CN108531488B

CN108531488B - Novel regulatory gene AurT and application thereof in increasing yield of aureofuscin

Info

Publication number: CN108531488B
Application number: CN201810275673.6A
Authority: CN
Inventors: 魏杰; 高军; 许德馨
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2018-03-30
Filing date: 2018-03-30
Publication date: 2022-06-14
Anticipated expiration: 2038-03-30
Also published as: CN108531488A

Abstract

The invention relates to a novel regulatory gene AurT and application thereof in improving the yield of aureofuscin. The technical scheme is as follows: extracting genomic DNA of the streptomyces aureofuscus, obtaining a new gene AurT by utilizing a PCR amplification technology, cloning the gene AurT into a high-efficiency expression plasmid vector pSET152, constructing a recombinant plasmid PBJAurT for intergeneric conjugative transfer of escherichia coli ET12567 and wild streptomyces aureofuscus, and transferring the PBJAurT to the chromosomal DNA of the wild streptomyces aureofuscus strain by an intergeneric conjugative transfer method to obtain a transformant. After screening, the obtained transformant is subjected to fermentation culture, and the change condition of the yield of aureofuscin is analyzed and determined by adopting an HPLC method, so that the fermentation yield of aureofuscin is improved by about 3.5 times.

Description

Novel regulatory gene AurT and application thereof in increasing yield of aureofuscin

Technical Field

The invention belongs to the field of molecular biology of food biotechnology, and particularly relates to a new regulatory gene AurT and a method for improving aureofuscin yield by using the new regulatory gene AurT.

Background

Aureofuscin (Aureofuscin) is a tetraene macrolide antifungal antibiotic produced by streptomyces aureofuscus, and has a chemical structure similar to that of natamycin reported in foreign literatures. According to research reports, aureofuscin has strong antibacterial effect on mould, various yeasts and filamentous fungi, but does not resist bacteria, is clinically used for treating fungal keratitis, has better curative effect than amphotericin B, and also has good curative effect on skin diseases caused by some fungi, mycotic vaginitis and the like. The fermentation yield of the aureofuscin is low, the extraction yield is not high, and the effect of improving the yield of the aureofuscin from the fermentation level in the early stage is not ideal, so that the research and the realization of the industrialized development from the molecular level are more deeply carried out, and the practical significance is realized for further increasing the yield of the aureofuscin.

Disclosure of Invention

The invention aims to obtain a regulatory gene AurT in streptomyces aureofuscus from a molecular level and improve the yield of aureofuscin by utilizing the regulatory gene AurT.

The technical scheme adopted by the invention is as follows: the new regulatory gene AurT has the nucleotide sequence as shown in SEQ ID No. 1.

The application of the new regulatory gene AurT in improving the yield of aureofuscin. The method comprises the following steps:

firstly, extracting a regulatory gene AurT in the genomic DNA of the streptomyces aureofuscus by utilizing a PCR amplification technology, cloning the gene AurT into an expression plasmid vector pSET152, and obtaining a recombinant plasmid PBJAurT containing the regulatory gene AurT. The method specifically comprises the following steps:

1) extracting total DNA of wild streptomyces aureofuscus;

2) by T₁And T₂Taking the total DNA as a template for PCR amplification as a primer to obtain 645bp AurT gene; the T is₁And T₂The sequence of (a) is:

T₁：5'-ATGGTGTCCACGGAGAGCAT-3'；

T₂：5'-CTAGTCGGCGCGGCCCGTCG-3'

PCR amplification System (20ul) T₁:1ul；T₂1 ul; 1ul of DNA template; 10 XPCR buffer:2 ul; dNTP mix 1.6 ul; ddH₂O:13ul PCR。

The PCR amplification reaction conditions are as follows: 94 ℃ for 5 min; 95 deg.C, 30sec, 55 deg.C, 30sec, 72 deg.C, 1 min; 30 cycles; 72 ℃ for 7 min; and keeping the temperature at 4 ℃.

