CN116333951A

CN116333951A - Genetically engineered bacterium for high yield of azalomycin F, construction method and application thereof

Info

Publication number: CN116333951A
Application number: CN202211018707.6A
Authority: CN
Inventors: 洪葵; 赵明侠; 朱冬青; 李欣玥; 张倩倩; 邓子新
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2023-06-27

Abstract

The invention discloses a genetically engineered bacterium for high yield of azalomycin F, and a construction method and application thereof, and belongs to the technical fields of genetic engineering and microorganisms. The genetically engineered bacteria for high-yield azalomycin F are streptomyces which over-express azl gene (SEQ ID NO. 1) and/or azl gene (SEQ ID NO. 2), and the construction method comprises the following steps: an over-expression plasmid with a strong promoter element for driving the expression of a target gene is constructed, and the constructed plasmid is transferred into a streptomycete host for producing azalomycin F by a conjugal transfer method. The invention carries out overexpression on endogenous genes azl and azl6 related to the biosynthesis of the azalomycin F by a genetic engineering means, so that the yield of the azalomycin F is greatly improved. The invention lays a foundation for improving the yield of the azalomycin F and reducing the production cost of the azalomycin F.

Description

Genetically engineered bacterium for high yield of azalomycin F, construction method and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and microorganisms, and particularly relates to a genetic engineering bacterium for high-yield azalomycin F, and a construction method and application thereof.

Background

Azalomycin F is a 36-membered macrolide compound isolated from a liquid medium of the variant of Streptomyces hygroscopicus azalomycin (Streptomyces hygroscopicus var. Azalomyceticus), and contains azalomycin F5a, F4a and F3a as main components. Streptomyces such as S.hygrosporius MSU/MN-4-75B, S.malaysiensis MJM1968 and the like are reported to be metabolized to generate azamycin F macrolides, and then Yuan Ganjun and the like are also separated from fermentation broth of Streptomyces sp.211726 to obtain 12 36-membered macrolides comprising azamycin F5a, F4a, F3a and 9 new analogues, and after the planar structures of the azamycin F5a, F4a and F3a, 13C and 1H nuclear magnetic resonance signal attributions are corrected and perfected on the basis of researches such as Chandra, iwasaki and the like, the relative configuration of the azamycin F5a, F4a, F3a and 7 new structural analogues thereof is reported for the first time.

Azamycin F compounds are reported to have broad spectrum antibacterial activity, especially against gram positive bacteria and fungi. The compounds are mainly bonded to bacterial and fungal cell membranes to change the permeability of the cell membranes to cause leakage of substances in the cells and death of the cells. It is reported by Yuan Ganjun that azamycin F5a, F4a and F3a have significant activity against methicillin-resistant staphylococcus aureus (MRSA), wherein azamycin F5a kills the methicillin-resistant staphylococcus aureus by binding to the polar head of cell membrane phospholipids targeting lipoteichoic acid. In addition, it was shown from the results of the study that azalomycin F also has moderate cytotoxic activity against human colon cancer cells HCT-116. Furthermore, yang Peiwen reports that the azalomycin F compound has better protection effect on tomato gray mold, rice blast, tobacco brown spot and pepper anthracnose. It can be seen that azalomycin F has the potential to be developed as a high-efficiency biological source bactericide.

Xu et al scanned the entire genome of Streptomyces 211726, and combined with experimental data mapped the possible biosynthetic gene cluster for azalomycin F, covering 23 open reading frames. Wherein the kit contains 8 polyketide synthase genes azlBCDEFGHA, 19 polyketide synthase modules are used for catalyzing and forming a polyketide skeleton, and 15 non-PKS genes azl-15 are contained at two sides of the polyketide synthase genes and are involved in the biosynthesis of azamycin F series compounds. Researchers believe that the azamycin F biosynthesis starts with 4-Guanidinobutyramide (4-guanylbutanamide), hydrolyzes the amide groups on the precursor to form carboxyl groups under the action of amidase Azl13, generates the compound 4-guanidinobutyric acid (4-Guanidinobutyric acid), activates under the action of ligase Azl4 to generate 4-guanidinobutyric CoA (4-Guanidinobutyric acid-CoA), connects to the PKS via an esterification reaction catalyzed by acyltransferase Azl5, and then completes extension of the carbon chain under the catalysis of the PKS, cyclizes and releases under the action of the thioesterase domain in the last polyketide synthase module to form the polyene macrolide backbone. Finally, the azalomycin F series compound is generated by the action of post-modification enzyme.

At present, the breeding of actinomycete high-yield strains mainly adopts a genetic breeding method, and the genetic breeding method mainly comprises two methods: one is the traditional (physical/chemical) mutation breeding technology, namely, the technology of ultraviolet mutagenesis, chemical mutagenesis, microwave mutagenesis, space mutagenesis, ion beam mutagenesis and the like is utilized to carry out mutagenesis treatment on the production strain, and then a large amount of screening is carried out to obtain the variant strain with better production performance. The method has great contribution to the breeding of antibiotic high-yield strains, and a plurality of excellent strains have been successfully bred at present. However, the method has great blindness in screening excellent mutant strains, the screening workload is very large, and the ideal effect is difficult to achieve in a short time. Another genetic breeding method for actinomycetes is as follows: modern molecular biology genetic breeding technology, namely, genetic modification of specific genes is adopted to further improve the productivity of strains. The method can directly carry out genetic transformation on genes affecting the level of the antibiotic produced by the strain, and has stronger purposiveness and simple genetic operation.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for high-yield azalomycin F, a construction method and application thereof, which are used for preparing azalomycin F compounds.

