CN105907778B

CN105907778B - Streptomyces fuscosporivii recombinant expression plasmid, engineering bacterium and application

Info

Publication number: CN105907778B
Application number: CN201610203779.6A
Authority: CN
Inventors: 刘浩; 孙春杰; 何希宏; 王海霞
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2015-11-30
Filing date: 2016-03-31
Publication date: 2019-12-31
Anticipated expiration: 2036-03-31
Also published as: CN105907778A

Abstract

The invention relates to a streptomyces fuscosporioides recombinant expression plasmid, engineering bacteria and application, wherein an expression vector pIMEP (pimM E), pimM (pimM E), vgb (vgb) is successfully constructed by cloning two genes (pimM and pimE) with positive regulation and control functions in a streptomyces fuscosporioides natamycin biosynthesis gene cluster and an exogenous hemoglobin gene vgb; the Streptomyces phaeoflavus engineering strain is obtained through a combined transfer experiment of Escherichia coli ETZ with expression vectors pIME:: pimE:: pimM:: vgb and Streptomyces phaeoflavus; the Streptomyces phaeoflavus engineering strain can improve the transcription of a natamycin biosynthesis gene cluster due to the additive effect of the overexpression of a regulatory gene pimM and a cholesterol oxidase gene pimE in the natamycin biosynthesis gene cluster and the oxygen-poor resistance of a hemoglobin gene vgb, thereby obviously improving the yield of natamycin.

Description

Streptomyces fuscosporivii recombinant expression plasmid, engineering bacterium and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a streptomyces limolividii recombinant expression plasmid, an engineering bacterium and application.

Background

Natamycin is a broad-spectrum antifungal polyene macrolide. It can effectively inhibit and kill mould, yeast and filamentous fungi, so that it can effectively reduce the damage of strong pathogenic mycomycin to human body. Compared with other antibacterial components, natamycin has very low toxicity to mammalian cells, and can be widely applied to diseases caused by fungi. Due to the characteristics of no toxicity, no teratogenicity, no harm and no residue to human bodies and the like of natamycin, in 1982, the FDA of the United states officially approves the use of natamycin as a food preservative. The Ministry of health of China officially approved the quality of natamycin as a food preservative in 1997. Now, natamycin has been widely used in the food industry, and besides, natamycin is also widely used in the fields of medical treatment, feed, grain storage and the like.

Currently, cholesterol oxidase is found in bacteria such as Streptomyces (Streptomyces), Brevibacterium (Brevibacterium), Pseudomonas (Pseudomonas), and Rhodococcus (Schizophyllum). In the natamycin biosynthesis gene cluster, pimE encodes cholesterol oxidase. Cholesterol oxidase genes have been isolated from Streptomyces and encode cholesterol oxidases that are members of the acetyl cholesterol oxidase family and catalyze the formation of cholesterol-17-ketosteroids and hydrogen peroxide. In 2007, Marta et al found that pimE encodes a functional cholesterol oxidase in Streptomyces natalis through gene knockout and complementation experiments, and the enzyme is involved in the biosynthesis of natamycin. The biosynthesis process of the natamycin does not need the participation of cholesterol, but the yield of the natamycin can be obviously improved by adding purified PimE protein or heterologous sterol oxidase into the pimE knockout engineering strain. Therefore, Marta et al speculate that cholesterol oxidase is a signal protein secreted to the outside in natamycin biosynthesis, and plays a positive control role in natamycin biosynthesis. The university of Zhejiang Sun Shihao paper indicates that the cholesterol oxidase gene also has similar effect in Streptomyces chatanogeus, and the cholesterol oxidase gene and two regulatory genes pimR and pimM are presumed to have synergistic regulation effect on the biosynthesis of natamycin.

The regulatory genes of the natamycin biosynthesis gene cluster are pimR and pimM, which respectively code two transcription factors. In Streptomyces natalis (Streptomyces natalensis), the PimM gene is the positive transcriptional regulator in the natamycin biosynthesis gene cluster. PimM, as a PAS regulatory protein, can activate transcription of a part of genes in a gene cluster. In Streptomyces natalis, the pimM knockout strain loses the synthesis capacity of natamycin, and the increase of the copy of the pimM gene can improve the yield of natamycin. The sumura paper of Zhejiang university indicates that, in Streptomyces chattanoogensis (Streptomyces chattanoogensis), a regulatory gene ScnRII having a function similar to that of the regulatory gene pimM of the natamycin biosynthesis gene cluster is also a positive regulatory gene, and the gene is likely to be also bound to the promoter regions of the eight genes in the natamycin biosynthesis gene cluster. In industrial production, the method for improving the yield of antibiotics by directly increasing the copy number of the pathway specific regulatory gene is widely applied to the construction of high-yield streptomyces strains.

