CN111690582B - Engineering bacterium for reducing neomycin impurity C and application thereof - Google Patents

Abstract

The invention discloses an engineering bacterium for reducing neomycin impurity C and application thereof, and also discloses a construction method of the engineering bacterium, wherein the preservation number of the obtained engineering bacterium is CGMCC No. 18778.

Description

Engineering bacterium for reducing neomycin impurity C and application thereof

Technical Field

The invention belongs to the field of microbial pharmacy, and particularly relates to construction and application of engineering bacteria for reducing and transforming neomycin impurity C components.

Background

Streptomyces (Streptomyces) is a gram-positive soil filamentous bacterium with high GC content, and belongs to the family Streptomycetaceae of actinomycetes in the kingdom Prokaryotae. In addition to having a complex developmental differentiation, it is also capable of producing abundant secondary metabolites such as various classes of antibiotics like macrolides, polyketides, aminoglycosides, etc. The antibiotics have different degrees of bacteriostatic and bactericidal capacities on various strains, can be used as anticancer drugs, herbicides, enzyme inhibitors, immunomodulators and the like, and play an important role in the fields of medical treatment, industry, agriculture, animal husbandry, fishery, environmental protection and the like.

Neomycin is an aminoglycoside antibiotic, which was first isolated from Streptomyces fradiae and Streptomyces leucinerea in 1949, and mainly binds to bacterial ribosome 30S subunit to inhibit bacterial protein synthesis, and is G ⁺ 、G ^- Bacteria, tubercle bacillus, etc. all have inhibitory action and have broad-spectrum antibacterial activity, see Waksman SA, Lechevalier ha. (1949), Neomycin, a new antibacterial active peptide induced bacterium-resistant bacterium, included bacteria organisms science.109 (2830): 305-307. In addition, studies have shown that neomycin is likely to be an antitumor drug with great potential for development, see Bottero V., (2013), Kaposi's sarcoma-associated human virus-positive primary effect kinetic formation in NOD/SCID microorganism inactivated by neomycin and polyamine blocking and antitumor's nuclear transfer Virology 87(21)：11806-11820；Hu GF(2001)Neomycin inhibits the angiogenic activity of fibroblast and epidermal growth factors.Biochemical&Biophysical Research Communication 287 (4): 870-874. Because neomycin has certain ototoxicity, the use of neomycin in the aspects of clinical oral administration, injection and the like is limited, and only a small amount of neomycin is prepared into ointment to be applied to skin for diminishing inflammation. At present, the compound antibiotic is mainly used as an antibiotic for livestock, can prevent and treat intestinal infection, white diarrhea, typhoid fever and respiratory diseases of livestock and poultry, can improve the utilization rate of feed, and promotes the growth of livestock and poultry and fish, so the compound antibiotic is also commonly used as a feed additive. In 1994, the pharmacokinetic reports of neomycin submitted to the FDA in the united states by Rooing and Fagerberg indicate its high safety to animals and thus become one of the most commonly used veterinary antibiotics abroad. Neomycin belongs to multicomponent antibiotics, the activity of the neomycin is closely related to the number of carried amino groups and the position of the carried amino groups, and the neomycin mainly comprises three components of neomycin A, neomycin B and neomycin C, wherein the main effective component neomycin B has strong activity and weak toxicity relative to other components; neomycin A is also called neomycin amine, is a precursor substance in a synthesis path, is quickly catalytically converted in the synthesis process and has low content, and is not regarded as a main byproduct in industrial production; neomycin C is a precursor for synthesizing neomycin B, the configuration of the neomycin C is different only at the position of amino sugar 5' (the neomycin B is in an L type), and the neomycin C is difficult to separate because of weaker biological activity and stronger toxicity, more yield accumulation in industrial production and similar structure and hydrophilicity to the neomycin B, and is regarded as an impurity in industrial production. The content of neomycin C in the existing neomycin production process is still higher, and the mutant strain screened by the strategies such as physical mutagenesis, chemical mutagenesis and the like applied in the prior art is time-consuming and labor-consuming, has blindness, has poor stability after mutation and cannot meet the requirement of reducing the content of the neomycin C component as an impurity at present. Therefore, the key problem to be solved urgently at present is to obtain the industrial production strain with high effective component yield, low impurity content and good genetic stability.