The first object of the present invention is to provide a genetically engineered bacterium which produces azalomycin F in high yield. The genetically engineered bacterium is obtained by over-expressing at least one of the following genes in a streptomycete host producing azalomycin F:

(a) azl4 gene (4-guidinobutanoate: coA ligase), the nucleotide sequence is shown in SEQ ID NO. 1;

(b) azl6 gene (TetR-family transcriptional regulator) and the nucleotide sequence is shown in SEQ ID NO. 2.

In one embodiment, the streptomyces producing azamycin F is a strain having a accession number of cctccc NO: streptomyces TKPJ3039 of M2013221, streptomyces TKPJ3039 has been disclosed in Chinese patent CN103232964A, "Streptomyces TKPJ3039, a high yield azalomycin F type compound strain, and its application, which was deposited in China center for type culture collection, 5 months and 20 days in 2013.

In one embodiment, the azl gene and/or azl gene described above are overexpressed in a Streptomyces host in a site-specific integrative vector containing a strong promoter. The site-specific integration vector is preferably a pSET152 or pIB139 plasmid. The strong promoter is preferably a constitutive promoter hrdBP, and the nucleotide sequence of the strong promoter is shown as SEQ ID NO. 3.

The second object of the present invention is to provide a construction method of genetically engineered bacteria for high production of azalomycin F, the construction method comprising the steps of: and constructing an over-expression plasmid with a strong promoter element for driving the expression of a target gene (azl gene and/or azl gene), and transferring the constructed plasmid into a streptomycete host for producing the azalomycin F by a joint transfer method to obtain the genetically engineered bacterium for producing the azalomycin F with high yield. The over-expression plasmid preferably uses pSET152 or pIB139 plasmid as a vector. The strong promoter is preferably the constitutive promoter hrdBP.

Further, the construction method comprises the following steps:

(1) Characterizing and screening the intensities of different promoters in streptomyces host bacteria for producing azalomycin F to obtain promoters with strong activity in the host bacteria;

(2) Constructing an over-expression plasmid containing the target gene (azl gene and/or azl gene), wherein the plasmid contains the strong promoter element selected in the step (1) and takes the arabidopsis resistance gene aac (3) IV as a marker;

preferably, the over-expression plasmid takes pSET152 or pIB139 plasmid as a vector;

in one embodiment, the strong promoter element of the overexpression plasmid of the azl gene and/or azl6 gene is the constitutive promoter hrdBp;

(3) Transferring the plasmid constructed in the step (2) into streptomyces host bacteria for producing azalomycin F;

(4) Screening: and selecting the zygote, then carrying out passage, screening strains with resistance to the arabinomycin, and carrying out colony PCR verification to obtain the integrated strains, thus obtaining the genetically engineered bacteria of the high-yield azalomycin F.

As one embodiment of the invention, in step (1), the intensities of the different promoters in the host bacteria are characterized and selected, comprising the steps of:

(1) transfer of plasmid containing promoter and kanamycin resistance Gene neo to Streptomyces

Transferring plasmids containing different promoters and kanamycin resistance genes neo into competent cells of escherichia coli ET12567/pUZ8002, selecting correct escherichia coli transformants, performing joint transfer with streptomyces host bacteria producing azamycin F, and transferring the plasmids into the streptomyces host bacteria producing azamycin F by a joint transfer method;

(2) screening

After subculturing the conjugative transfer on SFM medium at 28 ℃, picking up colonies growing on an arabinomycin resistance plate, carrying out colony PCR verification, and screening to obtain strains with target plasmids successfully integrated into a host bacterium genome.

Collecting spores of each strain, suspending in sterile water, and adjusting OD ₆₀₀ To the same level, gradient dilution was performed, and 10. Mu.L spore suspension was pipetted at a uniform spot on a kanamycin gradient concentration plate for resistance testing. The intensity of kanamycin resistance exhibited by the strain can directly characterize the activity intensity of the corresponding promoter.

Further, as an embodiment of the present invention, the method for constructing the genetically engineered bacteria for producing azalomycin F with high yield specifically comprises the following steps:

(1) Construction of a conjugal transfer plasmid containing the Gene of interest

The promoter hrdBP was inserted into the plasmid pSET152 to obtain a pWHU1288 vector, comprising the specific steps of: primers hrdB-pF and hrdB-pR were designed inside the hrdB gene of Streptomyces coelicolor M145, an NdeI cleavage site was added at the initiation codon ATG, the hrdB promoter (hrdBP) of Streptomyces coelicolor M145 was amplified by PCR, and the amplified 451bp DNA fragment containing hrdBP was digested with XbaI and BamHI as shown in SEQ ID NO.3, and inserted into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, SEQ ID NO. GenBank: AJ 414670.1) to obtain plasmid pWHU1288.

