WO2012119484A1 - Plasmid and method for using plasmid to increase yield of streptomyces antibiotic - Google Patents

Abstract

Provided are a plasmid and a method for using the plasmid to increase the yield of a streptomyces antibiotic. The method comprises the following steps: transferring an integrative expression plasmid into streptomyces, thus increasing the yield of the streptomyces antibiotic. The integrated expression plasmid is derived from vgbS gene and a regular vector, or from metK and vgbS genes and the regular vector, or from metK and adpA-c genes and the regular vector, or from metK, vgbS, and adpA-c genes and the regular vector. Also provided is an integrated expression plasmid pFVgb, an integrated expression plasmid pFMV, an integrated expression plasmid pFMA, and an integrated expression plasmid pFMVA. In the plasmid and the method for using the plasmid to increase the yield of streptomyces antibiotic of the present invention, one or a plurality of genes among three genes having a broad catalyzing effect on biosynthesis of the antibiotic are expressed simultaneously on one vector, and are transferred using conjunction transfer into streptomyces for highly-efficient expression, thus increasing the yield of the antibiotic.

Description

 Description of plasmids and methods for increasing the production of Streptomyces antibiotics using plasmids

 The present invention relates to a method and plasmid in the field of biomedical technology, and in particular to a plasmid and a method for increasing the production of a Streptomyces antibiotic using a plasmid.

Background technique

 With the application of cell biology and bioengineering technology in the field of microorganisms, especially antibiotic breeding, genetic engineering breeding technology has become a major means of improving strains, and has achieved great success in improving industrial production strains. Bioengineering breeding technology overcomes the randomness and blindness of traditional breeding techniques within a certain scope. It can use the knowledge of microbial secondary metabolic biosynthesis pathways to construct antibiotic high-yield strains by genetic engineering technology, which is helpful for secondary metabolites. Industrial production.

 In the field of microbial strain selection, the target gene is linked to the vector in vitro by genetic engineering technology and then transferred to the recipient cell, so that the foreign gene is stably expressed in the receptor. The main pathways include: 1 increasing the production of antibiotics by regulating the doubling or inactivation of genes, increasing the active efflux efficiency of antibiotics; 2 introducing foreign genes that increase the supply of precursors and cofactors in host cells, The biological substance of the active substance; 3 by activating or inhibiting the branch metabolism to change the active ingredient or ratio of the biologically active molecule, thereby increasing the accumulation of the optimal active ingredient, reducing or eliminating the accumulation of the low activity secondary component.

 AdpA is a global positive regulator widely present in Streptomyces and is the central transcriptional regulator of the A-factor regulatory network, which activates the expression of genes required for morphological differentiation and secondary metabolites. AdpA positively regulates melanin biosynthesis in Streptomyces coelicolor, St. Streptomyces lividans, Streptomyces antibioticus, and Streptomyces a verwi til is. There is no literature on the effect of the expression of the ao^I gene in antibiotic-producing strains on its antibiotic production.

 Zy ireosci a is a obligate aerobic filamentous Gram-negative bacterium. ^ The gene-encoded hemoglobin of Vitreoscillae has the property of binding oxygen to promote the transport of oxygen in microbial cells and increase oxygen. Utilization. For many cases of insufficient dissolved oxygen, Vgb expression promotes host growth, protein secretion, metabolite production, and enhanced compression resistance.

Integration of the Vitreoscilla hemoglobin gene into a heterologous host strain can increase recombinant protein production and fermentation yield the amount. After searching for the prior art, Wb gene can significantly promote the growth of Streptomyces cinnamensis and the synthesis of antibiotic monensin in antibiotic production (Wen Ying, Song Yuan. Vitreoscilla hemoglobin in Streptomyces cinnafolia)

基因的表达 使红霉素的产量提高 60% (Brunker, P. Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stabl e expression of the Vi treoscill hemoglobin gene (vhb ) . Microbiology 1998， 144 (9)： 2441-2448 )。 Effects of expression in { Streptomyces cinnamonensis^ on cell growth and antibiotic synthesis. Chinese Journal of Biotechnology, 2001, 17 (1 ) : 24-28 ).

