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CN-122012565-A - Apramycin genetically engineered bacterium with genetic stability, construction method and application thereof

CN122012565ACN 122012565 ACN122012565 ACN 122012565ACN-122012565-A

Abstract

The invention belongs to the technical field of genetically engineered bacteria, and particularly discloses an apramycin genetically engineered bacteria with genetic stability, and a construction method and application thereof. The construction method comprises the steps of taking plasmids containing xylE genes and tsnR genes as templates to obtain a linearization vector skeleton without promoters, inserting a promoter sequence of the Alternaria alternata into the linearization vector skeleton to enable the promoters to be located at the upstream of the xylE genes and tsnR genes, constructing a dual-reporter gene expression vector, transferring the expression vector into streptomyces through combination to obtain a binder, and carrying out color development screening through ultraviolet mutagenesis and catechol combination to obtain a genetically stable strain. The biological titer is improved by more than 40% compared with the original strain, the titer retention rate of more than 94.5% is maintained in continuous long-term passage experiments of 10 generations, and a powerful technical support is provided for the industrialized stable production of the mycoplasma-resistant active substance.

Inventors

  • ZHANG HUITU
  • WANG HAIKUAN
  • WEI LULU

Assignees

  • 天津科技大学

Dates

Publication Date
20260512
Application Date
20260228

Claims (10)

  1. 1. The construction method of the apramycin genetic engineering bacteria with genetic stability is characterized by comprising the following steps: Obtaining the sequence of the promoter of the Alternaria alternata, wherein the sequence is shown as SEQ ID NO. 1; Constructing a dual-reporter gene expression vector comprising a first reporter gene and a second reporter gene, wherein the first reporter gene is positioned downstream of the streptoverticillium indicum promoter, the second reporter gene is positioned downstream of the first reporter gene, the streptoverticillium indicum promoter drives transcription of the first reporter gene and the second reporter gene, the first reporter gene is catechol-2, 3-dioxygenase gene, and the second reporter gene is a thiostrepton resistance gene; Introducing the dual reporter gene expression vector into a host bacterium; screening to obtain a recombinant strain comprising the dual reporter gene expression vector, co-culturing the recombinant strain with a streptoverticillium indicum, such that the dual reporter gene expression vector is transferred into the streptoverticillium indicum by binding, thereby obtaining a binder; Carrying out ultraviolet mutagenesis treatment on the binder to obtain a mutagenized strain, culturing and passaging the mutagenized strain, spraying catechol before passaging, and screening according to the catechol chromogenic reaction to obtain the apramycin genetic engineering bacteria with genetic stability.
  2. 2. The construction method according to claim 1, wherein the construction process of the dual reporter gene expression vector is specifically as follows: Constructing a linearization vector skeleton containing a catechol-2, 3-dioxygenase gene and a thiostrepton resistance gene without a promoter by taking a plasmid PSET152 as a template; connecting the sequence of the Alternaria alternata promoter with the linearization vector skeleton to obtain a connecting product; and (3) transforming the connection product into competent cells of the escherichia coli for culture, picking up monoclonal, and obtaining the double-reporter gene expression vector through verification.
  3. 3. The construction method according to claim 2, wherein the sequence of the promoter of the streptoverticillium indicum and the linearized vector backbone are mixed in a molar ratio of 2:1-5:1 for ligation.
  4. 4. The method of construction according to claim 2, wherein the ligation reaction comprises ligation using homologous recombination, seamless cloning or DNA ligase mediated ligation.
  5. 5. The method of claim 1, wherein the host bacteria are selected from bacteria and yeasts.
  6. 6. The construction method according to claim 1, wherein the ultraviolet mutagenesis condition is that mutagenesis treatment is performed under a 15W ultraviolet lamp, and the irradiation time is 20-40 s.
  7. 7. A dual reporter gene expression vector comprising: A promoter sequence shown in SEQ ID NO. 1; the first reporter gene is positioned at the downstream of the promoter, and the sequence of the first reporter gene is shown as SEQ ID NO. 2; A second reporter gene positioned downstream of the first reporter gene, wherein the sequence of the second reporter gene is shown as SEQ ID NO. 3; Wherein the promoter drives expression of the first reporter gene and the second reporter gene simultaneously.
  8. 8. Use of the method according to any one of claims 1 to 6 or the dual reporter gene expression vector according to claim 7 for the evaluation and screening of mycoplasma-resistant active substance producing bacteria.
  9. 9. An engineered bacterium capable of producing an anti-mycoplasma active substance at a high yield, comprising the dual-reporter gene expression vector of claim 7 or obtained by the construction method of any one of claims 1 to 6.
  10. 10. The use of an engineered bacterium of claim 9 in the preparation of an anti-mycoplasma active substance, wherein said anti-mycoplasma active substance comprises apramycin, telithromycin.