The streptomyces fuscoporia is aerobic bacteria, and the density of the bacteria is rapidly increased along with the prolonging of the fermentation time. In the fermentation process, the thalli grow slowly due to insufficient dissolved oxygen, the expression amount of key enzymes for synthesizing natamycin is reduced, and the yield of natamycin is reduced. Researches show that the expression of VHb under the condition of oxygen deficiency can obviously promote the growth of escherichia coli and saccharomyces cerevisiae cells, improve the protein synthesis capacity and increase the yield of target products. In addition, the vgb gene is also applied to microorganisms such as bacillus, Erwinia, and cephalosporium chrysogenum, so that the yield of biochemical products such as amylase, vitamin C, cephalosporin C and the like is successfully improved. It has also been reported that the gene is successfully expressed by introducing it into plants. Therefore, the gene is also introduced into the streptomyces fuscospora, and the engineering strain obtains the oxygen-poor resistance through the expression of the gene, so that the yield of the natamycin is improved.

Furthermore, according to the Tulingui report that "intermittent fed-batch fermentation increases natamycin yield", the natamycin yield was increased from 1.5g/L to 2.3g/L by maintaining the glucose concentration at 2% with an initial glucose concentration of 40g/L and intermittent glucose supplementation. Lijing le of Zhejiang university reports 'spatial breeding, process optimization and industrial scale-up research of natamycin high-producing strains', through comparing different control conditions, the optimal control conditions on the 30L fermentation scale are that the pH value in the fermentation process is 5.8-6.2, the whole stirring speed is 400rpm, the ventilation volume is 1.0vvm, and the temperature is 28 ℃. The streptomyces fuscoporia is an aerobic bacterium, so that the requirement of the bacteria on dissolved oxygen can be met by providing a rotating speed to improve the dissolved oxygen in the fermentation process. In the fermentation process, how to control the fermentation conditions is also of great significance to improve the yield of natamycin.

Disclosure of Invention

The invention aims to solve the technical problem of providing a streptomyces limolii recombinant expression plasmid.

Another technical problem to be solved by the invention is to provide an engineering bacterium containing the streptomyces limolividans recombinant expression plasmid.

The invention also aims to provide application of the engineering bacteria.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a streptomyces fuseoflavus recombinant expression plasmid is a recombinant expression vector pIME obtained by respectively inserting a regulatory gene pimM, a cholesterol oxidase gene pimE and an exogenous hemoglobin gene vgb into a vector pIME containing an erythromycin promoter, wherein the sequence of the recombinant expression vector pIME is a sequence shown in a sequence table <400> 17; the recombinant expression plasmid is used for the serial expression of three genes, an erythromycin promoter is arranged in front of each gene, the nucleotide sequence of pimM is a sequence shown in a sequence table <400>1, the nucleotide sequence of pimE is a sequence shown in a sequence table <400>2, the nucleotide sequence of vgb is a sequence shown in a sequence table <400>3, and the sequences of pimE/pimM/vgb and the promoter are sequences shown in a sequence table <400> 16; wherein the regulatory gene pimM and the starting vector pIMP are subjected to enzyme digestion by using BamH I/EcoR I, the pimE and the starting vector pIMP are subjected to enzyme digestion by using BamH I, and the vgb and the starting vector pIMP are subjected to enzyme digestion by using Kpn I; the fragment containing the erythromycin promoter and the regulatory gene (Perm:: pimM) and the starting vector pIME:: pimE were digested with EcoR I, and the fragment containing the erythromycin promoter and the hemoglobin gene (Perm:: vgb) and the starting vector pIME:: pimE:: pimM were digested with Mun I/Spe I.

Preferably, in the streptomyces fusciproflumii recombinant expression plasmid, the promoters of the regulatory gene pimM, the cholesterol oxidase gene pimE and the exogenous hemoglobin gene vgb are all erythromycin promoter ermE (Perm).

An engineering bacterium (Streptomyces gilvosporus/pIMEP:: pimE:: pimM:: vgb) containing the Streptomyces fuscoporia recombinant expression plasmid has a preservation number of CGMCC No. 11790.

The genome of the engineering bacteria is integrated with a recombinant expression vector pIME (pimE) pimmM (vgb), the capacity of high-yield natamycin is achieved, and the yield of fermentation for 120 hours reaches 10.136 g.