Wherein the structure of neomycin A, also known as neomycin amine, is shown in the yellow circle (top left) and the 5' "position on the amino sugar of neomycin C and neomycin B, R and L, respectively, are shown in the red circle (bottom left and bottom right). The conversion of neomycin C to neomycin B is catalyzed by the SAM-dependent epimerase NeoN. The structure of neomycin impurity C is shown below:

in the biosynthetic pathway of neomycin, the by-product neomycin C is a precursor of neomycin B. In 2014, Kudo et al showed by in vitro experiments that the conversion of neomycin C to neomycin B was effected by the SAM (S-adenosyl-L-methionine) -dependent epimerase NeoN, converting the R-type amino group to the L-type amino group, see Kudo F, Hoshi S, Kawashima T, Kamachi T, Eguchi T. (2014.) the Characterisation of a radial S-adenosyl-L-methione epoxide, NeoN, in the last step of neomycin B biosynthesis, J Am Chem Soc.136 (39): 13909-15. SAM has an activated methyl group, is a coenzyme participating in the methyl transfer reaction, plays an important role in the metabolism in the cells of organisms, and is a methyl donor for more than 100 different methyl transfer enzyme catalytic reactions in vivo. In the secondary metabolism of Streptomyces, SAM accumulation can improve the actinopurpurin yield in Streptomyces lividans TK23 to inhibit sporulation, and can also improve the transcription of global regulatory gene adpA in Streptomyces griseus to promote streptomycin biosynthesis, SAM plays a crucial role in the biosynthesis and regulation pathways of secondary metabolites, see Kim DJ, Huh JH, Yang YY, Kang CM, Lee IH, Hyun CG, Hong SK, Suh JW. (2003). J bacteriol.Accumulantion of S-adenosyl-L-methionine production process of actinomycin but not genes plasmid coding in Streptomyces lividans TK23.185 (2): 592-600, Shin SK, Xu D, Kwon HJ, Suh JW. (2006), S-adenosylmethionine activators adpA transcription and promoters biosynthesis sis in Streptomyces griseus. FEMS Microbiol Lett.259 (1): 53-9. Therefore, the reduction of the content of the by-product neomycin C by genetic engineering is a key point for the component optimization of neomycin. A complete neomycin biosynthesis gene cluster is cloned in streptomyces fradiae through a phi BT1 attB/attP-int system, a PCR targeting large-fragment editing method is applied to block a negative control gene neoI in the cloned neomycin biosynthesis gene cluster, and a strong promoter P is utilized _kasO* The transcription of the co-transcription unit of the key enzyme coding gene neoE-neoD is driven, the total output of neomycin in the obtained recombinant engineering strain Sf/pKCZ03 is increased by 35.78%, wherein the main effective component neomycin B is increased by 22.60%, but the neomycin C component is also increased by 13.18%, see Zheng J, Li Y, Guan H, Zhang J, Tan H. (2018), engineering of neomycin production by Y engineering the enteric biochemical gene cluster and feeding key precursors in streptomyces freadiae CGMCC 4.576.Appl Microbiol Biotechnology 103 (5): 2263-2275. The yield of the neomycin B and the content of the neomycin C are always in a dynamic change process, and the yield of the neomycin B and the content of the neomycin C cannot be simultaneously increased and reduced by means of single-factor variable transformation in a completely ideal mode.

With the development of genome sequencing technology and molecular biology, methods for large fragment cloning and editing are becoming more mature, for example: gibson in vitro isothermal one-step DNA assembly technology (Gibson, d.g., et al (2009)., Enzymatic assembly of DNA molecules up to sectional and cloned nucleic acids, nature Methods, 6(5), 343.), makes it easier to engineer microbial metabolism using genetic engineering techniques.

Disclosure of Invention

In view of the above situation, the present invention aims to obtain an engineered strain with reduced neomycin C impurity content by modifying neomycin synthesis related genes through genetic engineering means in order to reduce neomycin C impurity content. The engineering bacterium SF003 constructed in the specification can reduce the content of neomycin C to about 7.0%. The starting strain Streptomyces fradiae YC816 used in the present invention is derived from Yichang Sanxia pharmaceutical Co., Ltd.

In order to achieve the above purpose, the invention relates to the following technical steps:

(1) SAM synthetase-encoding Gene metK and epimerase-encoding Gene neoN were first highly expressed and constructed on an integration vector pSET152, see (Bierman M, Logan R, O' Brien K, Seno ET, Rao RN, Schoner BE. (1992.) Plasmid cloning vectors for the synthetic transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 116: 43-49.) stably inserted into the genomic DNA of Streptomyces fradiae to enhance SAM supply and expression of NeoN in the cells, and the engineered strain obtained by this construction reduced neomycin C content to 10.15%.