Description

Apramycin genetically engineered bacterium with genetic stability, construction method and application thereof Technical Field The invention relates to the technical field of genetically engineered bacteria, in particular to an apramycin genetically engineered bacteria with genetic stability, and a construction method and application thereof. Background Streptoverticillium indicum (Streptoalloteichus hindustanus) is an important pharmaceutical actinomycete capable of producing a variety of secondary metabolites with significant biological activity, mainly including the darkmycin complex (Nebramycin complex, containing apramycin, tobramycin, etc.), and telithromycin (Tallysomycin), etc. The active substances belong to aminoglycoside antibiotics or glycopeptide antibiotics, have remarkable killing effect on common Mycoplasma (Mycoplasma) and gram-negative bacteria of livestock and poultry by virtue of a unique action mechanism (such as inhibiting synthesis of bacterial ribosomal proteins or damaging DNA), and have irreplaceable application value in the fields of veterinary drug development and animal infectious disease prevention and treatment. However, in the industrial production of antibiotics, fermentation units and production costs are largely dependent on the genetic stability of the producing strain. Due to the huge cluster of secondary metabolite synthesis genes and complex regulation mechanism of the strain, the high-activity industrial strain is extremely easy to generate spontaneous mutation or metabolic flow redistribution when undergoing production links such as continuous passage, inclined plane preservation or seed expansion culture, so that the total antibacterial biological potency (Bio-potency) is obviously declined, namely the so-called 'strain degeneration', which severely limits the large-scale stable production of the mycoplasma-resistant active substance. The existing strain breeding and degradation monitoring technology has significant limitations in practical application. On the one hand, the traditional degradation detection mainly depends on shake flask fermentation and biological titer measurement (such as a tube dish method), the process generally takes 7 to 10 days, serious detection hysteresis exists, and degradation strains cannot be identified and removed in time in the seed preparation or flat growth stage, so that waste of production resources and severe fluctuation of fermentation units are often caused. On the other hand, the traditional mutation breeding technology lacks clear high-yield character phenotype indication, and the screening process is large in blindness and low in efficiency. Molecular mechanism studies have shown that in the cluster of the biosynthesis genes of Alternaria alternata, the key genes encoding the core backbone synthesis and the key modification enzyme gene (aprD 3) are located on specific transcriptional units (operons) on the genome. The transcriptional initiation activity of this particular operon has a decisive regulatory role, reflecting directly the overall level of metabolic flux in the biosynthetic pathway. Therefore, a real-time monitoring system capable of specifically responding to the transcription activity of the core operon is developed, the recessive metabolic flux is converted into a dominant visual phenotype, and the real-time monitoring system has important technical value for realizing high-fidelity screening of mycoplasma-resistant active substance high-yield strains and maintaining the genetic stability of the strains in real time in the industrial passage process. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a construction method of an apramycin genetic engineering bacterium with genetic stability, which converts invisible metabolic flux into a visualized colony phenotype by constructing a chromogenic and resistance double-reporting system driven by a biosynthetic gene cluster core promoter (PaprD), and further obtains the genetically stable strain through mutagenesis, thereby realizing real-time identification and optimization of the strain with high biological titer. In order to achieve the above purpose, the present invention adopts the following technical scheme: the first aspect of the invention provides a construction method of an apramycin genetic engineering bacterium with genetic stability, which comprises the following steps: Obtaining the sequence of the promoter of the Alternaria alternata, wherein the sequence is shown as SEQ ID NO. 1; Constructing a dual-reporter gene expression vector comprising a first reporter gene and a second reporter gene, wherein the first reporter gene is positioned downstream of the streptoverticillium indicum promoter, the second reporter gene is positioned downstream of the first reporter gene, the streptoverticillium indicum promoter drives transcription of the first reporter gene and the second reporter gene, the first reporter gene is cate