The construction method of the engineering bacteria comprises the following specific steps:

the streptomyces limolividans recombinant expression plasmid is firstly transformed into escherichia coli ETZ, and by utilizing a streptomyces limolividans-escherichia coli combined transfer experiment, streptomyces limolividans spore suspension and recombinant escherichia coli ETZ bacterial liquid containing a recombinant expression vector pIMP (pimE) (pimmM:vgb) are mixed and coated on MS (containing 5mM MgCl)₂) Plating, culturing in 28 deg.C incubator for 16-20h, spreading 1ml sterile water (containing nalidixic acid 0.4mg and adriamycin 1.5mg) for screening; the streptomyces flavus engineering strain containing the recombinant expression vector pIME:: pimE:: pimmM:: vgb can be obtained by continuous culture at 28 ℃.

The streptomyces fuseoflavus recombinant expression plasmid is a recombinant expression vector pIMEP containing a regulatory gene pimM of a natamycin biosynthesis gene cluster, a cholesterol oxidase gene pimE and an exogenous gene vgb, wherein pimE is pimM, and vgb is used for expressing the gene expression vector pIME.

Preferably, the construction method of the engineering bacteria comprises the steps of utilizing the streptomyces fusciparum engineering strain containing the resistance of the adriamycin, and screening transformants on an SS plate containing the resistance of the adriamycin, wherein the final concentration of the adriamycin in the plate is 15 mu g/ml; extracting a genome from a transformant with correct plate verification, and verifying whether the recombinant expression vector pIME:: pimE:: vmb is successfully inserted into the genome of streptomyces fusciparum by a PCR method.

The application of the engineering bacteria for high yield of natamycin is provided.

Preferably, in the application of the engineering bacteria, the fermentation conditions of the engineering bacteria are as follows: preparing fresh spore suspension, inoculating to seed culture medium at 2-5%, culturing at 28 deg.C and 200rpm for 2 days, inoculating to fermentation culture medium at 28 deg.C and 200rpm, and fermenting.

Preferably, in the application of the engineering bacteria, the seed culture medium comprises the following components in g/L: glucose 20, peptone 6, yeast extract 6, NaCl 10, pH 7.0, sterilized at 121 ℃ for 20 min.

Preferably, in the application of the engineering bacteria, the fermentation medium comprises the following components in g/L: glucose 40, soybean peptone 15, yeast extract 5, beeef extract powder 5, pH 7.5, sterilizing at 121 ℃ for 20min, wherein the preparation concentration of glucose is 50%, sterilizing at 115 ℃ for 30min, and fermenting and culturing conditions are as follows: fermenting and culturing at 28 deg.C and 200rpm in fermentation culture medium.

Preferably, the application of the engineering bacteria comprises supplementing a carbon source, regulating and controlling ventilation quantity and stirring rotation speed, and feeding 50% glucose solution at the flow rate of 2.5-3 g/L for 30-108 h in the fermentation process; controlling the pH value of the fermentation liquor to be 6; the initial ventilation volume is 4L/min, and the ventilation volume is increased to 6L/min after 7 h; the rotation speed is adjusted at corresponding different time points, and is not changed after the rotation speed is adjusted to 650rpm within 58 h.

The invention has the beneficial effects that:

the invention clones the regulatory genes pimE and pimM from streptomyces fuscosporus and the exogenous hemoglobin vgb gene by utilizing the genetic engineering technology, successfully expresses the genes and obtains the streptomyces fuscosporus engineering strain S.gilvosporus/pIME: (pimE:) and (vgb) with higher natamycin yield than the original strain of the streptomyces fuscosporus. The Streptomyces fuscosporensis engineering strain is applied to natamycin production, can produce more natamycin under the condition of consuming the same substrate, reduces the production cost and further improves the production benefit of enterprises.

Drawings

FIG. 1 is a plasmid map for constructing recombinant expression vector pIME:: pimM:: vgb.

FIG. 2 shows a transformant obtained by transforming a recombinant expression vector pIMEP pimE PImM vgb into Streptomyces gilvosporus (Streptomyces gilvosporus) according to the present invention.

FIG. 3 is a PCR-verified electrophoresis chart of a recombinant strain obtained by transforming Streptomyces gilvosporus (Streptomyces gilvosporus) with vgb, which is shown in the specification: all 3 randomly selected transformants can amplify specific bands with the size of 3230bp, and the results show that all the transformants are positive transformants, wherein 1 refers to negative control and no template is added; 2 is positive control, and the template is a constructed recombinant expression vector; 3 is a wild strain control, and the template is a streptomyces fuscosporium genome; 4, 5 and 6 are positive transformants, and the template is the genome of the corresponding transformant.