3) The AurT gene and the expression plasmid vector pSET152 are subjected to double enzyme digestion by BamHI and EcoRV restriction enzymes respectively, and are connected by T4 DNA ligase to construct a recombinant plasmid PBJAurT.

And (II) transforming the recombinant plasmid PBJAurT containing the regulatory gene AurT into escherichia coli ET12567 to obtain a transformant. The method comprises the following specific steps: the recombinant plasmid PBJAURT containing the regulatory gene AurT is transformed into an escherichia coli ET12567 competent cell, is coated on an LB culture medium containing 50ug/ml apramycin, and is cultured overnight at 37 ℃ to obtain a transformant.

And (III) carrying out heat shock on the spores of the streptomyces aureofuscus at 50 ℃ for 10min by using a method of escherichia coli-streptomyces conjugal transfer, pre-germinating the spores and the transformants obtained in the step (II) uniformly according to the weight ratio of 1:1, culturing the spores at 30 ℃ for 13-20 hours, covering the spores with 50ug/ml of apramycin and 25ug/ml of nalidixic acid, and continuously culturing the spores at 30 ℃ for 24-48 hours to obtain conjugal transfersomes.

And (IV) performing fermentation culture on the screened conjugal transferor in a fermentation culture medium. Preferably, the fermentation medium consists of: 0.2% of yeast extract powder, 1% of soluble starch and the balance of water. More preferably, the conjugal transferor is inoculated into the fermentation medium at an inoculum size of 10% and cultured with shaking at 29 ℃ and 220 rpm.

The invention has the beneficial effects that: the invention optimizes the PCR amplification system of the streptomyces aureofuscus genome DNA and obtains a new regulatory gene AurT. The AurT gene can be expressed in wild type streptomyces aureofuscus through an intergeneric conjugation transfer system. After multiple screening, transformants are obtained and cultured by fermentation, and the yield of aureofuscin is determined by an HPLC method. Compared with wild Streptomyces aureofuscus strain, the fermentation yield is increased by about 3.5 times, and the yield is stable in passage. The recombinant strain can improve the yield of aureofuscin, and the AurT gene can promote the biosynthesis of aureofuscin. The molecular level proves that the improvement of the AurT level of the regulatory gene in the streptomyces aureofuscus is an effective method for increasing the yield of aureofuscin.

Drawings

FIG. 1 is a diagram showing the construction of plasmid PBJAurT.

FIG. 2 is a diagram showing the results of PCR of AurT gene in genomic DNA of Streptomyces aureofuscus.

FIG. 3A is a chromatographic peak of aureofuscin as a standard substance determined by HPLC.

FIG. 3B is the aureofuscin-producing chromatographic peak of the original Streptomyces aureofuscus strain.

FIG. 3C is the chromatographic peak of aureofuscin produced by transformant fermentation.

Detailed Description

Example method (one) for increasing the yield of aureofuscin is as follows:

1. a PCR amplification technology is utilized to extract a regulatory gene AurT in the genomic DNA of the streptomyces aureofuscus, and the gene AurT is cloned into an expression plasmid vector pSET152 to obtain a recombinant plasmid PBJAurT containing the regulatory gene AurT. The method specifically comprises the following steps:

1) extracting total DNA of wild streptomyces aureofuscus;

2) by T₁And T₂Using total DNA as a template for PCR amplification to obtain 645bp of AurT gene; the T is₁And T₂The sequence of (a) is:

T₁：5'-ATGGTGTCCACGGAGAGCAT-3'

T₂：5'-CTAGTCGGCGCGGCCCGTCG-3'

PCR amplification System (20ul) T₁:1ul；T₂1 ul; 1ul of DNA template; 10 XPCR buffer:2 ul; dNTP mix 1.6 ul; ddH₂O:13ul。

The PCR reaction conditions were: 94 ℃ for 5 min; 95 deg.C, 30sec, 55 deg.C, 30sec, 72 deg.C, 1 min; 30 cycles; 72 ℃ for 7 min; and keeping the temperature at 4 ℃.

The PCR result of the obtained regulatory gene AurT is shown in FIG. 2, and as can be seen from FIG. 2, the genes AurT and 100bp DNA Marker are sequentially arranged from left to right, and the AurT shows a bright band at 645 bp.