Connecting a gene azl related to an azalomycin F biosynthesis precursor with a pWHU1288 vector by an enzyme digestion enzyme ligation method to construct an overexpression plasmid pMX301;

connecting an azalomycin F biosynthesis related regulatory gene azl with a pWHU1288 vector by an enzyme digestion enzyme linkage method to construct a target gene overexpression plasmid pMX303;

after transforming E.coli DH5 alpha competent cells, extracting plasmid enzyme digestion verification to obtain a conjugation transfer plasmid containing a target gene;

(2) Transfer of the conjugation to Streptomyces

Transferring the plasmid in the step (1) into competent cells of escherichia coli ET12567/pUZ8002, selecting a correct escherichia coli transformant and streptomyces griseus for joint transfer, and transferring the plasmid into the streptomyces griseus for producing the azadirachtin F by a joint transfer method to realize the over-expression of a target gene;

(3) Screening of genetically engineered strains

And (3) after picking the zygote and carrying out subculture on an SFM culture medium at 28 ℃, picking a colony growing on an Arabic resistance plate for colony PCR verification, and screening to obtain a strain successfully integrating a target gene, thus obtaining the genetically engineered bacterium of high-yield azalomycin F.

The third object of the invention is to provide the application of the genetically engineered bacterium in preparing azalomycin F.

A process for preparing azalomycin F comprising the steps of: inoculating the seed solution of the genetically engineered bacteria to a fermentation medium for fermentation.

In one embodiment, the fermentation medium consists of 10g of glucose, 35g of soluble starch, 2g of yeast powder, 4g of casein and 1L of deionized water, and the pH is adjusted to 7.2-7.4.

In one embodiment, the seed solution is a bacterial solution obtained by inoculating TSBY liquid culture medium into genetically engineered bacterial spore solution and culturing for 48-72 hours at 28 ℃ and 220 rpm.

In one embodiment, the fermentation conditions are 28 ℃, 220rpm, 10% of the container, and the fermentation period is 10-11 days.

The invention has the advantages and beneficial effects that: the invention carries out overexpression on endogenous genes azl and azl related to the biosynthesis of the azalomycin F by a genetic engineering means, and the yield of the obtained genetic engineering bacteria is respectively improved by 136.5 percent and 94.3 percent compared with that of the azalomycin F of a control strain. The invention provides a new idea for the industrial production of azalomycin F. The invention lays a foundation for improving the yield of the azalomycin F and reducing the production cost of the azalomycin F.

Drawings

Fig. 1: culturing on a basic culture medium at 28 ℃ in an incubator for 6 days, wherein the strength of the promoter in host bacteria is schematically shown;

fig. 2: culturing on a fermentation medium at 28 ℃ for 6 days in an incubator, wherein the strength of the promoter in host bacteria is schematically shown;

fig. 3: schematic diagram of recombinant plasmid containing strong promoter hrdBP for over-expressing target gene;

fig. 4: the recombinant strain PCR verifies agarose gel electrophoresis patterns; wherein lane M is a DNA molecular weight standard; lane 1 and Lane 2 are the products of PCR amplification by using the recombinant TKPJ3039, pMX301 and TKPJ3039, and the primers M13F and M13R, respectively, as templates;

fig. 5: HPLC detection of the fermentation product of the wild strain TKPJ 3039; among these, retention times 22.975min and 23.296min are azamycin F homologs;

fig. 6: the relative yields of the genetically engineered strain and the wild-type strain azalomycin F were compared.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below in connection with specific embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. It should be noted that, based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without making any inventive effort are within the scope of the present invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Molecular cloning tool E.coli DH 5. Alpha. And two parental ligation transfer tool E.coli ET12567/pUZ8002 are commercial products whose competent cells were prepared according to the guidelines for molecular cloning experiments. The collection number of the streptomyces azadirachta F-producing strain TKPJ3039 is CCTCC NO: m2013221, which is disclosed in Chinese patent CN103232964A, "Streptomyces TKPJ3039, a high yield of azalomycin F-type compound strain, and applications thereof.

DNA sequencing was performed by Wohan engine biotechnology Co.

The primers involved in the experimental process of the present invention are synthesized by Nanjing Jinsri biotechnology Co., ltd, as shown in Table 1 below.

TABLE 1 primers involved in the experiments of the present invention

The culture medium and the reagent involved in the experimental process of the invention are as follows:

LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

LA medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

2 XYT medium: 16g of tryptone, 10g of yeast extract, 5g of sodium chloride, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

AZ medium: 20g of soybean cake powder, 15g of bran, 0.02g of asparagine, 7.5g of soluble starch and K ₂ HPO ₄ 0.3g，KNO ₃ 1g，NaCl 0.5g，CaCO ₃ 1g, 15g of agar, adding distilled water to 1000mL, adjusting pH to 7.5 (adjusted by NaOH) to 115 ℃, sterilizing for 30min, and adding sterilized 5M CaCl when in use ₂ The final concentration was 120mM.

SFM medium: 20g of soybean cake powder, 20g of mannitol and 20g of agar, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

TSBY medium: sucrose 103g, oxoid tryptone soy soup powder 30g, yeast extract 5g, distilled water to 1000mL,115 ℃ and sterilizing for 30min.

MM medium: l-asparagine 0.5g, K ₂ HPO ₄ 0.5g, 0.2g of magnesium sulfate heptahydrate, 0.01g of ferrous sulfate heptahydrate, 5g of mannitol and 10g of agar, adding deionized water to 1000mL, and sterilizing for 30min at 115 ℃.

