO The TobR complex was observed, and as the concentration of TobR protein in the system increased, the band at point b gradually brightened, while the band at point a gradually darkened, indicating the presence of more P in the system. tobO The probe was blocked by TobR protein binding, and in vitro EMSA experiments proved that TobR protein binds to P. tobO The probes generated a direct interaction, suggesting that TobR may exert a regulatory effect by influencing the transcriptional level of tobO.

[0103] 3. Tb-△aprJ-△tobR showed increased carbamoyltobramycin production compared to the original strain Tb-△aprJ:

[0104] The construction process of the tobR gene inactivation mutant Tb-△aprJ-△tobR is described in [link to documentation]. Figure 5 Inactivation mutants were screened on MS plates containing spectinomycin and confirmed by PCR (electrophoresis results are shown in [link to electrophoresis results]). Figure 6 △tobR was fermented in fermentation medium for 6 days. After centrifugation of the fermentation broth, the supernatant was collected, diluted, derivatized, and analyzed by HPLC. The yield of carbamoyl tobramycin was 22.38% higher than that of the starting strain Tb-△aprJ (see...). Figure 7 The results suggest that tobR may be a negative regulator involved in the biosynthesis of carbamoyltobramycin.

[0105] 4. Overexpression of tobR in Tb-ΔaprJ leads to a decrease in carbamoyltobramycin production:

[0106] To further verify the function of tobR in the host *S. tenebrarius* and confirm its relationship with carbamoyl tobramycin yield, this invention also designed a tobR overexpression experiment. The expression vector pSpc8660-tobR was introduced into Tb-ΔaprJ to construct the overexpression strain Tb-ΔaprJ / ermE*p-tobR. Fermentation and HPLC analysis revealed that the carbamoyl tobramycin yield was reduced by 10.32% compared to the control Tb-ΔaprJ / pSpc8660 (see...). Figure 6 In summary, the results confirm that tobR is a negative regulator of carbamotobramycin biosynthesis in S. tenebrarius, and that inactivating the tobR gene in S. tenebrarius through genetic engineering can increase the yield of carbamotobramycin.

[0107] Based on the experimental results of Examples 4-6, we know that: inactivating the tobR gene in S. tenebrarius through genetic engineering increases the yield of carbamoyl tobramycin; overexpressing the tobR gene in the initial strain decreases the yield of carbamoyl tobramycin; therefore, the tobR gene is identified as a negative regulatory gene controlling tobramycin biosynthesis.

[0108] 5. Overexpression of tobO in Tb-ΔaprJ leads to increased production of carbamoyltobramycin:

[0109] To further verify the function of tobO in the host *S. tenebrarius* and confirm its relationship with carbamoyl tobramycin production, this invention designed a tobO overexpression experiment. The expression vector pSpc8660-tobO was introduced into Tb-△aprJ and Tb-△aprJ-△tobR, respectively, to construct overexpression strains Tb-△aprJ / ermE*p-tobO and Tb-△aprJ-△tobR / ermE*p-tobO. Fermentation and HPLC analysis revealed that the carbamoyl tobramycin production was increased by 36.25% and 16.33% compared to the controls Tb-△aprJ / pSpc8660 and Tb-△aprJ-△tobR / pSpc8660, respectively. The highest carbamoyl tobramycin production was observed in the hybrid mutant strain Tb-△aprJ-△tobR / ermE*p-tobO, at 3.76 g / L (see [link to original text]). Figure 6 In summary, the results confirm that tobO promotes the biosynthesis of carbamotobramycin in S. tenebrarius, and that overexpression of the tobO gene in S. tenebrarius through genetic engineering can increase the yield of carbamotobramycin.

[0110] 6. Effects of tobR gene deletion combined with tobO gene overexpression on cell growth and spore morphology differentiation:

[0111] By comparing the wet weight of the control strain Tb-△aprJ and the mutant strain Tb-△aprJ-△tobR / ermE*p-tobO in the fermentation medium after 144 h, the results showed that the biomass of Tb-△aprJ-△tobR / ermE*p-tobO was slightly increased compared with that of Tb-△aprJ. Figure 7 This indicates that the combination may have some impact on the growth of the strain. Furthermore, to determine whether the combined mutation regulates spore formation, the mutant strain Tb-△aprJ-△tobR / ermE*p-tobO and the control strain Tb-△aprJ were simultaneously streaked on MS agar plates and incubated at 37℃. Spore growth was observed over a 144-hour incubation period. Compared to Tb-△aprJ, the mycelial growth and spore morphology of the deletion mutant Tb-△aprJ-△tobR / ermE*p-tobO showed no significant difference.

