Gene-rcsB-deficient recombinant Serratia marcescens and application thereof

A Serratia marcescens and gene technology, applied in the biological field, can solve problems such as low yield and hindering the industrialization process of microbial fermentation

Active Publication Date: 2020-09-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are still certain defects in the existing biological methods, among which the low yield is the most important defect hindering the industrialization of microbial fermentation methods

Method used

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  • Gene-rcsB-deficient recombinant Serratia marcescens and application thereof
  • Gene-rcsB-deficient recombinant Serratia marcescens and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of Serratia marcescens engineering strain JNB5-1ΔrcsB

[0032] Specific steps are as follows:

[0033] Using the genome of Serratia marcescens JNB5-1 as a template, RcsB-DUF / RcsB-DUR (SEQ ID NO: 3 and SEQ ID NO: 4) and RcsB-DDF / RcsB-DDR (SEQ ID NO: 5 And SEQ ID NO: 6) are primers, the DNA fragment RcsB-U (SEQ ID NO: 7) and DNA fragment RcsB-D (SEQ ID NO: 8) are obtained by PCR amplification; the aacC3 resistance gene (SEQ ID NO :9); DNA fragment RcsB-U, DNA fragment RcsB-D, and aacC3 resistance gene were sequentially connected by overlap extension PCR to obtain DNA fragment RcsB-AacC3; DNA fragment RcsB-AacC3 and Kpn I digested linear After homologous recombination, the recombinant plasmid pUTmini was ligated to obtain the recombinant plasmid pUTmini-rcsB; the recombinant plasmid pUTmini-rcsB was transformed into Escherichia coli S17-1 to obtain the transformation product 1; the transformation product 1 was spread on LB solid medium (Contains 50μg·m...

Embodiment 2

[0034] Example 2: Production of Prodigiosin

[0035] Specific steps are as follows:

[0036] With Serratia marcescens JNB5-1 as a control, a single colony of Serratia marcescens engineered strain JNB5-1ΔrcsB obtained in Example 1 was inoculated into LB liquid medium (containing 50μg·mL -1 Apramycin and 50μg·mL -1 Clindamycin), shaking culture at 37°C and 180 rpm to the early logarithmic growth phase (OD 600 =0.6) to obtain a seed liquid; the seed liquid was inoculated into an LB liquid medium with an inoculum amount of 4% (v / v), and fermented at 30° C. and 180 rpm for 24 hours to obtain a fermentation liquid.

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Abstract

The invention discloses gene-rcsB-deficient recombinant Serratia marcescens and application thereof and belongs to the technical field of biology. The invention provides a Serratia marcescens engineering strain JNB5-1[delta]rcsB capable of high yielding prodigiosin. The Serratia marcescens engineering strain JNB5-1[delta]rcsB is obtained through knocking out a gene encoding a transcriptional regulation factor RcsB from Serratia marcescens JNB5-1. By inoculating an LB liquid culture medium with the Serratia marcescens engineering strain JNB5-1[delta]rcsB and carrying out fermentation for 24h, the yield of the prodigiosin in fermentation broth can reach up to 116.48mg/L and is 2.28 times that of wild type Serratia marcescens JNB5-1.

Description

Technical field [0001] The invention relates to a recombinant Serratia marcescens with rcsB gene deletion and an application thereof, and belongs to the field of biotechnology. Background technique [0002] Prodigiosin (PG) is a kind of methoxypyrrole skeleton structure substance with 3 pyrrole rings. It can be classified from Serratia, Pseudomonas, and Hexa (Hahella), Vibrio (Vibrio), and marine new bacteria (Zooshikella rubidus). [0003] Prodigiosin can damage the double-stranded DNA of cells with the cooperation of copper ions, and the concentration of copper ions in cancer cells is much higher than that in ordinary cells (the copper ion concentration in cancer cells is generally 3.5 times that of ordinary cells); Red pigment exhibits the most effective DNA damaging effect under the condition of pH 6.8, while cancer cells have a pH closer to 6.8 compared with ordinary cells (the pH of ordinary cells is around 7.4, and the pH of cancer cells is around 6.8); In addition, Prodig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/16C12R1/43
CPCC07K14/24C12P17/165
Inventor 饶志明潘学玮杨套伟尤甲甲易敢峰付维来徐美娟张显邵明龙
Owner JIANGNAN UNIV
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