FIG. 4 is a graph showing the time-dependent change of the ratio of natamycin yield to dry weight of cells of Streptomyces fuscospora engineering strains and wild strains after shake flask fermentation: as can be seen from the figure, the streptomyces fuscoporia engineered strain is more potent in the synthesis of natamycin than the wild type strain, wherein (□) refers to streptomyces fuscoporia (S. gilvosporus S139); (A. tangle-solidup.) refers to Streptomyces phaeosporus engineering strain (S. gilvosporus/pIMEP:: pimE:: pimM:: vgb).

FIG. 5 is a bacteriostatic circle experiment of fermentation broth obtained from day 4 after shaking fermentation of an engineering strain of Streptomyces phaeoflavus and a wild strain. Wherein, the left side is Streptomyces fuscoporia (S.gilvosporus S139), and the right side is the engineering strain of Streptomyces fuscoporia (S.gilvosporus/pIMEP:: pimE:: pimM:: vgb). The indicator strain is yeast. The results thereof were in agreement with those of FIG. 4.

Preservation information

The classification nouns are: streptomyces gilvosporus

The name of the depository: china general microbiological culture Collection center

The address of the depository: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 04 days 12 month 2015

The preservation number is as follows: CGMCC No.11790

Reference biological material (strain): s280

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description will be given with reference to specific embodiments.

Firstly, as shown in figure 1, constructing an expression vector pIME containing a regulatory gene pimM, a cholesterol oxidase gene pimE and a hemoglobin gene vgb, wherein pimE, pimM, vgb and a primer sequence are shown in a sequence table <400>4- <400> 15;

secondly, constructing the engineering strain of the streptomyces flaviviridis containing an expression vector pIME, pimE, vmb: the strain is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation date is as follows: the accession number of 12 months and 04 days in 2015 is as follows: CGMCC No. 11790;

thirdly, controlling the fermentation of the streptomyces fuscosporioides engineering strain and measuring the content of natamycin;

the invention uses the genome of a Streptomyces gilvosporus strain as a template, and clones a coding and regulating gene pimM, a coding cholesterol oxidase gene pimE and an exogenous vitreoscilla hemoglobin gene vgb in a natamycin biosynthesis gene cluster to an initial vector pIMEP, so that the coding and regulating gene pimM, the cholesterol oxidase gene pimE and the hemoglobin gene vgb are positioned behind an erythromycin promoter Perm of the vector. After colony PCR verification and enzyme digestion verification, an expression vector pIMEP (pimmM/pIMEP) is successfully constructed, and pimE/pIMEP (pimM E/pIMEP) is successfully constructed. Then, an expression vector pIME is used as a starting vector, an expression vector pIME is used as a template to amplify the gene Perm, pimM is used, and EcoRI single enzyme digestion is used for constructing a recombinant expression vector pIME, pimE and pimM. Then, using expression vector pIME:: pimM as starting vector, using expression vector pIME:: vgb as template to amplify gene Perm:: vgb, using Mun I/Spe I single enzyme digestion to construct recombinant expression vector pIME:: pimM:: vgb. The vector was transformed into E.coli ETZ competent cells. The Streptomyces limousii engineering strain S.gilvosporeum/pIMEP can be obtained by utilizing the combined transfer experiment of Escherichia coli ETZ containing recombinant expression vector pIME and vgb. Performing table fermentation test on the obtained streptomyces limolividii engineering strain, taking fermentation liquor and methanol, mixing the fermentation liquor and the methanol in a ratio of 1: mixing the components in a ratio of 9, diluting by proper times after ultrasonic treatment for 20min, and measuring the yield of the streptomyces fuscospora engineering strain natamycin by an HPLC method.

Example 1

Construction of expression vector pIME pimE pimmM vgb

(1) Construction of expression vector pIMEP:. pimE

The genome of Streptomyces fuscoporus (Streptomyces gilvosporus) is used as a template, the primers in the table 1 are used for amplifying a cholesterol oxidase gene pimE in a natamycin biosynthesis gene cluster, and the downstream primer pimE-R of the gene cluster carries a his tag.

The PCR amplification system using the primers of table 1 is: 2 × PCR Buffer (containing Mg)²⁺) Mu.l of dNTP (2.5mM), 1. mu.l of each of the forward primer pimE-F and the reverse primer pimE-R (10. mu.M), 1. mu.l of the template (Streptomyces phaeoflavus genomic DNA), 0.5. mu.l of KOD FxDNA (purchased from TOYOBO Co., Ltd., cat. No. KFX-101) polymerase, and sterile water were added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 100s, reaction for 35 cycles, and extension at 68 ℃ for 10 min.