Gene expression increases erythromycin production by 60% (Brunker, P. Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stabl e expression of the Vi treoscill hemoglobin gene (vhb ) . Microbiology 1998, 144 (9): 2441- 2448).

 S-adenosylmethionine (SAM) is synthesized by a catalytic reaction of adenosine triphosphate and L-methionine by SAM synthase. As an important intermediate metabolite in the biosynthesis process, the high expression of the SAM synthase gene ^i/r in many Streptomyces can effectively increase the production of its metabolites. In the Streptomyces coelicolor, the high expression of the SAM synthase gene / ffe can greatly increase the yield of the actin purplish ( Okamoto S , Lezhava A. Enhanced expressi on of S- Adenosylmethionine synthetase causes overproduct ion of act inorhodin in Streptomyces coelicolor k?> (2) . J Bacteriol , 2003, 185 (2): 601 - 609 ) ; heterologous expression of the S-adenosylmethionine synthase gene in Saccharopolyspora erythraea can increase erythromycin A production (Wang Y, Wang YG. Improved producti on of erythromycin A by expression of a heterologous gene encoding S- adenosylmethionine synthetase. Appl Microbiol Biotechnol , 2007, 75 (4) : 837-842 ); Streptomyces actuosus Φ SAM into the enzyme gene ffiei r Efficient expression promotes the synthesis of nosiheptide (Zhang XC, Fen MQ. Overexpression of yeast S-adenosylmethionine synthetase metK in Streptomyces ac tuosus leads to increased product ion of nosiheptide. Appl Microbiol Biotechnol, 2008, 78 (6): 991 - 995). '

However, the above prior art is only the expression of a single gene in certain microbial strains, and its effect on metabolites is still limited, and it is still very insufficient for the entire metabolic engineering. The complex metabolic pathway of the microbial strain itself and the complex regulation mode and specificity of the cell cycle determine that it is still not enough to change the direction of a certain reaction in the metabolic flow. The biochemical transformation of the microbial metabolic pathway can be carried out in multiple steps. , maximizing the accumulation of metabolites. In recent years, due to the development of DNA recombination technology, it has become possible to genetically engineer metabolic pathways. According to the deficiencies of the above techniques, the present invention designs a novel method and plasmid for improving the production of Streptomyces antibiotics, and constructs a plurality of genes with extensive positive regulation related to antibiotic synthesis and constructs them simultaneously on one vector, and the constructed plasmid can be constructed. Expression in different Streptomyces strains effectively increases the yield of various antibiotics. Summary of the invention

 SUMMARY OF THE INVENTION The object of the present invention is to overcome the deficiencies of the prior art and to provide a novel method for increasing the production of Streptomyces antibiotics and the plasmids used. The present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, wherein one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are simultaneously expressed on one vector, and the junction is transferred. Transfer into Streptomyces for efficient expression to increase the production of antibiotics.

In a first aspect, the present invention provides a method for increasing the production of antibiotics of Streptomyces i Streptomyces, comprising the steps of: transferring an integrated expression plasmid into Streptomyces, thereby increasing the production of Streptomyces antibiotics; said integrated expression plasmid is ^ZAS "Genes and conventional vectors are constructed, either constructed of ζτ^ί/Γ and b5" genes and conventional vectors, or constructed with the ac^-c gene and conventional vectors, or metK, and ad _P Ac The gene is constructed with a conventional vector.

 Preferably, the method for increasing the production of antibiotics of Streptomyces treptomyces comprises the steps of:

(a) constructing an integrated expression plasmid;

 (b) transferring the integrated expression plasmid into Streptomyces;

 (c) Fermentation of the Streptomyces obtained in step (b), obtaining antibiotics.

