CN113549643B - Method for improving synthesis of prodigiosin by Serratia marcescens through overexpression of gene psrB - Google Patents

Method for improving synthesis of prodigiosin by Serratia marcescens through overexpression of gene psrB Download PDF

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CN113549643B
CN113549643B CN202110800394.9A CN202110800394A CN113549643B CN 113549643 B CN113549643 B CN 113549643B CN 202110800394 A CN202110800394 A CN 202110800394A CN 113549643 B CN113549643 B CN 113549643B
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prodigiosin
psrb
serratia marcescens
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CN113549643A (en
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饶志明
潘学玮
杨套伟
尤甲甲
徐美娟
张显
邵明龙
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Jiangnan University
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Abstract

The invention discloses a method for improving serratia marcescens to synthesize prodigiosin through over-expression of a gene psrB, belonging to the technical fields of genetic engineering and microbial engineering. The invention obviously improves the capability of serratia marcescens in synthesizing the prodigiosin by over-expressing a DeoR family transcription regulatory factor PsrB (BVG 90_ 04085) coding gene BVG90_04085 (psrB) in serratia marcescens; the recombinant Serratia marcescens prepared by the method is fermented in a fermentation medium for 72 hours to produce the prodigiosin, and the prodigiosin producing capacity of the recombinant strain JNB5-1-PsrB is improved by 28.33% compared with that of a wild strain JNB5-1, and the yield is up to 6.84g/L.