(2) Secondly, the up-regulated gene neoGH and the resistance gene aphA in the neomycin biosynthetic pathway were identified as P _kasO High expression was performed under the drive of a promoter, which was constructed on the integration vector pSET152, to obtain an aphA-neoGH high expression strain having a neomycin C content reduced to 8.41%.

(3) Finally, the down-hybridizing module neoN-metK and the regulatory and resistance module aphA-neoGH are integrated and constructed on the carrier pSET152, and the engineering strain SF003 with the same high expression of the neoN-metK-aphA-neoGH 5 genes is obtained. The method reduces the content of neomycin C to 6.99%. The applicant carries out patent preservation on SF003 bacterial strains in the common microorganism center of China Committee for culture Collection of microorganisms (No. 3 Siro 1 of Beijing Korean-Yang district), the preservation number is CGMCC No.18778, the preservation time is 11 months and 1 day in 2019, and the strain is classified and named as Streptomyces fradiae.

The recombinant strain and the recombinant plasmid are all within the protection scope of the invention. Promoters as mentioned in the present inventionNot restricted to P _kasO* Also included are strong promoters commonly used in other Streptomyces by genetic manipulation or promoters with high fitness to neoN-metK and aphA-neoGH, such as erythromycin P _ermE* Promoter (Bibb MJ, White J, Ward JM, Janssen GR, the mRNA for the 23S rRNA methyl encoded by the ermE gene of Saccharomyces cerevisiae a transformed in the absence of a genetic promoter-binding site Mol Microbiol 14 (3): 533-. The high expression neoN-metK and aphA-neoGH are not limited to the sequences, but also include other amino acid sequences with equivalent efficacy, such as substitution, deletion or truncation, which can make the normal or high expression of the neoN-metK and the aphA-neoGH. Such high expression vectors include, but are not limited to, pSET152 as used herein, and other integrative or multicopy plasmids such as pKC1139(Kieser, t., Bibb, m.j., butterer, m.j., Chater, k.f., and Hopwood, d.a. practical Streptomyces Genetics, John ins Foundation, Norwich, United Kingdom), etc. are also within the scope of the present invention to enable normal or efficient expression of the desired transgene. The recombinant plasmid can be realized by protoplast transformation, chemical or electric shock transformation and the like, and the conjugal transfer technology is preferred in the invention.

More specifically, the present invention provides the following:

1. the modified Streptomyces fradiae is characterized by being represented by the preservation number of CGMCC No. 18778.

2. Application of the modified streptomyces fradiae in producing neomycin.

3. The modified streptomyces fradiae is applied to reducing the content of neomycin impurity C in neomycin production.

The invention reduces the content of neomycin impurity C from the source of the strain, can ensure that the product quality reaches the international highest standard, realizes higher selling price and economic benefit, simultaneously reduces the difficulty of extraction and separation and the discharge amount of wastewater, and can obtain better environmental protection social benefit.

Drawings

In order to facilitate understanding of the objects, technical solutions and presenting better results of the present invention, further description is provided by the accompanying drawings.

Fig. 1A is recombinant plasmid pSET 152: : p _kasO* Schematic diagram of-neoN-metK construction, FIG. 1B shows the detection of engineered strain SF001 (i.e., S.fragiae YC816/pSET 152:: P) by HPLC-ECD _kasO* -neoN-metK) and the relative content of neomycin B.

Fig. 2A is recombinant plasmid pSET 152: : p _kasO* FIG. 2B is a schematic diagram showing the construction of-aphA-neoGH, and the detection of the engineered strain SF002 (i.e., S.fragiae YC816/pSET 152:: P) by HPLC-ECD _kasO* Analysis of neomycin C content and of neomycin B relative content in aphA-neoGH).

Fig. 3A is recombinant plasmid pSET 152: : p _kasO* -aphA-neoGH-P _kasO* FIG. 3B is a schematic diagram showing the construction of-neoN-metK, and FIG. 3B is a diagram showing the detection of the engineered strain SF003 (i.e., S.fragiae YC816/pSET 152:: P) by HPLC-ECD _kasO* -aphA-neoGH-P _kasO* -neoN-metK) and the relative content of neomycin B.

Detailed Description

The invention is further illustrated by way of example and with reference to the accompanying drawings.