Primer sequences used in Table 1

The obtained DNA fragment pimE was digested with BamH I, recovered and ligated with the plasmid fragment pIMEP treated with the same endonuclease. The ligation product is transformed into escherichia coli JM109 competent cells, and is evenly coated on an LB plate with adriamycin resistance (50 mu g/ml), cultured overnight at 37 ℃, and single clone is selected for colony PCR verification and enzyme digestion verification, thus obtaining the expression vector pIMP (pimE).

(2) Construction of expression vector pIMEP

Similarly, the genome of Streptomyces gilvosporus (Streptomyces gilvosporus) is taken as a template, the primers in the table 2 are used for amplifying the regulatory gene pimM in the natamycin biosynthesis gene cluster, and the downstream primer pimM-R carries a his tag

The PCR amplification system is as follows: 2 × PCR Buffer (containing Mg)²⁺) Mu.l of dNTP (2.5mM), 1. mu.l of each of the forward primer pimM-F and the reverse primer pimM-R (10. mu.M), 1. mu.l of the template (Streptomyces phaeospora genomic DNA), 0.5. mu.l of KOD Fx DNA (purchased from TOYOBO Co., Ltd., product No. KFX-101) polymerase, and sterile water were added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 37s, reaction for 35 cycles, and extension at 68 ℃ for 10 min.

Primer sequences used in Table 2

The obtained DNA fragment, pimM, was similarly digested with BamH I/EcoR I, recovered and ligated with the plasmid fragment pIMEP treated with the same endonuclease. The ligation products were transformed into competent cells of Escherichia coli JM109, and uniformly spread on LB plates with resistance to apramycin (50. mu.g/ml), cultured overnight at 37 ℃, single clones were picked up, and colony PCR and enzyme digestion were performed to obtain the expression vector pIMEP:: pimM.

(3) Construction of expression vector pIMEP pimE pimM

The successfully constructed expression vector pIMEP is used as a template, and the successfully constructed expression vector pIMEP is used as a starting vector. The primers in Table 3 were used to amplify the pimM gene.

The PCR amplification system is as follows: 2 × PCR Buffer (containing Mg)²⁺) Mu.l of 10. mu.l of dNTP (2.5mM), 1. mu.l of each of the forward primer Perm-F and the reverse primer pimM-R (10. mu.M), 1. mu.l of the template (Streptomyces phaeoforth genomic DNA), 0.5. mu.l of KOD Fx DNA (purchased from TOYOBO Co., Ltd., product No. KFX-101) polymerase, and sterile water were added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 52s, reaction for 35 cycles, and extension at 68 ℃ for 10 min.

Primer sequences used in Table 3

The obtained DNA fragment Perm: (pimM) was digested with EcoR I, recovered, and ligated with the plasmid fragment pIMEP: (pimE) treated with the same endonuclease. The ligation products were transformed into competent cells of Escherichia coli JM109, and uniformly spread on LB plates with resistance to apramycin (50. mu.g/ml), cultured overnight at 37 ℃, single clones were picked up, and colony PCR and enzyme digestion were performed to obtain expression vectors pIME:: pimE:: pimmM.

(4) Construction of expression vector pIMP:: vgb

The vgb gene carrying the his tag was amplified using the primers of Table 4 using the plasmid vector puc57: (pLH217) as a template.

The PCR amplification system is as follows: 2 × PCR Buffer (containing Mg)²⁺) Mu.l of dNTP (2.5mM), 2. mu.l of each of the forward primer vgb-F and the reverse primer vgb-R (10. mu.M), 1. mu.l of the template (Streptomyces phaeoflavus genomic DNA), 0.5. mu.l of KOD Fx DNA (purchased from TOYOBO, cat. KFX-101) polymerase, and sterile water were added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 29s, reaction for 35 cycles, and extension at 68 ℃ for 10 min.

Primer sequences used in Table 4

The obtained DNA fragment vgb was digested with Kpn I, recovered and ligated with the plasmid fragment pIMEP treated with the same endonuclease. The ligation product is transformed into escherichia coli JM109 competent cells, and is evenly coated on an LB plate with adriamycin resistance (50 mu g/ml), cultured overnight at 37 ℃, and single clone is selected for colony PCR verification and enzyme digestion verification, thus obtaining the expression vector pIMEP:: vgb.

