CN-121450558-B - Attenuated synergistic host chassis capable of being widely used for pseudomonas aeruginosa phage production and construction method and application thereof

CN121450558BCN 121450558 BCN121450558 BCN 121450558BCN-121450558-B

Abstract

The invention discloses an attenuated synergistic host chassis capable of being widely used for pseudomonas aeruginosa phage production, and a construction method and application thereof, and belongs to the technical field of microorganisms. 68 phages separated from the pseudomonas aeruginosa model strain PAO1 can cover 97 percent (238/245) of 245 pseudomonas aeruginosa separated from a plurality of hospitals in comprehensive host range, and the aim of amplifying the clinical pseudomonas aeruginosa phages by a single host bacterium is fulfilled. By knocking out phage resistance system, prophage, virulence factor effector protein and antibiotic resistance related genes in PAO1, the yield of PAO1 phage is improved, and most of pollution of secreted prophage, virulence factor and antibiotic inactivating enzyme to phage is removed from the source. The related genes synthesized by key virulence factors such as O antigen, pili and core oligosaccharide are further knocked out, so that the pollution of the O antigen, pili and core oligosaccharide is reduced, lipopolysaccharide is greatly shortened, and the purification problem is solved.

Inventors

MA YINGFEI
GE HAOJIE
TAN XIN
GAO RUYUE

Assignees

深圳先进技术研究院

Dates

Publication Date: 20260508
Application Date: 20251230

Claims (3)

1. An attenuated synergistic host chassis widely used for producing pseudomonas aeruginosa phage is characterized in that the host chassis is obtained by sequentially knocking out phage resistance system genes, prophage genes, virulence factor effector protein genes and antibiotic inactivating enzyme genes from pseudomonas aeruginosa model strain PAO 1; The phage resistance system genes include genes encoding MTases, S subunits, REases, PA1371, PA1372, gajB and GajA; the prophage genes are genes for encoding PF phage proteins and key genes for encoding YMC phage proteins; the virulence factor effector protein genes comprise genes for transcription regulating factors Vfr, tesG, protease-IV, a lysophospholipase C precursor and a small protease PasP; the antibiotic inactivating enzyme genes comprise genes for encoding glutathione transferase FosA, vanW family proteins, acetylpolyamine amino hydrolase AphA, chloramphenicol acetyl transferase Cat and beta-lactam hydrolase OXA-50; The preservation number of the host chassis is CGMCC No.36006.
2. The attenuated synergistic host chassis for the production of pseudomonas aeruginosa phage of claim 1, wherein said host chassis further knocks out at least one of an O antigen assembly gene, a pilus synthesis gene, a core oligosaccharide assembly gene; When the O antigen ligase gene waaL is knocked out, the preservation number of the host chassis is CGMCC No.36007; when pilA synthetic gene pilA is knocked out, the preservation number of the host chassis is CGMCC No.36005; When pilus synthesis genes pilA and O antigen ligase genes waaL are knocked out simultaneously, the preservation number of the host chassis is CGMCC No.36003; When pilus synthesis genes pilA and O antigen ligase gene waaL and a core oligosaccharide assembly gene pa5001 are knocked out simultaneously, the preservation number of the host chassis is CGMCC No.36004.
3. Use of an attenuated synergistic host chassis of any of claims 1 to 2 for the production of phage of pseudomonas aeruginosa.

Description

Attenuated synergistic host chassis capable of being widely used for pseudomonas aeruginosa phage production and construction method and application thereof Technical Field The invention relates to the technical field of microorganisms, in particular to an attenuated synergistic host chassis widely applicable to pseudomonas aeruginosa phage production, and a construction method and application thereof. Background Pseudomonas aeruginosa has multiple drug resistance and opportunistic pathogenicity and cannot be used for phage production in common fermentation plants. The conventional common industrial chassis generally knocks out individual virulence genes, and constructs attenuated strains to realize application in common environments. For example, non-toxin-producing and non-pathogenic salmonella enteritidis C1106 constructed by celiac patent no An Baite is used for the industrial production of salmonella phages. However, in the phage production process, pseudomonas aeruginosa also releases a large amount of virulence factors, genetic materials, prophages, antibiotic inactivating enzymes and other polluted phages, and the direct use has great potential safety hazard. Therefore, phage generally needs to be purified by methods such as filtration, ultrafiltration, cesium chloride gradient centrifugation, dialysis and the like before clinical use, but the above purification method cannot remove prophages, and the lipopolysaccharide content in phage with lipopolysaccharide as a receptor is difficult to be reduced below a standard level. In addition, phages have host specificity, and no bacterial chassis is currently available for the production of all clinical Pseudomonas aeruginosa phages. Accordingly, the prior art is still in need of improvement and development. Disclosure of Invention In view of the shortcomings of the prior art, the invention aims to provide an attenuated synergistic host chassis which can be widely used for producing pseudomonas aeruginosa phage, and a construction method and application thereof, and aims to solve the problems that the existing phage has host specificity, the existing phage is mainly produced through the host bacteria, no bacteria can be widely used for safe production of pseudomonas aeruginosa phage, clinical pathogenic bacteria pseudomonas aeruginosa generally has various virulence factors and cannot be produced in a conventional fermentation workshop, and in addition, virulence factors released by the host bacteria, prophage and antibiotic inactivating enzyme can pollute the phage and are difficult to remove, and the clinical treatment effect is seriously affected. The 68 phage synthetic host range isolated with the Pseudomonas aeruginosa model strain PAO1 was found to cover about 97% (238/245) of a representative clinical isolate of Pseudomonas aeruginosa, demonstrating that PAO1 can be used as a universal production chassis for clinical Pseudomonas aeruginosa phages. Furthermore, the invention aims at improving the yield of pseudomonas aeruginosa phage, reducing chassis virulence and reducing impurity pollution in phage production process, and sequentially knocks out phage resistance system genes, prophage key genes, virulence factor effector protein genes, antibiotic inactivating enzyme genes and the like in PAO 1. Furthermore, the invention aims to reduce O antigen, pilus and core oligosaccharide pollution in the produced phage, and knocks out O antigen assembling genes, pilus synthesizing genes, core oligosaccharide assembling genes and the like of lipopolysaccharide. Specifically, the technical scheme of the invention is as follows: In a first aspect of the invention, an attenuated synergistic host chassis widely applicable to pseudomonas aeruginosa phage production is provided, wherein the host chassis is obtained by sequentially knocking out phage resistance system genes, prophage genes, virulence factor effector protein genes and antibiotic inactivating enzyme genes from pseudomonas aeruginosa model strain PAO 1; The phage resistance system genes include genes encoding MTases, S subunits, REases, PA1371, PA1372, gajB and GajA; the prophage genes are genes for encoding PF phage proteins and key genes for encoding YMC phage proteins; the virulence factor effector protein genes comprise genes for transcription regulating factors Vfr, tesG, protease-IV, a lysophospholipase C precursor and a small protease PasP; the antibiotic inactivating enzyme genes comprise genes for encoding glutathione transferase FosA, vanW family proteins, acetylpolyamine amino hydrolase AphA, chloramphenicol acetyl transferase Cat and beta-lactam hydrolase OXA-50; The preservation number of the host chassis is CGMCC No.36006. Optionally, the host chassis further knocks out at least one of an O antigen assembly gene, a pilus synthesis gene, a core oligosaccharide assembly gene. In a second aspect, the invention provides a method for constructing an attenuated synergistic host chassis widely