[0112] Accordingly, a method for increasing the yield of carbamotobramycin using the S. tenebrarius tobR and tobO genes can be obtained: Starting with the S. tenebrarius genes tobR and tobO or their expression products, a high-yield carbamotobramycin strain is obtained by deleting the Lrp / AsnC family transcriptional regulator tobR and overexpressing the TauD / TfdA family oxidase tobO (see the above examples for details), thereby obtaining a high-yield S. tenebrarius carbamotobramycin strain. The obtained corresponding high-yield engineered S. tenebrarius carbamotobramycin strain is then used for fermentation to produce carbamotobramycin.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for increasing the yield of carbamoyltobramycin, characterized in that, Through genetic engineering technology S. tenebrarius medium inactivation tobR Genes, and / or S. tenebrarius By overexpressing the encoding gene of the TauD / TfdA family oxidase, a high-yield strain of carbamoyl tobramycin was obtained and used for the fermentation production of carbamoyl tobramycin; tobR The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2; S. tenebrarius To knock out aprJ Genetic S. tenebrarius The encoding gene for the TauD / TfdA family oxidase is: tobO Gene, tobO The nucleotide sequence of the gene is shown in SEQ ID NO.

3. tobO The nucleotide sequence of the gene encodes the amino acid sequence of the protein, as shown in SEQ ID NO.

4.

2. The method for increasing the yield of carbamotobramycin according to claim 1, characterized in that, The above S. tenebrarius medium inactivation tobR The genetic method is as follows: S. tenebrarius Knockout tobR Gene.

3. The method for increasing the yield of carbamotobramycin according to claim 1, characterized in that, The above S. tenebrarius medium inactivation tobR The genetic approach is as follows: (1) with S. tenebrarius Using the genome as a template, primer pairs were designed for amplification. tobR Upstream and downstream homologous arm fragments of a gene; (2) tobR The upstream and downstream homologous arm fragments of the gene are fused together; (3) with tobR The fusion product of upstream and downstream homologous arm fragments of the gene is recombined with the knockout plasmid vector fragment to obtain... tobR Gene knockout plasmids; (4) tobR The gene knockout plasmid was introduced into E. coli ET12567 (pUZ8002) and then transferred via conjugation transfer. S. tenebrarius In the process, after two rounds of homologous recombination exchanges are completed, the missing cells are screened out. tobR Mutant strains of the gene.

4. The method for increasing the yield of carbamotobramycin according to claim 1, characterized in that, The S. tenebrarius Streptomyces strong promoter overexpression tobO Gene.

5. The method for increasing the yield of carbamotobramycin according to claim 1, characterized in that, overexpression tobO The genetic approach is as follows: (a) with S. tenebrarius Using the genome as a template, amplification tobO Gene fragments; (b) will tobO The gene fragment was recombined with an integrative overexpression vector containing a strong streptomyces promoter to obtain... tobO Overexpression plasmids; (c) will tobO The overexpression plasmid was first introduced into E. coli ET12567 (pUZ8002), and then introduced via conjugation transfer. S. tenebrarius Screening yielded integrated overexpression tobO Mutant strains of the gene.

6. The method for increasing the yield of carbamotobramycin according to claim 5, characterized in that, In step (c) S. tenebrarius For missing tobR Genetic S. tenebrarius The mutant strain obtained in step (c) is the deletion mutant. tobR Simultaneous overexpression of genes tobO Genetic S. tenebrarius Mutant strain.

7. A high-yield strain of carbamoyltobramycin, characterized in that, The strain was constructed using the following method: employing genetic engineering technology... S. tenebrarius medium inactivation tobR Genes, and / or S. tenebrarius By overexpressing the encoding gene of the TauD / TfdA family oxidase, a high-yield strain of carbamoyl tobramycin was obtained; S. tenebrarius To knock out aprJ Genetic S. tenebrarius ;in tobR The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2; the gene encoding the TauD / TfdA family oxidase is... tobO Gene, tobO The nucleotide sequence of the gene is shown in SEQ ID NO.

3. tobO The nucleotide sequence of the gene encodes the amino acid sequence of the protein, as shown in SEQ ID NO.

4.

8. The application of the high-yield carbamotobramycin strain according to claim 7 in the production of carbamotobramycin.

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