Description

Method for improving synthesis of prodigiosin by Serratia marcescens through overexpression of gene psrB
Technical Field
The invention relates to a method for improving serratia marcescens to synthesize prodigiosin by over-expressing a gene psrB, belonging to the technical fields of genetic engineering and microbial engineering.
Background
Prodigiosin (PG), a secondary metabolite produced by microorganisms, has various biological activities such as antibacterial activity, anti-diarrhea activity, anti-tumor activity, immunosuppression activity and the like, and has great application value in the fields of medicine development, environmental management, dye preparation and the like. Currently, the prodigiosin production method mainly comprises 2 methods such as a chemical synthesis method and a microbial fermentation method. Because of the defects of more reaction steps, low yield and the like in the synthesis of the prodigiosin by a chemical synthesis method, the large-scale production of the prodigiosin is difficult to realize. The microbial fermentation method for producing the prodigiosin has the advantages of environment friendliness, mild condition, low cost, easiness in industrial production and the like, so that the microbial fermentation method for producing the prodigiosin has become a research hotspot at home and abroad in recent years.
However, the existing biological methods still have certain defects, wherein the low yield is the most important defect for preventing the industrial process of the microbial fermentation method. For example, lee et al fermented to produce prodigiosin by inoculating Zooshikella ganghwensis KCTC 12044T into Marinebroth 2216 medium, but using this method fermentation for 24 hours only allowed prodigiosin to yield 15.40mg/L in the fermentation broth (see, in particular, lee, J.S., kim, Y.S., park, S., kim, J., kang, S.J., lee, M.H., et al (2011) Exceptional production ofboth prodigiosin and cycloprodigiosin asmajor metabolic constituents by a novel marine bacterium, zooshikella rubidus S1-1.appl. Environ. Microbiol.77, 4967-4973.); lee et al fermented to produce prodigiosin by inoculating Hahella chejuensis KCTC 2396T into Marinebroth 2216 medium, however, fermentation using this method only allowed for 28.10mg/L prodigiosin production in the fermentation broth (see, inter alia, lee, J.S., kim, Y.S., park, S., kim, J., kang, S.J., lee, M.H., et al (2011) Exceptional production ofboth prodigiosin and cycloprodigiosin asmajor metabolic constituents by a novel marine bacterium, zooshikella rubidus S1-1.Appl. Microbiol.77, 4967-4973.1).
Thus, there is an urgent need to find a microorganism that can produce prodigiosin at high yield to overcome the above-mentioned drawbacks.
Disclosure of Invention
The first object of the present invention is to provide a method for improving the synthesis of prodigiosin by Serratia marcescens; the method is to overexpress a DeoR family transcription regulatory factor BVG90_04085 (PsrB) encoding gene BVG90_04085 (psrB) in Serratia marcescens Serratia marcescens.
In one embodiment of the present invention, the amino acid sequence of the DeoR family transcription regulatory factor PsrB is shown in SEQ ID NO. 1.
In one embodiment of the invention, the over-expression is to integrate the psrB gene into a vector to obtain a recombinant plasmid, and then transfer the recombinant plasmid into Serratia marcescens to obtain a recombinant strain.
In one embodiment of the present invention, the nucleotide sequence of the psrB gene is shown as SEQ ID NO. 2.
In one embodiment of the present invention, the Serratia marcescens comprises Serratia marcescens JNB5-1.
In one embodiment of the invention, the vector comprises pUCP18.
The second object of the invention is to provide a recombinant strain for efficiently synthesizing prodigiosin.
In one embodiment of the invention, the recombinant strain is one that overexpresses the DeoR family transcription regulatory factor BVG90_04085 (PsrB) in serratia marcescens.
In one embodiment of the present invention, the amino acid sequence of the DeoR family transcription regulatory factor PsrB is shown in SEQ ID NO. 1.
In one embodiment of the invention, the expression is performed by integrating the psrB gene into a vector to obtain a recombinant plasmid, and then transferring the recombinant plasmid into Serratia marcescens to obtain a recombinant strain.
In one embodiment of the present invention, the nucleotide sequence of the gene psrB is shown as SEQ ID NO. 2.
In one embodiment of the invention, the Serratia marcescens comprises Serratia marcescens wild-type strain JNB5-1.
In one embodiment of the invention, the vector comprises pUCP18.
The third object of the invention is to provide a method for producing prodigiosin, which comprises the steps of inoculating the recombinant strain into a fermentation medium for culture to obtain fermentation liquor, and extracting prodigiosin from the fermentation liquor.
In one embodiment of the invention, the method comprises the following specific steps: inoculating the recombinant strain into LB liquid culture medium for culturing to obtain OD 600 The preparation method comprises the steps of (1) inoculating bacterial liquid with the bacterial liquid of which the number is between 0.4 and 0.8 into a fermentation medium, culturing at the temperature of between 28 and 32 ℃ and at the speed of between 160 and 200rpm to obtain fermentation liquid, collecting the fermentation liquid, and measuring the amount of the bacterial synthetic prodigiosin after the action of acid ethanol for 6 to 10 hours.
In one embodiment of the invention, the fermentation medium has a composition of: 1.5 to 2.5 percent of sucrose, 1.0 to 2.0 percent of beef extract and CaCl 2 0.75-1.25%, L-proline 0.5-1.0% and MgSO 4 ·7H 2 O 0.0025～0.0035％，pH 7.0。