Example 1: construction of neomycin neoN-metK Module

The conversion process of the by-product neomycin C to the target end product neomycin B requires the catalysis of SAM dependent epimerase NeoN, and the main function of the NeoN is to convert the R-type configuration at the 5' -position on the amino sugar of the neomycin into the L-type configuration. SAM is an important coenzyme participating in a methyl transfer reaction in a biological cell metabolic process, the SAM is low in content in cells and easy to degrade, SAM purchased in the market is expensive and very unstable, and is not suitable for long-time storage, a method for increasing the yield of antibiotics by adding SAM in a culture medium in industrial production is not economical, and some researches show that the method for externally adding SAM cannot effectively increase the yield of antibiotics, see Zhao XQ, Gust B, Heide L. (2010), S-adenosylmethionine (SAM) and antibacterial biosyntheses: effect of external addition of SAM and of overexpression of SAM biochemical genes on novobiocin production in Streptomyces 192 (4): 289-297. In addition, the catalytic activity of S-adenosyl-L-methionine synthetase is poor, the ability of converting into SAM by simply adding substrate L-methionine is limited, the inventor has tried to increase the supply ability of SAM to increase the yield of neomycin in the fermentation process by adding L-methionine in the previous work, the actual effect is not significant (data not shown), therefore, in conclusion, it is required to highly express SAM synthetase encoding gene metK and epimerase encoding gene neoN in the cell to realize the increase of SAM level and increase the conversion of L configuration.

First, using S.fradiae YC816 genomic DNA as a template, using primers neoN-F (par)/neoN-R (EcoRI) (SEQ ID NO.9 and SEQ ID NO.10 of the sequence listing) to amplify the gene encoding the epimerase neoN required for the neomycin synthetic pathway (SEQ ID NO.1 of the sequence listing), and using primers kasO-F (xbai) and kasOp-R (par) (SEQ ID NO.7 and SEQ ID NO.8 of the sequence listing) to amplify P _kasO* A strong promoter (the nucleic acid sequence of the promoter is shown in SEQ ID NO.6, Wang, W., et al (2013). An engineered promoter for streptomyces. appl Environ Microbiol, 79(14), 4484-4492), wherein kasOp-R (Pan) and neoN-F (Pan) need to be subjected to primer phosphorylation before PCR, and the promoter of the kasOp purified by XbaI digestion and the neoN fragment purified by EcoRI digestion are subjected to three-fragment co-ligation with the pSET152 cut by XbaI/EcoRI to obtain the vector pSET 152: : p _kasO* -neoN. Using S.fradiae YC816 genomic DNA as a template, amplifying a coding region of SAM synthetase coding gene metK and an RBS region (SEQ ID NO.2 of the sequence Listing) by using RBS (SAM) -F and RBS (SAM) -R (EcoRI) (sequence Listing SEQ ID NO.11 and SEQ ID NO.12), and carrying out enzyme digestion on the fragment and the pSET 152: : p _kasO* -neoN for Gibson ligation, obtaining the vector pSET 152: : p _kasO* -neoN-metK, the sequence of fragment neoN- (RBS) metK is shown in sequence table SEQ ID NO. 3.

And (3) PCR reaction system: 2 XPCR Buffer KOD FX Buffer 50. mu.L, 2mM dNTPs 20. mu.L, DMSO 5. mu.L, KOD FX (1U/. mu.L) 2. mu.L (reagents above are purchased from TOYOBO, Japan), 10. mu.M primers each 3. mu.L, genomic template 2. mu.L (1-50ng), ddH ₂ O19. mu.L, total reaction volume 100. mu.L. PCR cycling conditions: pre-denaturation: 94 ℃ for 3 min. Denaturation: 94 ℃, 30 sec; annealing:60 ℃ for 30 sec. Extension: 68 ℃, 1kb/min, 30 cycles; storing at 68 deg.C for 5min and 4 deg.C.

The plasmid pSET 152: : p _kasO* Introduction of NeoN-metK into E.coli ET12567/pUZ8002 (see Flett, F., ET al (1997) for E.coli ET12567/pUZ8002 strain information, High efficiency endogenous conjugate transfer of plasmid DNA from Escherichia coli to methyl DNA-restriction microorganisms FEMS Microbiol Lett, 155 (2): 223-: : p _kasO* -neoN: metK, this strain was named SF 001.