Azalomycin F fermentation medium: 10g of glucose, 35g of soluble starch, 2g of yeast extract, 4g of casein, 1000mL of deionized water, pH 7.2-7.4 and sterilizing at 115 ℃ for 30min.

Spore pre-germination cultureAnd (3) culturing: yeast extract 1%, casein amino acid 1%,0.01M CaCl ₂ 。

TES solution: 2.292g of tris (hydroxymethyl) aminoethane sulfonic acid (TES) powder was weighed, deionized water was added to 200mL, pH=8.0 was adjusted, and sterilization was performed for 20min at 121 ℃.

The technical scheme of the invention will be further elaborated in the following in connection with specific embodiments.

Example 1: construction of the tooling plasmid

The expression activities of constitutive strong promoters ermEp, ermEpA, hrdBp, SCO5768p and kasOp in actinomycete host cells were tested by constructing reporter plasmids pLXY37, pLXY38, pLXY39, pLXY40 and pLXY41 derived from site-specific integration vector pIB139 or pSET152, respectively, using kanamycin resistance gene neo as reporter gene.

The construction method comprises the following steps:

1. primers DQ195F and DQ195R are designed, commercial plasmid pHZ1358 (GenBank: AY 667410.1) is used as a template, 809bp DNA fragment containing kanamycin resistance gene neo is obtained by amplification and used as a reporter gene, and cloned to commercial blunt end vector pEasy-Blunt Zero Vector to obtain intermediate plasmid pLXY36, and enzyme digestion verification and sequencing comparison are correct; the pLXY36 was digested with NdeI and EcoRI, and 795bp DNA fragment containing the kanamycin resistance gene neo was recovered and inserted between the NdeI and EcoRI digestion sites of the commercialized vector pIB139 to obtain plasmid pLXY37, on which the neo reporter gene was under the control of the promoter ermEp, which was used to characterize the strength of the promoter ermEp under different host and different culture conditions.

2. Primers LYL F and LYL R were designed and a 75bp DNA fragment containing the ribosome binding region of ermEp was amplified using vector pIB139 as template. Wherein a mutation site is introduced into the primer LYL F, and the sequence of the original ribosome binding region is modified. Cloning the amplified product to a commercial blunt-end vector pEasy-Blunt Zero Vector to obtain an intermediate plasmid, and performing enzyme digestion verification and sequencing comparison to be correct; the plasmid pIB139A was obtained by digestion with BglII and EcoRI to recover 71bp DNA fragment containing the mutated ribosome binding region, replacing the original ribosome binding region between BamHI and EcoRI of the commercial vector pIB139, the promoter of which was altered compared to the promoter ermEp on pIB139, and the altered promoter was designated ermEpA to indicate the difference; the plasmid pLXY38, whose neo reporter gene is under the control of the promoter ermEpA, was obtained by digestion of pLXY36 with NdeI and EcoRI, recovery of 795bp DNA fragment containing the kanamycin resistance gene neo, insertion between the NdeI and EcoRI cleavage sites of pIB139A, and can be used to characterize the strength of the promoter ermEpA in different hosts and under different culture conditions.

3. The primers hrdB-pF and hrdB-pR are designed, the chromosomal DNA of Streptomyces coelicolor is used as a template (GenBank: AL 645882.2), the promoter hrdBP of the gene hrdB (namely SCO 5820) is amplified and cloned between the XbaI and BamHI cleavage sites of the vector pSET152, an over-expression vector pWHO 1288 containing hrdBP is constructed, and the cleavage verification and sequencing are correct; pLXY36 was digested with NdeI and EcoRI, and 795bp of DNA fragment containing the kanamycin resistance gene neo was recovered and inserted between the NdeI and EcoRI digestion sites of pWHU1288 to obtain plasmid pLXY39 whose neo reporter gene was under the control of the promoter hrdBp, which was used to characterize the strength of the promoter hrdBp in different hosts and under different culture conditions.

4. Primers DQ200F and DQ200R are designed, streptomyces coelicolor chromosomal DNA is used as a template, a promoter of SCO5768 is amplified, cloning is carried out between XbaI and BamHI cleavage sites of a vector pSET152, an over-expression vector pWHU1289 containing SCO5768p is constructed, and enzyme cleavage verification and sequencing comparison are correct; pLXY36 was digested with NdeI and EcoRI, and 795bp of DNA fragment containing the neo kanamycin resistance gene was recovered and inserted between the NdeI and EcoRI digestion sites of pWHU1289 to obtain plasmid pLXY40, the neo reporter gene of which was under the control of the SCO5768p promoter, which was used to characterize the strength of the SCO5768p promoter under different host and different culture conditions.

5. Designing primers DQ222F and DQ222R, amplifying a promoter of the Streptomyces coelicolor gene kasO, cloning the amplified primers between XbaI and BamHI cleavage sites of a vector pSET152, and constructing an overexpression vector pWHU1290 containing kasOp, and performing cleavage verification and sequencing comparison correctly; the plasmid ply41, whose neo reporter gene is under the control of the promoter kasOp, was obtained by digestion of ply36 with NdeI and EcoRI, recovery of 795bp DNA fragment containing the kanamycin resistance gene neo, insertion between the NdeI and EcoRI cleavage sites of pWHU1290, and can be used to characterize the intensity of the promoter kasOp under different host and different culture conditions.