A fourth object of the present invention is the use of said method for increasing prodigiosin synthesis or of said recombinant strain for the production of prodigiosin or of prodigiosin-containing products.
The fifth object of the present invention is that the method for improving prodigiosin synthesis or the recombinant strain is used in the fields of medicine development, environmental management, dye preparation, etc.
The beneficial effects are that:
1. the invention obtains recombinant Serratia marcescens with obviously improved prodigiosin synthesis capability by over-expressing psrB gene in Serratia marcescens; the recombinant Serratia marcescens is fermented in a fermentation medium for 72 hours, and the final prodigiosin yield can reach 6.84g/L, which is improved by 28.33% compared with a wild strain.
2. The method for improving the capability of serratia marcescens to synthesize the prodigiosin has good effect, does not influence the growth performance of serratia marcescens per se, and is suitable for large-scale industrial production.
Drawings
FIG. 1 shows the result of successful PCR verification of the construction of the recombinant plasmid pUCP 18-PsrB.
FIG. 2 analysis of the Synthesis capability of Serratia marcescens wild-type strain JNB5-1 and psrB gene overexpression strain JNB5-1-psrB prodigiosin.
FIG. 3 analysis of the growth capacities of Serratia marcescens wild-type strain JNB5-1 and psrB gene overexpression strain JNB5-1-psrB.
Detailed Description
The pUCP18 plasmid referred to in the examples below was purchased from the Biovector plasmid vector strain cell gene collection; serratia marcescens JNB5-1 referred to in the examples below was purchased from North Nanopsis under the product number BNCC336646.
Homologous recombination kits were purchased from south Beijing Norvigian Biotech Co., ltd.
The following examples relate to the following media:
fermentation medium: sucrose 2%, beef extract 1.5%, caCl 2 1%, L-proline 0.75% and MgSO 4 ·7H 2 O 0.03％，pH 7.0。
LB liquid medium: 10g/L NaCl, 10g/L tryptone and 5g/L yeast extract.
Example 1: construction of psrB Gene overexpression Strain JNB5-1-PsrB
According to the nucleotide sequence (shown as SEQ ID NO. 2) of a gene psrB of a serratia marcescens JNB5-1 transcription regulatory factor, carrying out PCR amplification by taking serratia marcescens JNB5-1 genome DNA as a template and taking PsrB-F and PsrB-R as primers to obtain a DNA fragment PsrB; the DNA fragment PsrB and pUCP18 plasmid linearized by EcoRI and HindIII endonuclease are subjected to homologous recombination and then transferred into Escherichia coli JM109 strain, and recombinant plasmid pUCP18-PsrB is obtained after colony PCR verification (the PCR verification result is shown in figure 1); the recombinant plasmid is transferred to Serratia marcescens JNB5-1 through electrotransformation to obtain a psrB gene overexpression strain JNB5-1-PsrB. The primer sequences used in the present invention are shown in Table 1.
TABLE 1 primer sequence listing
The PCR reaction system is as follows:
TABLE 2 mutant PCR reaction System
The PCR reaction conditions were: pre-denaturation at 98℃for 5min, denaturation at 98℃for 10s, annealing at 55℃for 5s and elongation at 72℃for 90s for 30 cycles.
Example 2: analysis of the ability of the psrB overexpressing Strain JNB5-1-PsrB to produce prodigiosin by fermentation
The wild strain JNB5-1 cultured overnight and the psrB gene overexpressing strain JNB5-1-psrB constructed in example 1 were inoculated into liquid LB medium, respectively, and cultured at 30℃and 180rpm to obtain a strain (OD) in the early logarithmic growth phase 600 Bacterial liquid of (0.6), then inoculating the bacterial liquid into 50mL of fermentation medium with an inoculum size of 4% (v/v), collecting bacterial cells at 30 ℃ and 180rpm in 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h and 108h after inoculation, standing the bacterial cells after collection for 8h by acidic ethanol with pH of 3.0, and measuring A by using a spectrophotometer 534 Wavelength at the lower wavelength, and according to the formula y= 1.1936X-0.001 (Y represents a 534 The absorbance obtained was measured below, X represents the prodigiosin yield) and the amount of prodigiosin synthesized by each strain at each time point was obtained (FIG. 2), and the effect of the overexpressed gene psrB on the prodigiosin synthesized by Serratia marcescens was analyzed.
As shown in FIG. 2, the result shows that the highest yield of prodigiosin of the strain JNB5-1-PsrB is improved by 28.33% compared with that of the strain JNB5-1 (5.33 g/L) obtained by fermenting the strain JNB5-1-PsrB by measuring the capacity of the psrB gene over-expression strain JNB5-1-PsrB, which shows that the capacity of synthesizing prodigiosin by Serratia marcescens can be remarkably improved by over-expression of the gene psrB.
Example 3: analysis of growth Capacity of wild-type Strain JNB5-1 and psrB overexpressing Strain JNB5-1-PsrB
The wild strain JNB5-1 cultured overnight and the psrB gene overexpressing strain JNB5-1-psrB constructed in example 1 were inoculated into a liquid LB medium, and cultured at 30℃and 180rpm to obtain a strain having an initial logarithmic growth phase (OD 600 Bacterial liquid of =0.6), then inoculated in an inoculum size of 4% (v/v) in 50mL fermentation medium at 30 ℃, 180rpm, and cells were collected at 0h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation, and a was measured using a spectrophotometer 600 And finally, the growth curves of the strains JNB5-1 and JNB5-1-PsrB are obtained by drawing at the lower wavelength. The results are shown in the graph3, compared with the wild-type strain JNB5-1, the growth of the psrB overexpression strain JNB5-1-psrB has no obvious change, which indicates that the overexpression gene psrB has no obvious influence on the growth of the strain.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Claims (10)