 Preferably, the Streptomyces is Streptomyces i Streptomyces sp.) ZYJ-6 CGMCC NO. 2394.

 Preferably, the conventional vector is a PIB139 vector.

 Preferably, the base sequence of the zz/ei gene is as shown in SEQ ID NO.

 Preferably, the base sequence of the gene is shown in SEQ ID NO.

 Preferably, the base sequence of the ac^1-c gene is as shown in SEQ ID NO.

 In a second aspect, the present invention also relates to an integrated expression plasmid pFVgb o constructed from a ^/^ gene and a conventional vector.

 Preferably, the conventional vector is a PIB139 vector.

 Further, the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFVgb, comprising the following steps: (a) Plasmid pJTU4405 contains whole gene synthesis vgbS, and Ndel and EcoRI restriction sites are introduced at both ends of gene wb5";

 (b) double digestion of pJTU4405 carrying the Vitreoscilla gene with Ndel and EcoRI;

(c) The wb5" gene was inserted into the corresponding site of the Streptomyces integrative vector PIB139 containing the erythromycin resistance gene strong promoter PermE* to obtain plasmid pFVgb.

In a third aspect, the invention also relates to an integrated expression constructed by fflei and "genes and conventional vectors" Plasmid pFMV.

 Preferably, the conventional vector is a PIB139 vector.

 Further, the present invention relates to a method for constructing the integrated expression plasmid pFMV according to claim 11, comprising the steps of: preparing a plasmid pFMetK plasmid; and then cleaving vgbS } pJTU4405 with Ndel and EcoRI and inserting the corresponding enzyme into pJTU968 At the site, the b5 gene fragment carrying the strong erythromycin promoter was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI to obtain plasmid pFMV.

 Preferably, the preparing the plasmid pFMetK plasmid comprises the following steps:

 (a) Designing the primer metKF2/metKR in the metK gene of Streptomyces coelicolor, and adding a Ndel restriction site at the ATG codon;

 The sequence of the primer metKF is: 5, - CAGGGAGCCATATGTCCCGT-3 ' ,

 The sequence of the primer metKR is: 5 ' -TCGCAAAGGCCACTGACAACA-3 ';

 (b) Inserting the amplified 1334 bp metK gene without the original promoter into the EcoRV site of pBlueScriptll SK (+) to construct a 4292 bp clone pJTU2524;

 (c) The promoterless metK gene fragment was excised from pJTU2524 using Ndel and EcoRI and inserted into the pIB139 vector containing the erythromycin resistance gene strong promoter PermE* to obtain plasmid pFMetK.

In a fourth aspect, the present invention also relates to an integrated expression plasmid pFMA constructed from the ζ^ί/Γ and _SC / 4- _C genes and a conventional vector.

 Preferably, the conventional vector is a PIB139 vector.

Further, the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFMA, comprising the steps of: inserting ad _P Ac from pJTU2520 into a corresponding site of plasmid pJTU968 carrying an erythromycin promoter using Ndel and EcoRI, The apA-c gene fragment carrying the strong erythromycin promoter was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI to construct plasmid pFMA.

 In a fifth aspect, the present invention also relates to an integrated expression plasmid pFMVA constructed from the ei r, ^ and atp/i-c genes and a conventional vector.

 Preferably, the conventional vector is a PIB139 vector.

 Further, the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFMVA, comprising the steps of: inserting apA-c carrying an erythromycin promoter from pJTU2522 into an EcoRI site of pFMV using Muni and EcoRI, Plasmid pFMVA.

The present invention has the following beneficial effects: The present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, and one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are taken. The manner in which expression is simultaneously carried out on a vector is transferred to Streptomyces by conjugation transfer for efficient expression to increase the production of antibiotics.