Example 2: characterization and selection of the intensities of the different promoters in the host bacteria

The pllxy 37, pllxy 38, pllxy 39, pllxy 40 and pllxy 41 constructed in example 1 were transferred into streptomycete TKPJ3039, respectively, to examine the expression intensities of the constitutive strong promoters ermeep, ermEpA, hrdBp, SCO5768p and kasOp in host cells, respectively.

1. Transformation of E.coli ET12567/pUZ8002

The plasmid was transformed into E.coli ET12567/pUZ8002 competent cells according to the molecular cloning protocol, briefly described as follows: e.coli ET12567/pUZ8002 competent cells were added with 1. Mu.L of the objective plasmid, gently mixed, and ice-bathed for 30 minutes; heat-shock at 42 ℃ for 90 seconds; ice bath for 3 minutes; 1mL of LB medium is added; shaking culture at 37 ℃ for 45 minutes; centrifuging at 6000rpm for 3 minutes; the supernatant was discarded, and 300. Mu.L of LB medium was added to suspend the pellet; coating on LA flat plates containing apramycin, kanamycin and chloramphenicol with final concentrations of 50 mug/mL, 50 mug/mL and 25 mug/mL respectively, and air-drying; the culture was carried out in an incubator at 37℃overnight. Single colonies were picked and transferred to LB medium containing final concentrations of arabinomycin, kanamycin and chloramphenicol of 50. Mu.g/mL, 50. Mu.g/mL and 25. Mu.g/mL, respectively, and shake cultured overnight at 37 ℃; the plasmid was extracted and detected by agarose gel electrophoresis.

2. Indirect transfer of E.coli ET12567/pUZ8002 to Streptomyces TKPJ3039 genus

Reference is made to the cloning and functional analysis of genes related to the biosynthesis of azalomycin F3a, from Streptomyces laboratory Manual and from the university of Wuhan, ma Yanling, doctor graduation paper, a brief procedure is as follows:

coli ET12567/pUZ8002 containing the objective plasmid was inoculated in 5mL LB medium containing 50. Mu.g/mL of apramycin, 50. Mu.g/mL of kanamycin and 25. Mu.g/mL of chloramphenicol, and cultured overnight at 37 ℃; coli cultured overnight was transferred to 5mL of fresh, 50. Mu.g containing arabinomycin at a ratio of 1:20, respectivelyLB medium/mL, chloramphenicol 25. Mu.g/mL and kanamycin 50. Mu.g/mL, shaking culture at 37℃to OD ₆₀₀ 0.4-0.6. 1mL of the supernatant was collected by centrifugation, the cells were washed twice with fresh LB medium containing no antibiotic, and the cells were suspended in 500. Mu.L of 2 XYT medium for use.

Spores of Streptomyces TKPJ3039 were suspended in 1mL of a 0.05M TES solution at pH 8.0, and the mixture was shaken, dispersed and mixed well, centrifuged at 10000rpm for 1min, and the supernatant was removed. This procedure was repeated once. Spores were resuspended with 700 μl fresh TES. Heat-shock for 10min at 50 ℃, cooling to room temperature, adding 500 mu L of spore pre-germination culture medium, and shake culturing at 37 ℃ for 1-2 hours for later use.

Mixing the treated standby escherichia coli suspension and the spore suspension subjected to pre-germination, performing shake culture at 28 ℃ for 1h, and coating the mixture on an AZ culture medium plate. After 17 hours of incubation in an incubator at 28℃the incubator was covered with 1mL of sterile water containing apramycin (final concentration 75. Mu.g/mL) and nalidixic acid (final concentration 25. Mu.g/mL), and after 5-7 days of incubation at 28℃the presence or absence of the conjugal transfer was observed.

3. Adapter transfer verification

The single colony of the zygote appearing on the conjugative transfer plate was picked up and inoculated onto SFM medium containing apramycin (50. Mu.g/mL), cultured at 28℃for 5-7 days, transferred to TSBY liquid medium containing apramycin (25. Mu.g/mL), shake-cultured at 28℃for 48 hours, and the total DNA was collected. The method for extracting the chromosome DNA is described in the manual of Streptomyces experiment, and the brief flow is as follows: placing 500 μl of mycelium into sterilized 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and discarding supernatant; washing the thalli twice by using sterile water, centrifuging and discarding the supernatant; adding 500 μl lysozyme (2 mg/mL), incubating at 37deg.C for 30min, and mixing at intervals of 2min; adding 4% SDS 25. Mu.L, and drying at 55deg.C for 5-10min to transparent; adding 300 mu L of phenol-chloroform, stirring and mixing uniformly by vortex, and centrifuging at 12000rpm for 5min; taking 700 mu L of upper liquid, adding 70 mu L of sodium acetate (3M), uniformly mixing, adding 770 mu L of isopropanol, slightly reversing the upper liquid for 5-6 times, and standing at-40 ℃ for 20min; centrifuging at 12000rpm for 5min, discarding supernatant, adding 75% ethanol 1mL, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, discarding upper layer, and repeating the steps once; oven drying at 55deg.C, adding 40-60 μl of sterile ultrapure water, dissolving, and storing at-40deg.C.

