Self-splitting engineering modified bacterium

Abstract

The present invention relates to a self-cleaving engineered bacterium comprising a metabolic pathway for producing polyhydroxyalkanoate (PHA); the perforin gene and the endolysin gene are expressed under the control of a promoter. According to the method, on the basis of not influencing the dry cell weight and PHA yield of the engineering strain, the self-splitting of the engineering strain after PHA accumulation is completed is realized, the downstream extraction step of PHA biosynthesis is greatly simplified, and the extraction time is shortened.

Claims

1. A self-lysing engineered bacterium comprising: 1) Metabolic pathways for the production of polyhydroxyalkanoates (PHAs); and 2) Perforin gene and endolysin gene expressed under the control of promoter.

2. The self-lysing engineered bacteria according to claim 1, wherein: 1) The self-lysing engineered bacteria contain endogenous and / or exogenous PHA synthesis genes; 2) The perforin gene and endolysin gene are derived from the perforin-endolysin system of a bacteriophage, preferably the bacteriophage is an Escherichia coli T-series phage, more preferably T1 to T7 phage, most preferably T4 phage; and / or 3) The perforin gene and endolysin gene are independently linked to the same or different promoters.

3. The self-lysing engineered bacteria according to claim 1 or 2, wherein: 1) The promoter is a promoter of a PHA granule-binding protein derived from a natural PHA-producing bacterium, preferably a PhaP promoter, more preferably a PhaP1 promoter derived from Ralstonia eutropha, Halomonas bluephagenesis, or Halomonascampaniensis, or a PhaP2 promoter derived from Halomonas bluephagenesis or Halomonascampaniensis, even more preferably a PhaP1 promoter derived from Halomonas, and most preferably the nucleotide sequence of the promoter is shown in SEQ ID NO: 1; and / or 2) The self-lysing engineered bacteria also express endogenous or exogenous PhaR protein.

4. The self-lysing engineered bacterium according to any one of claims 1 to 3, wherein the amino acid sequence encoded by the perforin gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the endolysin gene is shown in SEQ ID NO: 3, preferably the nucleotide sequence of the perforin gene is shown in SEQ ID NO: 4, and the nucleotide sequence of the endolysin gene is shown in SEQ ID NO:

5.

5. The self-lysing engineered bacterium according to any one of claims 1 to 4, wherein the upstream of the perforin gene and the endolysin gene further independently include a ribosome binding site, preferably the ribosome binding site is selected from the group consisting of RBS10, RBS15, RBS25 and RBS30, wherein the nucleotide sequence of RBS10 is shown in SEQ ID NO: 6, the nucleotide sequence of RBS15 is shown in SEQ ID NO: 7, the nucleotide sequence of RBS25 is shown in SEQ ID NO: 8, and the nucleotide sequence of RBS30 is shown in SEQ ID NO:

9.

6. The self-lyzing engineered bacterium according to any one of claims 1 to 5, wherein the starting strain of the self-lyzing engineered bacterium is selected from the group consisting of Halomonas, Pseudomonas, Escherishia (e.g., E. coli EphaCAB), Corynebacterium glutamicum, Ralstonia eutropha, Aeromonas, Bacilllus, Alcaligenes latus, and Alcaligenes eutropus, more preferably the microorganism is Halomonas, and even more preferably the Halomonas is Halomonas bluephagenesis, Halomonasaydingkolgenesis, Halomonas campaniensis, Halomonas lutescens, Halomonashydrothermalis, Halomonas sp.KM1, Halomonas elongata, and Halomonas. smyrnensis, still more preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 (Culture Collection Number CGMCC No. 4353), Halomonas aydingkolgenesis M1 (Culture Collection Number CGMCC No. 19880), Halomonas campaniensis LS21 (Culture Collection Number CGMCC No. 6593), Halomonasbluephagenesis TD27, Halomonas bluephagenesis TDB141, Halomonas bluephagenesis TDB141ΔAC, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis WZY278 or Halomonas bluephagenesis CYL0307.

7. The self-lysing engineered bacterium according to any one of claims 1 to 6, wherein: 1) The expression is on a plasmid or genome; 2) The endogenous plasmid has been knocked out in the self-lysing engineered bacteria; and / or 3) The self-lying engineered bacteria is a recombinant Halomonas, and / or the plasmid used in the expression is a pSEVA plasmid (preferably pSEVA341, pSEVA321 or pSEVA241), and / or the plasmid used in the expression is a toxin-antitoxin system plasmid, more preferably the toxin-antitoxin system plasmid is pHbPBC.

8. The self-lysing engineered bacteria according to any one of claims 1 to 7, wherein the PHA comprises at least one of PHB, P4HB, P3HB4HB3HV, PHV, PHBHHX, P34HB, P3HO3HH, PHO, PHP, and PHBVHHX; preferably one or more of PHB, P34HB, PHBHHX, and PHBV.

9. A method for producing polyhydroxyalkanoate (PHA), comprising fermenting and culturing the self-lysing engineered bacteria according to any one of claims 1 to 8.

10. The method according to claim 9, wherein the method does not include a step of lysing cells by treatment with lysozyme.

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