 The Streptomyces sp. CGMCC NO. 2394 ZYJ-6 of the present invention has been submitted to the General Microbiology Center of the China Microbial Culture Collection Management Committee, and its deposit registration number is CGMCC N0. 2394; the deposit address is No. 1 Courtyard of Beichen West Road, Chaoyang District, Beijing. No. 3, Institute of Microbiology, Chinese Academy of Sciences. The Streptomyces CGMCC NO. 2394 ZYJ-6 has been disclosed ((Zhou YJ, Li JL, Zhu J, Chen S, Bai LQ, Zhou XF, Wu HM, Deng ZX. Incomplete β -Ketone Processing as a Mechanism for Polyene Structural Variation in the FR-008/Candicidin Complex. Chemi stry & Biology, 2008, 15 : 629-638. The Streptomyces CGMCC NO. 2394 ZYJ-6 is directed to produce a single component of virulence FR-008- III The yield is 120-130% of the yield of the wild-type strain, and FR-008-ΠΙ is the most medicinal component of the three main components of the bacteriocin, which can be produced on a large scale. The high-purity mycotoxin effective component has significant industrial application value.

 The PIB 139 is described in "11:^50:1 CJ, Hughes- Thoraas ZA, Martin CJ, Bohm L Mironenko T, Deacon M, Wheatcroft M, Wirtz G, Staunton J, Leadlay PF. Increasing the efficiency of heterologous promoters in actinomycetes J Mol Microb Biotech, 2002, 4 (4): 417-426.

 The strain PJTU4405 is described in Chinese Patent Application No. 201010148837. 2, Publication No. CN 101805742 A, Japanese Patent Publication No. 2010- 8-18, the disclosure of the patent document of the disclosure of the disclosure of the disclosure of the patents of the patent of The strain PJTU4405 was prepared by the following method: changing the codon used frequently in Streptomyces to the codon with the highest frequency in Streptomyces, keeping the amino acid sequence unchanged, and generating a new gene Wb5 Ndel and EcoRI cleavage sites were introduced at the wb5M end of the gene, respectively, in which 7 nucleotides were added at the 5' end of the ^b5t-heidate sequence to form a restriction endonuclease Ndel site and a protective base. Add 8 nucleotides at the 3' end of the ^/λ5 nucleotide sequence to form a restriction endonuclease EcoRI site and a protective base. The 5 5" whole gene was synthesized and the full length was 456 bp. The synthetic 456 bp b 3⁄4 DNA was inserted into the Smal site of the vector pGH to generate the plasmid pJTU4405, which was sequenced to confirm the synthesis.

 The E. coli ET12567/pUZ8002 is disclosed in "Paget, MS, Chamberl in, L., Atrih, A., Foster, SJ, and Buttner, MJ. Evidence that the extracytoplasmic function sigma factor σ E is required for normal cell wall structure In Streptomyces coelicolor A3 (2) . J. Bacteriol, 1999 , 181 : 204 - 211 .

DRAWINGS Figure 1 is a construction diagram of an expression plasmid.

 Figure 2 shows the PCR validation of the »ei gene in strains ZYJ-6/pFMetK, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA.

 Figure 3 is a PCR verification of the wb5" gene in strains ZYJ-6/pFVgb, ZYJ-6/pFMV, ZYJ-6/pF VA. Figure 4 shows strains ZYJ-6/pFAdpA, ZYJ-6/pFMA, ZYJ-6 PCR verification of ap I-c gene in /pFMVA. Figure 5 shows spectrophotometric analysis of strains ZYJ-6 and ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA,

ZYJ-6/pFMV, ZYJ-6/pF A, ZYJ-6/pFMVA yield changes of FR-008-III in different culture times.

 Figure 6 is a quantitative analysis of the yield change of the oxytocin FR-008-ΙΠ in the starting strain ZYJ-6 and the high-yield strain ZYJ-6/pFMVA.

detailed description

 The invention is further illustrated below in conjunction with specific embodiments. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to conventional conditions, such as those described in molecular clones of Sambrook et al.: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. Recommended conditions.