The reporter plasmids pllxy 37, pllxy 38, pllxy 39, pllxy 40 and pllxy 41 were derived from vector pIB139 or pSET152, and the binding regions of the universal primers M13F and M13R were located upstream and downstream of the multiple cloning site, so that primers M13F and M13R were synthesized for PCR to verify the correctness of the zygotes, the sequences of which are shown in table 1. PCR was performed using the 2 XTaq Master Mix (Dye Plus) kit from Nanjinouzan Biotech Co., ltd. The 20. Mu.L PCR system included: template, 1 μl; primer F, 1. Mu.L; primer R, 1. Mu.L; sterile deionized water, 7 μl;2 XTaq Master Mix, 10. Mu.L. PCR reaction procedure: 95 ℃ for 3min;95 ℃,15s,60 ℃,15s,72 ℃,60s,30 cycles; 72℃for 5min. The PCR products were analyzed by agarose gel electrophoresis.

4. Promoter screening

Collecting spores of each strain, suspending in sterile water, adjusting the concentration of spores to be consistent as mother solution, and then carrying out gradient dilution. Suction 10 ^-2 -10 ^-5 The 10 mu L uniform spot of spore suspension with different dilution factors is subjected to resistance strength test on a kanamycin gradient concentration flat plate (the culture medium is a basic culture medium MM or an azamycin F fermentation culture medium), and the culture is carried out at 28 ℃ for 6 days, the result is shown in figure 1, and the activity strength of the corresponding promoter can be directly characterized by the kanamycin resistance of the strain. The final concentration of MM medium plate kanamycin was in the order: 0 μg/mL, 2.5 μg/mL, 5.0 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL; the final kanamycin concentration in the plates of the fermentation medium was 0. Mu.g/mL, 10. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 150. Mu.g/mL, 200. Mu.g/mL, 250. Mu.g/mL, 300. Mu.g/mL, 400. Mu.g/mL, 500. Mu.g/mL, 650. Mu.g/mL.

As can be seen from the results of FIG. 1, the expression intensities of the promoters on MM medium plates were in that order: hrdBP, kasOp > SCO5768p, ermEpA > ermEp. As can be seen from the results of fig. 2, the expression intensities of the promoters in the growth environment of the fermentation medium are in order: hrdBp > KasOp, SCO5768p > ermEpA, ermEp.

It was concluded that the promoter hrdBP activity was strongest in the azalomycin F-producing strain TKPJ3039. Overexpression of the biosynthesis genes of the azalomycin F compounds takes a plasmid pWHU1288 containing an hrdBP promoter as a vector to construct a target gene overexpression plasmid.

Construction of the plasmid pWHU1288 as vector: the PCR amplification of hrdB promoter in Streptomyces coelicolor M145 includes the following specific steps: primers hrdB-pF and hrdB-pR were designed inside the hrdB gene of Streptomyces coelicolor M145, an NdeI cleavage site was added at the initiation codon ATG, the hrdB promoter (hrdBP) of Streptomyces coelicolor M145 was amplified by PCR, and the amplified 451bp DNA fragment containing hrdBP was digested with XbaI and BamHI as shown in SEQ ID NO.3, and inserted into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, SEQ ID NO. GenBank: AJ 414670.1) to obtain plasmid pWHU1288.

Example 3: azamycin F high-yield strain and construction method thereof

In order to obtain genetically engineered bacteria with high yield of azalomycin F, the synthesis related genes azl, azl5, azl13 and azl of the initial unit of the azalomycin F, the synthesis regulation related genes azl6 and azl12 of the azalomycin F and the transport related gene azl10 are adopted.

1. Construction of overexpression plasmid of target Gene

The total DNA of the azalomycin F producing strain TKPJ3039 is used as a template, and primers DQ201F and DQ201R are used for PCR amplification to obtain a 2596bp DNA fragment containing azl. Wherein PCR was performed using 2X Hieff Canace from Shanghai, assist, santa Clay ^TM PCR Master Mix high-fidelity enzyme premix system. The 20. Mu.L PCR system included: template, 1 μl; primer F, 1. Mu.L; primer R, 1. Mu.L; DMSO,1 μl; sterile deionized water, 6 μl; 2X Hieff Canace ^TM PCR Master Mix, 10. Mu.L. PCR reaction procedure: 98 ℃ for 3min;98 ℃,10s,60 ℃,20s,72 ℃,90s (30 s/kb), 30 cycles; 72℃for 5min. The DNA fragment obtained by PCR amplification was isolated and purified by agarose gel electrophoresis and a gel recovery kit of Omega Bio-Tek company, and PCR amplification was performed using the purified DNA fragment as a template and MX01F and MX01R as primers to obtain a 1467bp DNA fragment containing the target gene azl (SEQ ID NO. 1), re-isolated and purified, digested with NdeI and EcoRI, and re-isolated and purified. Vector pWHU1288 was digested with NdeI and EcoRIAnd (5) separating and purifying. The exogenous fragment and the vector fragment were ligated using T4 ligase. The ligation system was transformed into DH 5. Alpha. Competent cells, plasmid was extracted from the plasmid extraction kit of Omega Bio-Tek company, and verified by digestion and DNA sequencing. The correct plasmid was designated pMX301, an over-expression plasmid of azl4, and strain and plasmid DNA were maintained.