The nucleotide sequence of the obtained regulatory gene AurT is shown as SEQ ID NO: 1, the amino acid sequence is shown as SEQ ID NO: 2.

3) carrying out BamHI and EcoRV restriction enzyme double digestion on AurT gene and expression vector pSET152 respectively, and carrying out T₄And (3) connecting the DNA with ligase to construct a recombinant plasmid PBJAurT.

2. The recombinant plasmid PBJAURT containing the regulatory gene AurT is transformed into an escherichia coli ET12567 competent cell, is coated on an LB culture medium containing 50ug/ml apramycin, and is cultured overnight at 37 ℃ to obtain a transformant.

3. By utilizing an escherichia coli-streptomycete conjugal transfer method, heat shock is carried out on streptomycete aureofuscus spores at 50 ℃ for 10min, pre-germination is carried out, the spores and the transformant obtained in the step 2 are uniformly mixed according to the weight ratio of 1:1, the spores and the transformant are cultured for 13-20 hours at 30 ℃, then the spores and the transformant are covered by 50ug/ml apramycin and 25ug/ml nalidixic acid, and the spores and the transformant are continuously cultured for 24-48 hours at 30 ℃ to obtain the conjugal transfer. This allows the recombinant plasmid containing the regulatory gene AurT to be transferred into the chromosomal DNA of a wild-type strain of Streptomyces aureofuscus.

And (3) selecting a single colony, respectively carrying out colony PCR and enzyme digestion detection, and carrying out agarose gel electrophoresis to show that a bright band is obviously formed at a position of about 645bp, namely, selecting the recombinant plasmid.

4. The conjugative transfersomes were transferred to a plate containing apramycin and nalidixic acid, subcultured, and the selected conjugative transferants were inoculated into a fermentation medium (0.2% yeast extract powder, 1% soluble starch) at an inoculum size of 10%, and shake-cultured at 29 ℃ and 220 rpm.

(II) detection

And (4) determining and analyzing the yield change condition of the aureofuscin by using an HPLC method. The results are shown in FIGS. 3A-3C. FIG. 3A represents a aureofuscin standard; FIG. 3B represents the original S.aureofuscus strain; FIG. 3C represents the transformant fermentation broth, which shows about a 3.5-fold increase in fermentation yield of Aureomycin as measured by chromatographic peak analysis compared to the original S.aureofuscus strain.

<110> Liaoning university

<120> new regulatory gene AurT and application thereof in increasing yield of aureofuscin