 Example 1. Construction of an integrated expression plasmid for increasing the production of Streptomyces antibiotics

 The gene expression vector PIB139 according to the present embodiment is a Streptomyces integrative vector containing a strong promoter of erythromycin resistance gene PermE*, which can be transferred into Streptomyces by the two parents between Escherichia coli and Streptomyces. After point-specific recombination, it is integrated on the chromosome of Streptomyces and replicates and inherited along with the chromosomes. Figure 1 is a diagram showing the construction of all expression plasmids involved in this example.

 1. 1 Construction of plasmid pFMetK

PCR amplification of the /^ί gene without the original promoter in Streptomyces coelicolor M145, the specific steps include: designing the primer metKF2/metKR in the ffei gene of Streptomyces coelicolor, and adding a Ndel restriction enzyme at the ATG codon The 4334 bp/^ i/r gene amplified without the original promoter was inserted into the EcoRV site of _P BlueS _C riptn SK(+) to construct a 4292 bp clone pJTU2524; no promoter was used with Ndel and EcoRI The 7?ei r gene fragment was excised from PJTU2524 and inserted into the pIB139 vector containing the erythromycin resistance gene strong promoter PermE* to obtain plasmid pFMetK.

 The sequence of the primer metKF2/metKR is:

 Met F (5, -CAGGGAGCCATATGTCCCGT-3 ' );

raetKR (5' - TCGCAAAGGCCACTGACAACA-3, )<, 1. Construction of 2 plasmid pFVgb

 Plasmid PJTU4405 contains a full-length synthetic wb5" with a length of 456 bp, and Ndel and EcoRI restriction sites were introduced at both ends of the gene. The pJTU4405 carrying the Vitreoscilla gene was digested with Ndel and EcoRI.

"Gene (441 bp) The corresponding site of the Streptomyces integrative vector PIB139 containing the erythromycin resistance gene strong promoter PermE* was inserted to obtain plasmid pFVgb.

 1. 3 plasmid pFMV construction

PCR amplification of the aDPA-c gene in Streptomyces coelicolor, the specific steps include: designing the primer aDPAc2F/adpAclR, adding a Ndel restriction site at the ATG codon; inserting the 1556 bp ao^!-c amplified fragment The plasmid pJTU2520 was obtained from the EcoRV site of pBlueScriptl SK (+); the apA-c was excised from _P JTU2520 by Ndel and EcoRI and inserted into the corresponding site of plasmid pIB139 carrying the erythromycin promoter to obtain a plasmid. pFAdpA.

 The sequence of the primer adpAc2F/adpAc lR ' is:

 adpAc2F ( 5 ' -GGCTTAGCCATATGAGCCAC- 3 ' );

 adpAclR (5 ' - CGTTCATGCGGGCCACTTTA- 3, ).

 1. Construction of plasmid pFMV containing two genes of metK and ^bS"

 The vgbS PJTU4405 was excised into the corresponding restriction sites of pJTU968 by Ndel and EcoRI, and the w^S" gene fragment carrying the strong promoter of erythromycin was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI. Plasmid pFMV.

 1. 5 Construction of plasmid pFMA containing two genes of ingqing adpA-c

 The apA_c was cleaved from pJTU2520 with Ndel and Eco PI and inserted into the corresponding site of the plasmid PJTU968 carrying the erythromycin promoter, and the atp-c gene fragment carrying the strong promoter of erythromycin was amplified by Muni and Eco^. The plasmid pFMA was constructed by inserting into the EcoRI site of the pFMetK plasmid.

 1. 6 Construction of plasmid pFMVA containing metJ, and c

The ac/^-c carrying the erythromycin promoter was excised from _P JTU2522 into the EcoRI site of pFMV using Muni and EcoRI to obtain plasmid pFMVA.

 Example 2. Screening and verification of transfer of plasmid into Streptomyces host and junction transfer

 The constructed plasmid was transformed into E. coli ET12567/pUZ8002, and the transformant was selected to culture E. coli ET12567/pUZ8002 carrying the transfer plasmid in liquid LB medium. The final concentration of the liquid LB medium was 25 μg/μ. L-chloramphenicol, 50 μg/μL kanamycin and 30 μg/μL apramycin.