(5) Construction of expression vector pIME pimE pimmM vgb

The successfully constructed expression vector pIMP:: vgb is taken as a template, and the successfully constructed expression vector pIMP:: pimE:: pimM is taken as an initial vector. Perm:: vgb gene was amplified with the primers of Table 5.

The PCR amplification system is as follows: 2 × PCR Buffer (containing Mg)²⁺) Mu.l of 10. mu.l of dNTP (2.5mM), 1. mu.l each of the forward primer Perm-F and the reverse primer vgb-R (10. mu.M), 1. mu.l of the template (Streptomyces phaeospora genomic DNA), KOD Fx DNA (purchased from TOYOBO, Ltd., product number K)FX-101) polymerase 0.5. mu.l, sterile water was added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 43s, reaction for 35 cycles, and extension at 68 ℃ for 10 min.

Primer sequences used in Table 5

The obtained DNA fragment Perm:: vgb was digested with Mun I/Spe I, recovered and ligated with the plasmid fragment pIMEP:: pimE:: pimM treated with the same endonuclease. The ligation products were transformed into competent cells of Escherichia coli JM109, and uniformly spread on LB plates with resistance to apramycin (50. mu.g/ml), cultured overnight at 37 ℃, single clones were picked up, and subjected to colony PCR and enzyme digestion to obtain expression vectors pIME:: pimM:: vgb.

LB culture medium:

tryptone: 10.0g, yeast extract: 5.0g, NaCl: 10.0g, dissolving in deionized water, diluting to a constant volume of 1.0L, adjusting pH to 7.0-7.2, and adding 1.5% agar powder into solid culture medium. Sterilizing at 121 deg.C for 20 min.

Example 2

Construction of Streptomyces flaviviridae engineering strain containing expression vector pIME pimE pimmM vgb

1. Combined transfer of E.coli-S.fuscoporia

(1) The correct recombinant plasmid pIME:: pimM:: vgb is transformed into escherichia coli ETZ competent cells, after the cells are cultured in a shaker at 37 ℃ for 45min, 100 mul of fermentation liquor is uniformly coated on an LB plate containing resistance of kanamycin (with the final concentration of 50 mug/ml), tetracycline (with the final concentration of 15 mug/ml), chloramphenicol (with the final concentration of 25 mug/ml) and apramycin (with the final concentration of 50 mug/ml), and after the bacteria grow out, a single clone is selected and inoculated in a liquid LB culture medium containing the same four antibiotics for colony PCR verification and enzyme digestion verification.

(2) Inoculating the single clone with correct verification to a liquid LB culture medium containing the same four antibiotics, and culturing until OD is reached₆₀₀The value is between 0.4 and 0.6. The cells were collected in a 1.5ml EP tube, washed 2 to 3 times with liquid LB medium, and suspended in 1.5ml EP in 0.5ml LB medium for further use.

(3) Fresh Streptomyces fuscoporia spores were collected with sterilized cotton swabs in 1.5ml EP tubes, incubated with 0.5ml 2 XYT broth in a water bath at 50 ℃ for 10min, removed and cooled to room temperature.

(4) The treated E.coli cells and the Streptomyces phaeoflavus spore suspension were mixed and MS (containing 5mM MgCl) was applied₂) The plate was incubated at 28 ℃ for 16 to 20 hours in an incubator, and then 1ml of sterile water (containing 0.4mg of nalidixic acid and 1.5mg of apramycin) was applied. The culture was continued at 28 ℃ until colonies grew.

(5) Approximately 50-200 transformants grew per plate (FIG. 2). Transformants were transferred to SS plates with resistance to apramycin.

2. Verification of transformants

And (3) inoculating fresh spores of the obtained transformant of the streptomyces limolii engineering strain into a seed culture medium, and after culturing for two days, centrifuging the bacterial liquid to extract a genome. PCR verification is carried out by taking the genome of the transformant as a template and the genome of the wild strain as a control, and taking the expression plasmid pIMP, pimE, pimmM and vgb as a positive control.

The PCR reaction system is as follows: 10 XPCR Buffer 2. mu.l, dNTP (10mM) 0.4. mu.l, forward primer pimE-F and reverse primer pimM-R (10. mu.M) 0.4. mu.l each as shown in Table 6, template 1. mu.l, Taq DNA polymerase (purchased from Fermentas, Inc., cat. EP0405) 0.4. mu.l, sterile water was added to a final volume of 20. mu.l.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 96 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 194s, reaction for 35 cycles, and re-extension at 72 ℃ for 10 min.