<160> 2

<210> 1

<211> 645

<212> DNA

<213> Streptomyces aureofuscus regulatory gene AurT

<400> 1

ATGGTCTCCACGGAGAGCATCCTCGCGTTCGCGGCGATGTCGCTACTGGTGATCGTGATT 60

CCGGGGCCGAGCGTGCTGTTTGTGATCGGCAGAGCCCTTGCGCACGGCCGCCGCACGGCG 120

CTCGCGACGGTCCTCGGCAACCTGGTCGGCTCGTACCTCCTGGTGACGGCTGTGGCGTGG 180

GGCCTCGGCGCGCTGGTGGAAGGTTCGGTGGCGGTGTTCACGGGCGTGAAGCTGGCCGGC 240

GCGGCGTATCTCGTCTACCTCGGCGTGCGCGCGTTCCGGCACCGCAAGGAGATGCGCGCG 300

GCGGACATGGAGGCCCCGGCGGGTGAGCGGCGCGGCGATCTGCGCACGATCCTGGACGGG 360

ATTTTCGTGGGCGTCACCAATCCGAAGGGCGTCGTCTTCTTCGCGGCGGTGCTGCCGCAG 420

TTCGTGAACCACTCGGCCGGCCGGGTTCCCCTCCAGATGATGGTGTTGGGGCTGGTCCCG 480

GTCGCCATCGGCATGATCACGGACACCCTGTGGGGCCTGGGTGCCGCGGCGGCCCGTTCG 540

TGGTTCGCCCGCTCGGACCGCCGCCTGTCGATGGTCGGCGGGGCGGGCGGCTTCGCGATG 600

ATCGGGCTGGGCGTGACCGTGGCAGCGACGGGCCGCGACGACTAG 645

<210> 2

<211> 214

<212> amino acid

<213> Streptomyces aureofuscus regulatory gene AurT

<400> 2

MVSTESILAFAAMSLLVIVIPGPSVLFVIGRALAHGRRTALATVLGNLVGSYLLVTAVAW 60

GLGALVEGSVAVFTGVKLAGAAYLVYLGVRAFRHRKEMRAADMEAPAGERRGDLRTILDG 120

IFVGVTNPKGVVFFAAVLPQFVNHSAGRVPLQMMVLGLVPVAIGMITDTLWGLGAAAARS 180

WFARSDRRLSMVGGAGGFAMIGLGVTVAATGRDD 214

Claims

1. The regulatory gene AurT is characterized in that the nucleotide sequence of the new regulatory gene AurT is shown as SEQ ID NO. 1.

2. Use of the regulatory gene AurT of claim 1 for increasing aureofuscin production.

3. Use according to claim 2, characterized in that the method comprises the following steps:

1) Extracting a regulatory gene AurT in the genomic DNA of the streptomyces aureofuscus by utilizing a PCR amplification technology, and cloning the gene AurT into an expression plasmid vector pSET152 to obtain a recombinant plasmid PBJAurT containing the regulatory gene AurT;

2) transforming the recombinant plasmid PBJAurT containing the regulatory gene AurT into escherichia coli ET12567 to obtain a transformant;

3) thermally shocking streptomyces aureofuscus spores at 50 ℃ for 10min by using an escherichia coli-streptomyces conjugal transfer method, uniformly mixing the spores with the transformant obtained in the step 2) after pregermination, culturing the spores at 30 ℃ for 13 to 20 hours, covering the spores with 50ug/ml apramycin and 25ug/ml nalidixic acid, and continuously culturing the spores at 30 ℃ for 24 to 48 hours to obtain conjugal transfersomes;

4) and (4) performing fermentation culture on the screened conjugal transferor in a fermentation culture medium.

4. The use according to claim 3, wherein step 1) is specifically:

1) extracting the total DNA of wild type streptomyces aureofuscus;

2) by T₁And T₂Taking the total DNA as a template for PCR amplification as a primer to obtain an AurT gene; the T is₁And T₂The sequence of (a) is:

T₁：5'-ATGGTGTCCACGGAGAGCAT-3'

T₂：5'-CTAGTCGGCGCGGCCCGTCG-3'

5. Use according to claim 4, characterized in that in step 2), the PCR amplification system (20ul) T₁:1ul; T₂1ul, 1ul DNA template, 2ul 10 XPCR buffer, 1.6ul dNTP mix, ddH₂O13 ul PCR; the PCR amplification reaction conditions are as follows: 94 ℃ for 5 min; 95 deg.C, 30sec, 55 deg.C, 30sec, 72 deg.C, 1 min; 30 cycles; 72 ℃ for 7 min; keeping the temperature at 4 ℃.

6. The use according to claim 3, wherein step 2) is specifically: the recombinant plasmid PBJAURT containing the regulatory gene AurT is transformed into an escherichia coli ET12567 competent cell, is coated on an LB culture medium containing 50ug/ml apramycin, and is cultured overnight at 37 ℃ to obtain a transformant.

7. The use according to claim 3, characterized in that in step 3), the spores of Streptomyces aureofuscus are heat-shocked at 50 ℃ for 10min, pre-germinated and then mixed with the transformant obtained in step 2) in a weight ratio of 1: 1.

8. The use according to claim 3, wherein in step 4) the fermentation medium consists of: 0.2% of yeast extract powder, 1% of soluble starch and the balance of water.

9. The use according to claim 3, wherein in step 4), the conjugal transferor is inoculated into the fermentation medium at an inoculum size of 10% and cultured with shaking at 220rpm and 29 ℃.