The cells were collected after incubation at 37 ° C for 8 hours, and the cells were washed 3 times with fresh LB medium for use. 0. The pH of the pH is 8. 0. 0. 05. Then, it was heat-shocked in a water bath at 55 ° C for 10 min. After cooling to room temperature, it was mixed with E. coli cells in an equal amount of 10 ⁸ : 10 ⁸ and then applied to the SFM medium plate. The composition of the medium plate was: 2 %. Agarose (m/v), 2% mannitol (m/v), 2% soy cake powder (m/v), the pH of the culture plate was 7. 2~7. 5, dried and placed at 30 ° C The culture is carried out in an incubator.

 After 12 hours, the plate was covered with 1 ml of sterile water containing nalidixic acid and apramycin, the final concentration of nalidixic acid on the plate was 50 ng/mL, and the apramycin was 30 ng/mL. The transfer zygote can be seen after 30 days of culture.

 Resistance was determined by inoculating a single junction transfer from the overlay onto the apramycin resistant plate. Using the total DNA of the alternative strain as the PCR template, the pFetK, pFMA, pFMV, and pFMVA plasmids all contain the y7?ei r gene, and the plasmid was transformed into ZYJ-6/pFMetK, ZYJ-6/pFMV, ZYJ-6. The primer used for PCR verification of /pFMA, ZYJ-6/pFMVA strain was metKTF/metKTR, and the PCR product was 986 bp (see Figure 2). In Fig. 2, 1 is a marker, 2, 3, 4, and 5 are strains ZYJ-6/pFMetK, ZYJ-6/pFMV, ZYJ-6/pFMA, and ZYJ-6/pFMVA chromosomes can be amplified by a template of 986 bp. Amplification band.

 The pZVgb, pFMV, pFMVA three plasmids all contain the gZ^ gene, and the ZYJ_6/pFVgb, ZYJ-6/pFMV, ZYJ-6/pFMVA strains obtained by the transformation of the plasmid were verified by PCR to be vgbTF/vgbTR, and the PCR product was 436 bp (see Figure 3). In Fig. 3, 1 is marker, 2, 3, and 4 are strains ZYJ-6/pFVgb, ZYJ-6/pFMV, and ZYJ-6/pFMVA chromosomes can be used to amplify a 436 bp amplification band.

 The ppdpA, pFMA, pFMVA three plasmids all contain the adpA gene, and the ZYJ-6/pFAdpA, ZYJ-6/pF A, ZYJ6-/pFMVA strains obtained by the transformation of the plasmid were verified by PCR as adpATF/adpATR, PCR products. It is 620 bp (see Figure 4). In Fig. 4, 1 is marker, 2, 3, and 4 are strains ZYJ-6/pFAdpA, ZYJ-6/pFMA, and ZYJ-6/pFMVA chromosomes are used as templates to amplify a 620 bp amplification band.

 Primer sequence:

 metKTF: 5' GAACAGACCCACGGGCTCGG 3'

 metKTR: 5' TGTCCCGTCGCCTGTTCACC 3'

 vgbTF: 5' GTGGACCAGCAGACCATCAA 3'

 vgbTR: 5' ACTCGACCGCCTGGGCGTAC 3'

 adpATF: 5' CGCAGGGACTGGAGGCGATC 3'

adpATR: 5' CACCCGCTGGGTGATCAGCC 3' PCR reaction system: 0. 1 μ _β template DNA, primers 50 pmol each, 4 μΐ DMSO, 4 μΐ dNTP, 5 μΐ PCR buffer and 1 unit Tag DNA polymerase. The polymerase is a product of Japan T0Y0B0, plus pure Water to

The cycling conditions of the PCR reaction were: 94 ° C, 5 min; 94 ° C, 30 s; 57 ° C, 30 s; 72 ° C, 80 s (30 cycles); 72 ° C extension for 5 min, 4 ° C End the reaction.