1.A method for improving the synthesis of prodigiosin by Serratia marcescens, which is characterized in that a DeoR family transcription regulatory factor PsrB is overexpressed in Serratia marcescens; the amino acid sequence of the DeoR family transcription regulatory factor PsrB is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the over-expression is a recombinant plasmid obtained by integrating the psrB gene into a vector, and then transferring the recombinant plasmid into Serratia marcescens to obtain a recombinant strain.
3. The method of claim 2, wherein the nucleotide sequence of the psrB gene is set forth in SEQ ID No. 2.
4. The method of claim 2, wherein the serratia marcescens comprises serratia marcescens wild-type strain JNB5-1.
5. A method as recited in claim 2, wherein said vector comprises pUCP18.
6. A recombinant strain for efficiently synthesizing prodigiosin is characterized in that the recombinant strain is a DeoR family transcription regulatory factor PsrB with an amino acid sequence shown as SEQ ID No.1 overexpressed in Serratia marcescens.
7. A method for producing prodigiosin, which comprises inoculating the recombinant strain of claim 6 into a fermentation medium, culturing to obtain a fermentation broth, and extracting prodigiosin from the fermentation broth.
8. The method according to claim 7, characterized in that the method comprises the following specific steps: inoculating the recombinant strain of claim 6 into a culture medium to obtain OD 600 The preparation method comprises the steps of (1) inoculating bacterial liquid with the bacterial liquid of which the number is between 0.4 and 0.8 into a fermentation medium, culturing at the temperature of between 28 and 32 ℃ and at the speed of between 160 and 200rpm to obtain fermentation liquid, collecting the fermentation liquid, and using acid ethanol to act for 6 to 10 hours to determine the amount of the synthetic prodigiosin of the bacterial strain.
9. The method of claim 7, wherein the fermentation medium comprises sucrose 1.5-2.5%, beef extract 1.0-2.0%, caCl 2 0.75-1.25%, L-proline 0.5-1.0% and MgSO 4 ·7H 2 O 0.0025～0.0035％。
10. Use of the method of any one of claims 1-5 or the strain of claim 6 for the production of prodigiosin or for the production of prodigiosin-containing products.
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Citations (4)

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CN111621458A (en) * 2020-06-30 2020-09-04 江南大学 BVG90_11450 gene-deleted serratia marcescens engineering bacterium
CN111778298A (en) * 2020-07-27 2020-10-16 中国热带农业科学院热带生物技术研究所 Application of Serratia marcescens ITBB B5-1 in prodigiosin production

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102002469A (en) * 2010-09-28 2011-04-06 嘉兴学院 Bacterial strain for producing prodigiosin and method thereof
CN110846339A (en) * 2019-10-18 2020-02-28 江南大学 Method for improving acid stress resistance of serratia marcescens
CN111621458A (en) * 2020-06-30 2020-09-04 江南大学 BVG90_11450 gene-deleted serratia marcescens engineering bacterium
CN111778298A (en) * 2020-07-27 2020-10-16 中国热带农业科学院热带生物技术研究所 Application of Serratia marcescens ITBB B5-1 in prodigiosin production

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Title
刘思航 ; 邹宜均 ; 常菲菲 ; 陈芳芳 ; 徐辉 ; 乔代蓉 ; 曹毅 ; .一株高产灵菌红素粘质沙雷氏菌的分离鉴定及发酵条件优化.应用与环境生物学报.2018,(01),第28-34页. *

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