The detailed method of the bond transfer is as follows: the clone containing the recombinant plasmid was picked up and cultured in liquid LB medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.0) to OD ₆₀₀ 0.4 to 0.6. The collected thalli are washed twice by a fresh LB culture medium without antibiotics, and the thalli are suspended in a 2 XYT culture medium (tryptone 16g/L, yeast powder 10g/L, NaCl 5g/L and pH 7.0) in equal volume for later use. Culturing Streptomyces fradiae (S.fradiae YC816) in a slant culture medium (glucose 1.0%, beef powder 0.2%, dipotassium hydrogen phosphate 0.05%, DL-aspartyl 0.05%, agar 1.8-2.5% (2.2%), pH 7.5-8.0, 121 ℃, and sterilizing for 30 min), culturing for 4-5 days at 28 ℃, washing spores of the Streptomyces fradiae twice with a 2 XYT culture medium after the spores are produced, suspending the spores in the 2 XYT culture medium, thermally shocking for 10 min in a water bath kettle at 50 ℃, and cooling to room temperature for later use. mu.L of E.coli ET12567/pUZ8002 containing recombinant plasmid and 500. mu.L of heat-shocked Streptomyces fradiae spore suspension were mixed well and finally spread onto MS solid medium (mannitol 2%, soy flour 2%, agar 2%, pH natural, 115 ℃, 30min sterilized) plates. The correct zygotes were obtained by screening and PCR validation.

Respectively carrying out liquid fermentation on the wild type strain and the recombinant strain, carrying out centrifugal filtration on fermentation liquor, and then carrying out ion chromatography quantitative analysis (HPLC-ECD).

The fermentation process comprises the following steps:

the seed culture medium formula comprises: TSB 30g/L, pH natural, 121 ℃, 30min sterilization.

The fermentation medium formula comprises: 8.0 percent of rice flour, 1.4 percent of glucose, 3.0 percent of soybean meal powder, 0.05 percent of disodium hydrogen phosphate, 0.01 percent of potassium dihydrogen phosphate, 0.3 percent of corn steep liquor, 0.5 percent of ammonium sulfate, 0.45 percent of calcium carbonate, 0.02 percent of amylase and 0.45 percent of sodium chloride, natural pH, and sterilization is carried out at 121 ℃ for 30 min.

The strain is inoculated in a TSB liquid culture medium, after 24 hours of culture, 10 percent of thalli are respectively transferred to 50mL (500mL triangular flask) of fermentation culture medium, the fermentation is carried out for 6.5 days at 35 ℃, then the thalli are removed by centrifugation, and the supernatant is taken for HPLC-ECD analysis.

The HPLC-ECD detection conditions were as follows:

an Agilent zorbax-SB C18(5 μm, 4.6X 250mm) chromatography column was used; the mobile phase is 25 mL TFA +6mL NaOH (50%, W/W) with constant volume of 1000mL and flow rate of 0.7 mL min ^-1 The flow rate of the post-column derived solution is 0.5M NaOH solution and 0.5 mL/min ^-1 The column temperature is 30 ℃, the sample injection amount is 20 mu L, and the electrochemistry adopts a pulse ampere detection mode: e ₁ ＝+0.1V，E ₂ ＝+0.8V，E ₃ ＝-0.6V，t ₁ ＝290ms，t ₂ ＝150ms，t ₃ ＝60 ms，t _s ＝180ms，t _{General assembly} ＝500ms。

After HPLC-ECD analysis, data statistics shows that compared with a wild type strain, the neomycin engineering strain SF001 can reduce the neomycin C content from 18.52% to 10.15%, and the neomycin B yield is close to that of the original strain, as shown in figure 1. In the next step, research work for further reducing the content of the impurity neomycin C still needs to be carried out.

Example 2: construction of the neomycin aphA-neoGH Module

The neoGH gene in the neomycin gene cluster is presumed to be the most directly regulated gene of the neomycin biosynthesis pathway, but the mechanism of action is not clear. Earlier studies showed that the knockout of positive regulatory genes afsA-g and neoR both resulted in a decrease in the transcriptional levels of the regulatory gene neoGH and the resistance gene aphA, while the high expression of these two genes resulted in a significant increase in Neomycin production compared to wild-type, see Meng X, Wang W, Xie Z, Li P, Li Y, Guo Z, Lu Y, Yang J, Guan K, Lu Z, Tan H, Chen Y., (2017). Neomycin biosynthesis peptides regulated genetic byAfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387, Sci China Life Sci.60 (9): 980-991. Therefore, we speculate that neoGH may activate key structural genes related to neomycin biosynthesis, especially neoE-D, which contains neoN, and since high expression of resistance genes can improve cell tolerance and aphA and neoGH belong to a co-transcriptional unit, aphA and neoGH are located at P _kasO* Simultaneously high expression is carried out, so as to further enhance the efficiency of the transformation of neomycin C to neomycin B and increase the secretion of products to the outside of cells.