 Example 3, strain fermentation and extraction and detection of antibiotics

 The starting strain ZYJ-6 and the obtained ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA were sequentially liquid. Fermentation and antibiotic testing. The strain ZYJ-6 was used as the host, and the yield of the FR-008 III component was used as the detection standard.

 The strain was first activated on SFM plates and cultured for 30 days for 30 days. Then inoculated in 25 ml liquid medium TSBY ( 10. 3% sucrose), wherein the liquid medium TSBY is prepared by: tryptone (TSB) 30 g, Difco yeast powder 5 g, sucrose 340 g (34%) or 103 g ( 10. 3 %), to a volume of 1000 ml, sterilized.

 The cells were incubated at 30 ° C for 24 hours, sampled, centrifuged, collected, dried, and weighed to determine the difference in bacterial density between the strains. It was inoculated into 50 ml of liquid medium YEME at a volume ratio of about 1/100, and cultured at 30 ° C for 84 hours on a shaker.

The preparation method of liquid medium YEME is as follows: Take Digco yeast powder 3 g, Difco peptone 5 g, Oxoid malt powder 3 g, sucrose 103 g, glucose 10 g, then dilute to 1000 ml, sterilize, add 2 before use a sterile 2. 5 M MgCl ₂ · 60 solution in ml.

 The fermentation broth was taken at different culture times for extraction and detection of antibiotics. The fermentation broth was taken at 36 h, 48 h, 60 h, 72 h and 84 h, respectively, then 2 volumes of n-butanol were added, shaken and extracted, and the supernatant was centrifuged, and the antibiotics in the broth were extracted three times. The extracts were combined at a wavelength of 380 nm with a spectrophotometer (PerkinElmer®) and a non-antibiotic-producing mutant HJ-5 of Streptomyces FR-008 (Yirong Zhang, Linquan Bai and Zixin Deng. Functional characterization of the The first two actinomycete 4'-amino- 4-deoxychorismate lyase genes. Microbiology, 2009, 155: 2450 - 2459) was used as a blank control. The experiment was repeated three times.

 Figure 5 shows the strain ZYJ-6 and the six strains constructed ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA Yield analysis of oxytocin.

Six strains were tested in parallel with the starting strain, and the results showed that the first five strains increased the production of myostatin by 90% (pFMetK), 130% (pFVgb), 80% (pFAdpA), 130% ( pFMV), 150% (pFMA) (see Figure 5); Figure 6 shows that the expression of the integrated plasmid pFMVA of three genes in ZYJ-6 will eventually The production of nystatin increased by 2.1 times, and the yield increased from 424ug/ml (ZYJ-6) to 1301ug/ml.

 (ZYJ-6/pFMVA).

 In summary, the present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, and one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are simultaneously expressed on one vector. In a manner, the transfer is transferred to Streptomyces for efficient expression to increase the production of antibiotics.