Primer sequences used in Table 6

The results are shown in FIG. 3, compared with the Streptomyces fuscoporia wild strain, the recombinant expression vector pIME:: pimE:: pimmM:: vgb, Streptomyces fuscoporia engineering strain can amplify a 3230bp band, which indicates that the recombinant expression vector pIME:: pimM:: vgb has been successfully inserted into the genome of Streptomyces fuscoporia. Wherein, 1 refers to negative control, and no template is added; 2 is positive control, and the template is a constructed recombinant expression vector pIME, pimmM and vgb; 3 is a wild strain control, and the template is a streptomyces fuscosporium genome; 4, 5 and 6 are positive transformants, and the template is the genome of the corresponding transformant.

The formula of the culture medium is as follows:

MS culture medium:

mannitol: 20.0 g; soybean meal: 20.0 g; agar: 15.0 g; after dissolving in deionized water, the volume is adjusted to 1.0L. Sterilizing at 121 deg.C for 20 min.

Seed culture medium:

glucose: 20.0g, peptone: 6.0g, yeast extract: 6.0g, NaCl: 10.0g, dissolving in deionized water, diluting to 1.0L, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.

Example 3

Fermentation control of streptomyces fuscoporia engineering strain and determination of natamycin content

1. Shaking table fermentation control of streptomyces flavosporus engineering strain

The screened Streptomyces fuscoporia engineering strain (Streptomyces gilvosporus/pIMEP:: pimE:: pimM:: vgb) CGMCC No.11790 and the wild strain (S. gilvosporus S139) were streaked on SS plates and cultured at 28 ℃ for 8-12 days. Fresh spores were washed with sterile water to prepare a fresh spore suspension. Inoculating spore suspension into 50ml triangular flask containing 20ml seed culture medium at 2% -5%, and culturing at 28 deg.C and 200rpm for 2 days. Then, the seed culture medium is transferred into a 250ml triangular flask containing 50ml of fermentation culture medium according to the inoculation amount of 5 percent, and the fermentation culture is carried out for 5 days at the temperature of 28 ℃ and at the rpm of 200.

2. Fermentation control of Streptomyces phaeoflavus engineering strain 5L fermentation tank

The screened S.fuscoporia engineered strain (S.gilvosporus/pIMEP:: pimE:: pimM:: vgb) and the wild type strain (S.gilvosporeu S139) were streaked on SS plates and cultured at 28 ℃ for 8-12 days. Fresh spores were washed with sterile water to prepare a fresh spore suspension. Inoculating spore suspension into 500ml triangular flask containing 150ml seed culture medium at 2% -5%, and culturing at 28 deg.C and 200rpm for 2 days. Then, the seed culture medium is transferred into a 5L fermentation tank according to the inoculation amount of 5 percent, and the total volume of the fermentation liquid is 3L. The initial aeration rate of fermentation is 4L/min, the pH value is controlled at about 6.0 in the whole fermentation process, and the temperature is 28 ℃. And 50% glucose is dripped at the flow rate of 2.5g-3.0g per hour at 30h (the residual sugar of the fermentation liquor is lower than 2%), and the dripping is stopped at 108 h. The fermentation process control during the whole fermentation process is as follows:

(1) the rotation speed is 250rpm within 0-7h, and the ventilation volume is maintained at 4L/min;

(2) the rotating speed is 250rpm for 7-8h, and the ventilation volume is maintained at 6L/min;

(3) the rotating speed is 300rpm for 8-12h, and the ventilation volume is maintained at 6L/min;

(4) the rotating speed is 350rpm for 12-16h, and the ventilation volume is maintained at 6L/min;

(5) the rotating speed is up to 400rpm within 16-20h, and the ventilation volume is maintained at 6L/min;

(6) the rotating speed is 450rpm for 20-24h, and the ventilation volume is maintained at 6L/min;

(7) the rotating speed is 500rpm for 24-30h, and the ventilation volume is maintained at 6L/min;

(8) the rotation speed is 550rpm within 30-38h, and the ventilation volume is maintained at 6L/min;

(9) the rotation speed is 600rpm within 38-50h, and the ventilation volume is maintained at 6L/min;

(10) the rotating speed is 625rpm within 50-58h, and the ventilation volume is maintained at 6L/min;

(11) the rotation speed is 650rpm within 58-120h, and the ventilation volume is maintained at 6L/min.

Seed culture medium:

glucose: 20.0g, peptone: 6.0g, yeast extract: 6.0g, NaCl: 10.0g, dissolving with deionized water, diluting to 1.0L, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.