The overexpression plasmids of azl, azl, azl10, azl, azl, 13, azl and adpA (plasmid schematic is shown in FIG. 3) were constructed in the same way, respectively, and the strain and plasmid DNA were kept ready for use. The construction process is briefly described as follows:

taking total DNA of the azalomycin F producing strain TKPJ3039 as a template, and carrying out PCR amplification by using primers DQ201F and DQ201R to obtain 2596bp DNA fragment containing a gene azl; purifying the DNA fragment as a template, and carrying out PCR amplification by taking MX02F and MX02R as primers to obtain 1058bp DNA fragment containing target gene azl (SEQ ID NO. 4); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX302, an overexpression plasmid of azl 5.

PCR amplification is carried out by using total DNA of the azamycin F producing strain TKPJ3039 as a template and primers DQ202F and DQ202R to obtain 891bp DNA fragment containing a gene azl; purifying the DNA fragment as a template, and carrying out PCR amplification by taking MX03F and MX03R as primers to obtain 633bp DNA fragment containing target gene azl (SEQ ID NO. 2); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX303, an overexpression plasmid of azl.

Taking total DNA of the azalomycin F producing strain TKPJ3039 as a template, and carrying out PCR amplification by using primers DQ204F and DQ204R to obtain a 1003bp DNA fragment containing a gene azl; purifying the DNA fragment as a template, and carrying out PCR amplification by taking MX05F and MX05R as primers to obtain 812bp DNA fragment containing target gene azl (SEQ ID NO. 5); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX305, an overexpression plasmid of azl10.

Taking total DNA of the azalomycin F producing strain TKPJ3039 as a template, and carrying out PCR amplification by using primers DQ206F and DQ206R to obtain a 977bp DNA fragment containing a gene azl; the DNA fragment was purified and used as a template, and PCR amplification was performed using MX07F and MX07R as primers. Obtaining 827bp DNA fragment containing target gene azl (SEQ ID NO. 6); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX307, an over-expression plasmid of azl.

PCR amplification is carried out by using total DNA of the azamycin F producing strain TKPJ3039 as a template and primers DQ207F and DQ207R to obtain 2589bp DNA fragment containing a gene azl; purifying the DNA fragment as a template, and carrying out PCR amplification by taking MX08F and MX08R as primers to obtain 829bp DNA fragment containing target gene azl (SEQ ID NO. 7); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX308, an overexpression plasmid of azl 13.

PCR amplification is carried out by using total DNA of the azamycin F producing strain TKPJ3039 as a template and primers DQ207F and DQ207R to obtain 2589bp DNA fragment containing a gene azl; the DNA fragment was purified and used as a template, and PCR amplification was performed using MX09F and MX09R as primers. Obtaining 1470bp DNA fragment containing target gene azl (SEQ ID NO. 8); cutting with NdeI and EcoRI, connecting with a vector pWHU1288 treated by the same enzyme, and transforming escherichia coli DH5 alpha; the transformants were transferred, the plasmids were extracted, digested and sequenced, and the correct plasmid was designated pMX309, an overexpression plasmid of azl 14.

In addition, using Streptomyces coelicolor M145 total DNA as a template, amplifying 1229bp DNA fragment using primers adpA-F and adpA-R, comprising 1197bp gene adpA, encoding a functionally defined global transcription regulatory factor, related sequences such as gene sequence number GenBank in NCBI database: 1098226. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pllxy 55, the over-expression plasmid for the gene adpA.

2. Indirect transfer of over-expressed target gene plasmid from escherichia coli to streptomycete TKPJ3039 genus

The over-expression plasmid is respectively transformed into competent cells of escherichia coli ET12567/pUZ8002, transformants are picked and cultured, and correct transformants and streptomycete TKPJ3039 are selected for joint transfer of two parents.

The specific method of this step is shown in step 2 in example 2.

3. Phenotypic selection and genotyping of Gene overexpression Strain

Picking single colony of the apramycin resistant zygote, transferring the single colony to an SFM plate containing apramycin (50 mug/mL) for culture; colonies growing well on the apramycin resistant plates were selected, transferred into TSBY liquid medium containing apramycin (25. Mu.g/mL) for cultivation, and the thalli were collected to extract total DNA.

PCR amplification was performed using the total DNA of the overexpressed strain as template, with primers M13F and M13R, and the correct S.sp.zygote strain TKPJ3039:: pMX301, TKPJ3039:: pMX302, TKPJ3039:: pMX303, TKPJ3039:: pMX305, TKPJ3039:: pMX307, TKPJ3039: pMX308, TKPJ3039:: pMX309 and TKPJ3039:: the DNA fragments theoretically to be generated by pLXY55 were of the following sizes: 1956bp, 1548bp, 1122bp, 1305bp, 1317bp, 1959bp and 1718bp. The agarose gel electrophoresis analysis accords with the theoretical value, the obtained zygote is proved to be correct, and the strain is preserved for standby. Wherein TKPJ3039:: pMX301 and TKPJ3039:: pMX303 agarose gel electrophoresis analysis is shown in FIG. 4,

lanes

1 and 2.

The specific method of this step is shown in step 3 in example 2.

Example 4: fermentation of over-expressed strains and detection of fermentation products

1. Seed culture: the over-expressed strain obtained in the step 3 of example 3 was activated on SFM plates containing apramycin (50. Mu.g/mL), and a proper amount of spore liquid was collected and inoculated into 5mL of TSBY liquid medium containing apramycin (25. Mu.g/mL) and cultured at 28℃and 220rpm for 2 to 3 days to obtain seed liquid.

