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Method for improving serratia marcescens to synthesize prodigiosin through overexpression gene psrB

A technology of Serratia marcescens and prodigiosin, applied in the field of genetic engineering and microbial engineering, can solve the problems of low yield and hindering the industrialization process of microbial fermentation, and achieve good results and good capabilities

Pending Publication Date: 2021-10-26
JIANGNAN UNIV
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Problems solved by technology

[0003] However, there are still certain defects in the existing biological methods, among which the low yield is the most important defect hindering the industrialization process of microbial fermentation methods
For example, Lee et al. produced prodigiosin by inoculating Zooshikella ganghwensis KCTC 12044T into Marinebroth 2216 medium for fermentation. However, using this method to ferment for 24 hours, the yield of prodigiosin in the fermentation broth could only reach 15.40 mg / L (see references for details: Lee, J.S., Kim, Y.S., Park, S., Kim, J., Kang, S.J., Lee, M.H., et al. (2011). Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novelmarine bacterium, Zooshikella rubidus S1-1.Appl.Environ.Microbiol.77,4967-4973.); Lee et al. produced prodigiosin by inoculating Hahella chejuensis KCTC 2396T into Marinebroth 2216 medium for fermentation, but , using this method to ferment for 24h, only the output of prodigiosin in the fermented liquid can reach 28.10mg / L (see references for details: Lee, J.S., Kim, Y.S., Park, S., Kim, J., Kang, S.J.,Lee,M.H.,et al.(2011).Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium,Zooshikella rubidus S1-1.Appl.Environ.Microbiol.77,4967-4973.1)

Method used

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  • Method for improving serratia marcescens to synthesize prodigiosin through overexpression gene psrB
  • Method for improving serratia marcescens to synthesize prodigiosin through overexpression gene psrB
  • Method for improving serratia marcescens to synthesize prodigiosin through overexpression gene psrB

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Experimental program
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Embodiment 1

[0034] Embodiment 1: Construction of psrB gene overexpression strain JNB5-1-PsrB

[0035] According to the nucleotide sequence of Serratia marcescens JNB5-1 transcription regulator PsrB coding gene psrB (as shown in SEQIDNO.2), with Serratia marcescens JNB5-1 genomic DNA as template, with PsrB-F and PsrB-R as primers, PCR amplification was carried out, and the DNA fragment PsrB was amplified; the DNA fragment PsrB was subjected to homologous recombination with the pUCP18 plasmid linearized with EcoRI and HindIII endonuclease, and then transformed into the Escherichia coli JM109 strain, After colony PCR verification, the recombinant plasmid pUCP18-PsrB was obtained (see the PCR verification results in figure 1 ); the recombinant plasmid was transferred to Serratia marcescens JNB5-1 by electroporation to obtain psrB gene overexpression strain JNB5-1-PsrB. The primer sequences used in the present invention are shown in Table 1.

[0036] Table 1 Primer sequence list

[0037] ...

Embodiment 2

[0042] Example 2: Analysis of the ability of psrB overexpression strain JNB5-1-PsrB to produce prodigiosin by fermentation

[0043]The wild-type strain JNB5-1 cultivated overnight and the psrB gene overexpression strain JNB5-1-PsrB constructed in Example 1 were respectively inoculated in liquid LB medium, cultivated at 30°C and 180rpm to obtain the initial logarithmic growth phase (OD 600 =0.6), then inoculated in 50mL fermentation medium with 4% (v / v) inoculum size, 30°C, 180rpm, 0h, 12h, 24h, 36h, 48h, 60h, 72h, The bacteria were collected at 84h, 96h and 108h, and after the collected bacteria were left to stand for 8 hours in acidic ethanol with pH 3.0, the A 534 under the wavelength, and according to the formula Y=1.1936X-0.001 (Y means A 534 The absorbance value measured below, X represents the prodigiosin output) to obtain the amount of prodigiosin synthesized by each bacterial strain at each time point ( figure 2 ), to analyze the effect of the overexpressed gene psr...

Embodiment 3

[0045] Example 3: Analysis of growth ability of wild-type strain JNB5-1 and psrB overexpression strain JNB5-1-PsrB

[0046] The wild-type strain JNB5-1 cultivated overnight and the psrB gene overexpression strain JNB5-1-PsrB constructed in Example 1 were inoculated in liquid LB medium, cultivated at 30°C and 180rpm to obtain the initial logarithmic growth phase (OD 600 =0.6), then inoculated in 50mL fermentation medium with 4% (v / v) inoculum size, collected at 0h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation at 30°C and 180rpm Bacteria, using a spectrophotometer to measure A 600 Finally, the growth curves of strains JNB5-1 and JNB5-1-PsrB were drawn. The result is as image 3 As shown, compared with the wild-type strain JNB5-1, the growth of the psrB overexpression strain JNB5-1-PsrB did not change significantly, indicating that the overexpression gene psrB had no significant effect on the growth of the strain.

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Abstract

The invention discloses a method for improving serratia marcescens to synthesize prodigiosin through an overexpressed gene psrB, and belongs to the technical field of gene engineering and microbial engineering. According to the invention, the prodigiosin synthesis capability of the serratia marcescens is remarkably improved by overexpressing an encoding gene BVG90_04085 (psrB) of a DeoR family transcriptional regulation factor PsrB (BVG90_04085) in the serratia marcescens; when the recombinant serratia marcescens prepared by the method disclosed by the invention is fermented for 72 hours in a fermentation culture medium to produce prodigiosin; the prodigiosin production 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 reaches 6.84 g / L.

Description

technical field [0001] The invention relates to a method for improving the prodigiosin synthesis of Serratia marcescens by overexpressing gene psrB, and belongs to the technical field of genetic engineering and microbial engineering. Background technique [0002] Prodigiosin (PG), a class of secondary metabolites produced by microorganisms, has various biological activities such as anti-bacteria, anti-dysentery, anti-tumor and immunosuppression, and is used in the fields of pharmaceutical development, environmental governance and dye preparation. It has great application value. Currently, the production methods of prodigiosin mainly include chemical synthesis and microbial fermentation. Due to the shortcomings of many reaction steps and low yield in the synthesis of prodigiosin by chemical synthesis, it is difficult to realize the large-scale production of prodigiosin. The production of prodigiosin by microbial fermentation has the advantages of environmental friendliness,...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/31C12N1/21C12P17/16C12R1/43
CPCC12N15/74C07K14/24C12P17/165Y02E50/10
Inventor 饶志明潘学玮杨套伟尤甲甲徐美娟张显邵明龙
Owner JIANGNAN UNIV
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