Claims

Claim A method for increasing the production of Strep _P tomyces antibiotics, comprising the steps of: transferring an integrated expression plasmid into Streptomyces, thereby increasing the production of Streptomyces antibiotics; said integrated expression plasmid is a wb5" gene Constructed with a conventional vector, or constructed as a y^i and wb5" gene and a conventional vector, or a Hei/r and acp-c gene constructed with a conventional vector, or fflei T, and ₃ φ Ι- The c gene is constructed with a conventional vector.  2. A method of increasing the production of Streptomyces antibiotics according to claim 1, comprising the steps of:  (a) constructing an integrated expression plasmid;  (b) transferring the integrated expression plasmid into Streptomyces;  (c) Fermentation of the Streptomyces obtained in step (b), obtaining antibiotics.  The method for increasing the production of a Streptomyces antibiotic according to claim 1, wherein the Streptomyces is Streptomyces sp. ZYJ-6 CGMCC NO. 2394.  A method for increasing the production of a Streptomyces antibiotic according to claim 1, wherein the conventional vector is a PIB139 vector.  The method for increasing the production of a Streptomyces antibiotic according to claim 1, wherein the nucleotide sequence of the ^ίΤΓ gene is as shown in SEQ ID NO.  The method for increasing the production of a Streptomyces antibiotic according to claim 1, wherein the base sequence of the gene is as shown in SEQ ID NO.  The method for increasing the production of a Streptomyces antibiotic according to claim 1, wherein the base sequence of the ao^I-c gene is as shown in SEQ ID NO.  8. An integrated expression plasmid pFVgb constructed from a gene and a conventional vector.  9. The integrated expression plasmid pFVgb according to claim 8, wherein the conventional vector is a PIB139 vector  10. A method of constructing an integrated expression plasmid pFVgb according to claim 8, comprising the steps of:  (a) Plasmid pJTU4405 contains the whole gene synthesis vgbS, and Ndel and EcoRI restriction sites were introduced at both ends of the gene; (b) digesting pJTU4405 with Vibrio (Vi treoscilla gene) with Ndel and EcoRI; (c) The b5" gene was inserted into a corresponding site of the Streptomyces integrative vector PIB139 containing the erythromycin resistance gene strong promoter PermE* to obtain a plasmid pFVgb.  11. An integrated expression plasmid pFMV constructed from the z/ei r and wb5" genes and a conventional vector.  The integrated expression plasmid pFMV according to claim 11, wherein the conventional vector is a pIB139 vector <=  A method for constructing the integrated expression plasmid pFMV according to claim 11, comprising the steps of: preparing a plasmid pFMetK plasmid; and then cleaving the vgbS pJTU4405 into the corresponding cleavage site of PJTU968 by Ndel and EcoRI. Then, a gene fragment carrying a strong erythromycin promoter was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI to obtain a plasmid pFMV.  The method for constructing the integrated expression plasmid pFMV according to claim 13, wherein the preparation of the plasmid pFMetK plasmid comprises the following steps:  (a) Designing the primer metKF2/metKR in the metK gene of Streptomyces coeli color, and adding a Ndel restriction site at the ATG codon;  The sequence of the primer metKF is: 5, -CAGGGAGCCATATGTCCCGT- 3, ,  The sequence of the metkR substance metKR is: 5 ' -TCGCAAAGGCCACTGACAACA- 3 ';  (b) Inserting the amplified 1334 bp metK gene without the original promoter into the EcoRV site of pBlueScriptll SK (+ ) to construct a 4292 bp clone pJTU2524; (c) The promoterless metK gene fragment was excised from _P JTU2524 with Ndel and EcoRI and inserted into the pIB139 vector containing the erythromycin resistance gene strong promoter PermE* to obtain plasmid _P FMetK. 15, one by

The ao^!-c gene was integrated with a conventional vector to construct the expression plasmid pFMA.  The integrated expression plasmid pFMA according to claim 15, wherein the conventional vector is a PIB139 vector  17. A method of constructing the integrated expression plasmid pFMA according to claim 15, comprising the steps of: inserting apA-c k pJTU2520 into the erythromycin-producing promoter with Ndel and EcoRI. The corresponding site of the plasmid pJTU968 was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI, and the plasmid pFMA was constructed by inserting the ac^4-c gene fragment carrying the erythromycin strong promoter.  18. An integrated expression plasmid pFMVA constructed from the ^, "and ao^l-c gene and a conventional vector.  The integrated expression plasmid pFMVA according to claim 18, wherein the conventional vector is a pIB139 vector 20. A method of constructing an integrated expression plasmid pFMVA according to claim 18, wherein The following steps were included: The apA_c carrying the erythromycin promoter was excised from pJTU2522 and inserted into the EcoRI site of pFMV using Muni and EcoRI to obtain plasmid pFMVA.