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CN-122012499-A - Promoter library suitable for bifidobacterium breve and application

CN122012499ACN 122012499 ACN122012499 ACN 122012499ACN-122012499-A

Abstract

The invention discloses a promoter library suitable for bifidobacterium breve and application thereof, belonging to the technical fields of genetic engineering and microbial engineering. The invention screens and obtains the promoter P C9 with bi-directional transcription characteristic and the inducible promoter responding to linoleic acid. Among them, promoter P C3‑LA can respond to LA to increase activity significantly, and P B5 has strongest activity. The invention also constructs a promoter library with gradient strength, can realize controllable expression of target proteins in microorganisms of bifidobacterium, provides elements for promoter engineering of bifidobacterium breve, and has wide application value.

Inventors

  • CHEN HAIQIN
  • ZHANG CHANGYI
  • CHANG LULU
  • YANG BO
  • ZHAO JIANXIN
  • CHEN WEI

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260129

Claims (10)

  1. 1. A promoter, characterized by being (a) or (b): (a) The nucleotide sequence of the bidirectional promoter P C9 is shown as SEQ ID NO. 9; (b) Promoter P C6 capable of raising gene expression strength has nucleotide sequence shown in SEQ ID No. 6.
  2. 2. The promoter library is characterized by comprising a promoter P C6 、P C9 、P C1 、P C11 with the nucleotide sequences shown as SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.1 and SEQ ID NO.11 from high to low in intensity, and a promoter P C2-LA 、P C4-LA 、P C3-LA with the nucleotide sequences shown as SEQ ID NO.30, SEQ ID NO.32 and SEQ ID NO.31 from high to low in expression intensity in response to linoleic acid.
  3. 3. The promoter library is characterized by comprising promoters P B5 、P B6 、P B2 、P B7 、P B12 with high intensity from high to low, and the nucleotide sequences of the promoters P B5 、P B6 、P B2 、P B7 、P B12 are respectively shown as SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.18, SEQ ID NO.23 and SEQ ID NO. 28.
  4. 4. A recombinant plasmid carrying the promoter of claim 1 or the promoter in the promoter library of any one of claims 2 to 3.
  5. 5. The recombinant plasmid according to claim 4, wherein the promoter is linked downstream only to the gene of interest.
  6. 6. The recombinant plasmid according to claim 4, wherein the promoter P C9 is simultaneously linked to the target gene at the upstream and downstream.
  7. 7. The bifidobacterium genetically engineered bacterium is characterized in that pNZ123 is used as a vector, and a promoter with a nucleotide sequence shown as any one of SEQ ID NO. 1-36 is used for regulating and controlling the expression of target proteins.
  8. 8. The bifidobacterium genetically engineered bacteria of claim 7, wherein the bifidobacterium includes, but is not limited to, bifidobacterium breve, bifidobacterium longum, bifidobacterium bifidum, or bifidobacterium animalis.
  9. 9. The promoter of claim 1 or the promoter in the promoter library of any one of claims 2 to 3 for regulating gene expression.
  10. 10. The use according to claim 9, wherein the use is for sustained stable expression of a regulated gene with a constitutive promoter or for increased expression at a specific time point in response to a linoleic acid regulated gene with an inducible promoter.

Description

Promoter library suitable for bifidobacterium breve and application Technical Field The invention relates to a promoter library suitable for bifidobacterium breve and application thereof, belonging to the technical fields of genetic engineering and microbial engineering. Background Bifidobacterium breve is an intestinal probiotic that positively affects the human body through a variety of mechanisms including, but not limited to, enhancing intestinal barrier function, inhibiting the growth of harmful pathogens, regulating the immune system, and promoting nutrient absorption and metabolic processes. Probiotic strains belonging to the genus bifidobacterium, including bifidobacterium breve, have been used in the food industry as prophylactic and therapeutic agents. With the mechanical analysis of bifidobacterium breve probiotics focused on metabolic pathways, some genes of key enzymes were mined, for example, the genes of key enzymes bbi for CLA conversion were identified in bifidobacterium breve CCFM683 with high CLA yield, and the genes of key aromatic lactate dehydrogenase Aldh were identified in bifidobacterium breve CCFM1025 with depression relief in mice. The development and validation of key enzyme genes for the probiotic mechanism of bifidobacterium breve is a current research hotspot, and thus more genetic manipulation tools are also needed to promote gene expression within bifidobacterium breve. The promoter is a sequence capable of specifically combining with RNA polymerase, and assists gene transcription in a host body and further plays a role after translation, so that the promoter is widely applied to the fields of gene editing, metabolic engineering, synthetic biology and the like. The promoters are divided into constitutive promoters and inducible promoters, the constitutive promoters can stably start transcription without being influenced by environment, the inducible promoters need to be assisted by inducers to regulate transcription, and the constitutive promoters and the inducible promoters have important functions because gene operations need to be regulated and expressed besides stable expression genes. However, the endogenous promoter resources of bifidobacterium breve are very limited, and most of currently available promoters are derived from other lactic acid bacteria, or require specific inducers to initiate gene transcription, or are poorly applicable to most bifidobacterium breve. Therefore, development and establishment of a promoter suitable for bifidobacterium breve and a promoter library are necessary for achieving controllable expression of a target gene in bifidobacterium genetically engineered bacteria. Disclosure of Invention [ Technical problem ] The invention aims to solve the technical problem of screening widely-available constitutive promoters and inducible promoters regulated by linoleic acid, so as to construct a promoter library suitable for bifidobacterium breve and provide elements for promoter engineering of bifidobacterium breve. Technical scheme The invention provides a bidirectional promoter P C9, the nucleotide sequence of which is shown as SEQ ID NO. 9. The invention also provides a promoter P C6 capable of improving the expression intensity of the target protein, and the nucleotide sequence of the promoter P C6 is shown as SEQ ID NO. 6. The present invention provides a library of promoters comprising 4 constitutive promoters and 3 inducible promoters. The constitutive promoter has the nucleotide sequence shown as SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.1 and SEQ ID NO.11 from high to low in strength, and the inducible promoter can respond to the induction of linoleic acid, has the nucleotide sequence shown as SEQ ID NO.30, SEQ ID NO.32 and SEQ ID NO.31 from high to low in strength in sequence of P C2-LA、PC4-LA、PC3-LA. The invention also provides a recombinant plasmid carrying the promoter. In one embodiment, the plasmid includes, but is not limited to, pNZ123. In one embodiment, the promoter is linked downstream only to the gene of interest. In one embodiment, the promoter is linked simultaneously upstream and downstream of the gene of interest. In one embodiment, the gene of interest includes, but is not limited to, an endogenous gene or an exogenous gene. The invention also provides a bifidobacterium genetic engineering bacterium, which takes pNZ123 as a vector and takes a promoter with a nucleotide sequence shown as any one of SEQ ID NO.1-36 to regulate and control the expression of target proteins. In one embodiment, the bifidobacteria include, but are not limited to, bifidobacterium breve, bifidobacterium longum, bifidobacterium bifidum, or bifidobacterium animalis. In one embodiment, the bifidobacterium is selected from the group consisting of bifidobacterium breve (Bifidobacterium breve) CCFM683, bifidobacterium breve FJSWX M7, bifidobacterium breve CCFM1310, bifidobacterium breve CCFM1400, bifidobacterium longum subspecies infantis (Bifidobacterium long