Fermentation medium:

glucose: 40.0g, soy peptone: 15.0g, yeast extract: 5.0g, beef extract powder: 5.0g, dissolving with deionized water, diluting to a constant volume of 1.0L, adjusting pH to 7.5, sterilizing at 121 deg.C for 20min, wherein the concentration of glucose is 50%, and sterilizing at 115 deg.C for 30 min.

SS culture medium:

glucose: 15.0 g; peptone: 5.0g, yeast extract: 3.0g, malt extract powder: 3.0g, agar powder: 15.0g, dissolving with deionized water, diluting to 1.0L, and sterilizing at 121 deg.C for 20 min.

3. Determination of natamycin content in fermentation liquor

And after the fermentation is finished, mixing 1ml of fermentation liquor with 9ml of methanol, uniformly mixing, performing ultrasonic treatment for 20min, centrifuging for 15min at 8000r/min, filtering by using an organic membrane of 0.22 mu m to obtain a sample to be detected, and quantitatively analyzing the natamycin by using HPLC.

Conditions for HPLC analysis: mobile phase: 70:30 parts of methanol and water; flow rate: 0.700 ml/min; the detection wavelength is 303 nm; the sample amount is 10 mul; the column temperature was 30 ℃.

The shaker data are shown in FIG. 4: the ratio of the yield of natamycin to the dry weight of cells of the S.fuscoporus engineering strain (S.gilvosporus/pIMEP:: pimE:: pimM:: vgb) after shaking fermentation and the starting strain (S.gilvosporus S139) changes with time. From FIG. 4, it can be seen that the yield of natamycin from the S.fuscosporeus/pIMEP:: pimE:: pimM:: vgb) engineered strain of S.fuscoporia was higher than that from the wild strain, and this difference was most significant at day 4. The calculation result shows that the yield of natamycin produced by the fermentation liquor unit thalli of the streptomyces limosporus engineering strain (S.gilvosporus/pIMEP:: pimE:: pimmM:: vgb) on the 4 th day is improved by 56 percent compared with the original strain. Therefore, the additive effect of the regulatory gene pimmM and the cholesterol oxidase gene pimE in the natamycin biosynthesis gene cluster and the expression of the exogenous gene vgb are proved to be capable of obviously improving the yield of natamycin in streptomyces fuscosporioides. FIG. 5 is a zone of inhibition experiment with day 4 broth, indicating that the strain was yeast. The figure shows more visually that the Streptomyces fuscosporeus engineered strain (S.gilvosporus/pIMEP:: pimE:: pimM:: vgb) has higher natamycin synthesis capacity than the wild strain. The data of the wild strain and the engineering strain after 5L fermentation tank culture are shown in Table 7, the yield of natamycin is increased after the engineering strain is fermented on the tank due to the additive effect of pimM and pimE overexpression and exogenous gene vgb expression, and the growth rate is gradually reduced at the later stage; however, at 120h, the yield of natamycin is still improved by 15.6 percent compared with that of the wild strain, and reaches 10.136 g/L. Therefore, the synthesis time of natamycin is greatly shortened due to the superposition effect of the three.

TABLE 7 comparison of natamycin yields from starting and engineered strains

The above detailed description of the streptomyces fuscospora recombinant expression plasmid, the engineered bacterium and the use thereof with reference to the specific embodiments is illustrative and not restrictive, and several examples can be cited according to the limited scope, so that variations and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A streptomyces fusciparum recombinant expression plasmid is characterized in that: the recombinant expression vector pIME is obtained by inserting a regulatory gene pimM, a cholesterol oxidase gene pimE and an exogenous hemoglobin gene vgb into a vector pIMEP containing an erythromycin promoter, wherein the sequence of the recombinant expression vector pIME is pimM E, and the sequence of the recombinant expression vector vgb is shown in a sequence table SEQ ID NO. 17.

2. An engineered bacterium comprising the streptomyces fusciparum recombinant expression plasmid of claim 1, wherein the engineered bacterium comprises: the preservation number is CGMCC No. 11790.

3. Use of the engineered bacteria of claim 2 for the production of natamycin.

4. The use of the engineered bacteria of claim 3, wherein: comprises supplementing carbon source, regulating and controlling aeration quantity and stirring speed, and feeding 50% glucose solution at a flow rate of 2.5-3 g/L for 30-108 h in the fermentation process; controlling the pH value of the fermentation liquor to be 6; the initial ventilation volume is 4L/min, and the ventilation volume is increased to 6L/min after 7 h; the rotation speed is adjusted at corresponding different time points, and is not changed after the rotation speed is adjusted to 650rpm within 58 h.