2. Shaking and fermenting: the seed solution obtained in step 1 was inoculated into a 250mL shake flask containing 25mL of azalomycin F fermentation medium at an inoculum size of 10%, and fermented at 28℃for 10 days at 220 rpm.

3. And (3) fermentation liquid treatment: centrifuging the fermentation broth obtained in the step 2 at 3500rpm for 15min, separating thalli and supernatant, adding equal volume of methanol into thalli for soaking, centrifuging to collect organic phase, mixing with the fermentation broth supernatant, fixing volume to 45mL with methanol, and mixing well. 1mL of the sample was centrifuged at 12000rpm for 2min, and the supernatant was filtered through a 0.22 μm organic microporous filter membrane.

Hplc detection: azamycin F was detected using an Shimadzu SPD-M20A/LC-20AT chromatograph. C18 chromatography column: shim-pack VP-ODS, 250X 4.6mm,5 μm; column incubator: 25 ℃; pump mode: binary high pressure gradients; HPLC total flow rate: 1.0mL/min; setting the sample injection amount to be 20 mu L; full wavelength detection, total detection duration of 30min. The mobile phase A is deionized water, and the mobile phase B is methanol. Elution was performed according to the following conditions: 0-2min; the mobile phase B is maintained at 70% for 2-25min, and the mobile phase B rises from 70% to 90%;25-28min; mobile phase B was reduced from 90% to 70%, and maintained at 70% for 28-30 min. The HPLC detection results are shown in FIG. 5, with peaks of retention times 22.975min and 23.296min being various homologs of azalomycin F, based on standard. The peak area was integrated, the yields of the wild-type strain and the overexpressed strain were converted according to a standard curve, the change in the yield of azalomycin F of the overexpressed strain was calculated, and repeated three times, and the statistical result is shown in FIG. 6. The yield of azalomycin F of the wild strain was 5.01g/L, for which 100% the yields of azalomycin F of the strain TKPJ3039 over-expressing gene azl: pMX301 and the strain TKPJ3039 over-expressing gene azl: pMX303 were increased by 136% and 94%, respectively. The yield of the azalomycin F of other strains is not increased obviously.

The foregoing has described embodiments of the present invention. After reading the above description of the present invention, any person skilled in the art may make various changes and modifications within the technical scope of the present invention, and all those changes and modifications are covered in the scope of the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims

1. A genetically engineered bacterium for high yield of azalomycin F is characterized in that: the genetically engineered bacterium is obtained by over-expressing at least one of the following genes in a streptomycete host producing azalomycin F: the nucleotide sequence of the azl gene is shown as SEQ ID NO.1, and the nucleotide sequence of the azl gene is shown as SEQ ID NO. 2.

2. The genetically engineered bacterium for high production of azalomycin F of claim 1, wherein the genetically engineered bacterium is characterized in that: the streptomyces generating the azamycin F is a compound with a preservation number of CCTCC NO: m2013221 Streptomyces TKPJ3039.

3. The genetically engineered bacterium for high production of azalomycin F of claim 1, wherein the genetically engineered bacterium is characterized in that: azl4 genes and/or azl6 genes are overexpressed in Streptomyces hosts in a site-specific integrative vector containing a strong promoter.

4. The genetically engineered bacterium for high production of azalomycin F of claim 3, wherein: the site-specific integration type vector is pSET152 or pIB139 plasmid.

5. The genetically engineered bacterium for high production of azalomycin F of claim 3, wherein: the strong promoter is a constitutive promoter hrdBP, and the nucleotide sequence of the strong promoter is shown as SEQ ID NO. 3.

6. The method for constructing genetically engineered bacteria of high-yield azalomycin F according to any one of claims 1-5, wherein the method is characterized by comprising the following steps: the method comprises the following steps: and constructing an over-expression plasmid for driving target gene expression by a strong promoter element, and transferring the constructed plasmid into a streptomycete host for producing the azalomycin F by a conjugal transfer method to obtain the genetically engineered bacterium for producing the azalomycin F with high yield.

7. The construction method according to claim 6, wherein: the method comprises the following steps:

(2) Constructing an over-expression plasmid containing the target gene, wherein the plasmid contains the strong promoter element selected in the step (1) and takes the arabidopsis resistance gene as a marker;

8. The construction method according to claim 7, wherein: in the step (1), the intensity of different promoters in host bacteria is characterized and screened, and the method comprises the following steps:

(2) screening

Collecting spores of each strain, suspending in sterile water, and adjusting OD ₆₀₀ To the same level, carrying out gradient dilution, sucking 10 mu L of spore suspension to be uniformResistance strength test is carried out on a kanamycin gradient concentration plate, and the strength of kanamycin resistance shown by the strain is characterized by the activity strength of the corresponding promoter.

9. Use of the genetically engineered bacterium of high-yield azalomycin F of any one of claims 1-5 in preparing azalomycin F.

10. A process for the preparation of azalomycin F, characterized in that: the method comprises the following steps: inoculating the genetically engineered seed solution of high-yield azalomycin F of any one of claims 1-5 onto a fermentation medium for fermentation.