First, using S.fradiae YC816 genomic DNA as a template, using primers neoH-F (par)/aphA (EcoRI) -R (SEQ ID NO.13 and SEQ ID NO.14 of the sequence Listing) to amplify the transcription unit aphA-neoGH region of the resistance gene and the regulatory gene (SEQ ID NO.4 of the sequence Listing), and using primers kasO-F (xbai) and kasOp-R (par) (SEQ ID NO.7 and SEQ ID NO.8 of the sequence Listing) to amplify P _kasO* The strong promoter (the nucleic acid sequence of this promoter is shown in SEQ ID NO.6, supra), wherein kasOp-R (Pan) and neoH-F (Pan) require primer phosphorylation prior to PCR, and the XbaI-cleaved and purified kasOp promoter and EcoRI-cleaved and purified aphA-neoGH fragment was subjected to triple-fragment co-ligation with the XbaI/EcoRI-cleaved pSET152 to obtain the vector pSET 152: : p is _kasO* -aphA-neoGH. The plasmid is introduced into a streptomyces fradiae YC816 original strain by means of conjugative transfer, and a recombinant strain S.fradiae YC816/pSET152 is obtained: : p is _kasO* The yield of the neomycin B in the engineering bacteria is 92.45 percent of that of the original strain, and the content of the neomycin C in the engineering bacteria is reduced from 18.84 percent to 8.41 percent (see figure 2) through fermentation and HPLC-ECD analysis detection, wherein the nomenclature of the-aphA-neoGH is SF 002. The conjugation transfer method, fermentation method and HPLC-ECD analysis method were the same as in example 1.

Example 3: construction of neomycin neoN-metK-aphA-neoGH Module

In combination with the analysis of the above results, we expressed the neoN-metK and aphA-neoGH modules in combination in order to obtain a composition-optimized engineered bacterium. First, with plasmid pSET 152: : p is _kasO* PCR amplification with primers kasO-F (XbaI) and aphA (XbaI) -R (SEQ ID NO.7 and NO.15 of the sequence Listing) using-aphA-neoGH as a template to obtain P _kasO* -aphA-neoGH fragment anddigested with XbaI, plasmid pSET 152: : p is _kasO* XbaI digestion with-neoN-metK followed by P _kasO* -ligation of aphA-neoGH fragments, obtaining the vector pSET152 for combined expression of 5 genes: : p _kasO* -neoN-metK-P _kasO* -aphA-neoGH, fragment P thereof _kasO* -neoN-metK-P _kasO* The sequence of-aphA-neoGH is shown in the sequence table SEQ ID NO. 5. The plasmid is introduced into a starting strain streptomyces fradiae YC816 through conjugative transfer to obtain a recombinant strain S.fradiae YC816/pSET 152: : p _kasO* -neoN-metK-P _kasO* -aphA-neoGH, named SF 003.

The research result shows that the content of neomycin C in the strain is reduced from 17.38% to 6.99%, the reduction amplitude is up to 59% on average, and the yield of neomycin B is close to that of the original strain (figure 3). The conjugation transfer method, fermentation method and HPLC-ECD analysis method were the same as in example 1. In conclusion, the integration operation by reducing the impurity module and the regulatory-resistance module is an effective strategy for reducing the neomycin C impurity and optimizing the neomycin component.

TABLE 1 plasmids and strains

Kieser，T.，Bibb，M.J.，Buttner，M.J.，Chater，K.F.，and Hopwood，D.A.(2000). Practical Streptomyces Genetics(The John Innes Foundation).

Paget，M.S.，Chamberlin，L.，Atrih，A.，Foster，S.J.，and Buttner，M.J.(1999) Evidence that the extracytoplasmic functionσfactorσ ^E is required for normal cell wall structure in Streptomyces Coelicolor A3(2).J.Bacteriol.181， 204-211.

Sambrook，J.，and Russell，D.W.(2001)MolecularCloning：A Laboratory Manual，3rd Ed.，Appendix A3.7，Cold Spring Harbor Laboratory Press，NY.

TABLE 2 primer sequences

Claims (3)

1. The modified Streptomyces fradiae (Streptomyces fradiae) is characterized by being represented by the preservation number CGMCC No. 18778.

2. Use of the modified Streptomyces fradiae of claim 1 in the production of neomycin.

3. Use of the modified Streptomyces fradiae of claim 1 for reducing neomycin impurity C content in neomycin production.

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