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KR-102963558-B1 - LACTOBACILLUS REUTERI TRANSFORMANTS AND METHODS OF PREPARATION THEROF

KR102963558B1KR 102963558 B1KR102963558 B1KR 102963558B1KR-102963558-B1

Abstract

The present invention provides a transformant of Lactobacillus reuteri. More specifically, the present invention can effectively reduce cell lysis in the production of 1,3-propanediol using a transformant of Lactobacillus reuteri in which the prophage producing a complete viral particle is deleted.

Inventors

  • 박성훈
  • 칼파나 싱

Assignees

  • 울산과학기술원

Dates

Publication Date
20260512
Application Date
20230419

Claims (7)

  1. A Lactobacillus reuteri DSM 20016 transformant having a deletion in the gene encoding prophage Φ3 consisting of the sequence of SEQ No. 1.
  2. A Lactobacillus reuteri DSM 20016 transformant of claim 1, wherein at least a portion of the gene encoding prophage Φ4 consisting of the sequence of SEQ ID NO. 2 is further deleted.
  3. A Lactobacillus reuteri DSM 20016 transformant according to claim 1, wherein a gene encoding adh (alcohol dehydrogenase) 2 or a gene encoding adh 6 is further deleted.
  4. A Lactobacillus reuteri DSM 20016 transformant according to claim 1, comprising a recombinant promoter P ldhL operably linked to a gene encoding PduQ (1,3-propanediol oxidoreductase).
  5. A composition for producing 1,3-propanediol comprising the Lactobacillus reuteri DSM 20016 transformant of claim 3, The above Lactobacillus reuteri DSM 20016 transformant comprises a recombinant promoter P ldhL operably linked to a gene encoding PduQ (1,3-propanediol oxidoreductase), a composition for producing 1,3-propanediol.
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  7. delete

Description

Lactobacillus reuteri transformants and methods of preparation therefrom The present invention relates to a transformant of Lactobacillus reuteri, which can be utilized in the field of 1,3-propanediol production. Lactobacillus reuteri is a lactic acid bacterium (LAB) found in various natural environments, including the human gastrointestinal tract. LABs are GRAS (Generally Regarded As Safe) microorganisms and are beneficial probiotics that can be used as food additives or feed additives. Lactobacillus reuteri is suitable for producing metabolites such as lactic acid, reuterin, 3-hydroxypropioic acid (3-HP), and 1,3-propanediol (1,3-PDO), but its use is currently limited due to strain instability, specifically cell lysis, during the production of these metabolites under conditions of cell growth or stress. For example, during the production of 1,3-propanediol, Lactobacillus reuteri exhibits severe cell lysis in the later stages of fermentation. Cell lysis becomes even more severe, particularly when two alcohol dehydrogenase genes (adh2 and adh6) that promote ethanol production are removed to enhance 1,3-propanediol production. This strain instability acts as a serious obstacle to the development of Lactobacillus reuteri strains and fermentation processes using them. Figure 1 illustrates the results of the genome analysis of L. reuteri from DSM 20016. Functionally complete prophages (Φ3 and Φ4: green) and incomplete prophages (Φ1, Φ2, and Φ5: orange diamonds), RM-systems, and their gene sequences are shown. Blue: DNA methyltransferases/methyltransferases; Orange: Proteins with specific domains; Red: Putative restriction enzymes; Gray: Genes with unclassified functions. Figure 2 shows the experimental results for adh removal. (A) Cell growth profile at the flask scale. (B) Virus production quantified in plaque forming units (Pfu) (Pfu/ml). Figure 3 shows the results of an experiment on the growth of L. reuteri strains (LR1) reinfected with phage lysates. (a) Glycerol-free. (b) Glycerol-free. Control group: No lysate added. 0.5% (v/v) LR2/LR3 phage lysates were added to LR1 cultures (with or without glycerol) at 0.3–0.4 OD600. Figure 4 shows the experimental results of prophage activation in LR1 strains by various byproducts of glucose-glycerol fermentation. Figure 5 shows the results of characterizing L. reuteri strains with prophages removed. (A, B) Growth profiles of LR2 (red circle), LR8 (green triangle), and LR9 (light purple square). Cell growth was investigated under conditions of the absence (A) and presence (B) of mitomycin C (0.5 µg/mL). (C) Phage production of LR2, LR8, and LR9. (D, E) Growth profiles of LR3 (green circle), LR10 (brown triangle), and LR11 (light purple square). Cell growth was investigated under conditions of the absence (D) and presence (E) of mitomycin C (0.5 µg/mL). (F) Phage production of LR2, LR8, and LR9. In (C, F), phage production was measured by the Pfu/ml of the cell supernatant grown in a medium containing mitomycin C. Figure 6 shows the results of promoter experiments for enhancing PduQ expression. (A) Crude-cell enzymatic activity of PduQ expressed under P gly , P nisin , or P ldhL promoters. pNZ8148 was used as a control vector. (B) Effects of PduQ expression on cell growth and metabolites. Measured at the end of the flask experiment (after 10 hours). Figure 7 shows the results of experiments on the effects of prophage removal and PduQ++ overexpression on 1,3-PDO production in fed-batch bioreactor experiments. (A,B) LR2, (C,D) LR9. In both strains, PduQ was overexpressed by the P ldhL promoter. Figure 8 shows the results of experiments on the effects of prophage removal and PduQ++ overexpression on 1,3-PDO production in fed-batch bioreactor experiments. (A,B) LR3. (C,D) LR11. In both strains, PduQ was overexpressed by the P ldhL promoter. Figure 9 shows the experimental results of changes in foreign gene introduction efficiency following the removal of the prophage and restriction-modification (RM) system. (A) Comparison of efficiency in various LR strains using high copy number (pNZ8148) and medium copy number (pSIP411) plasmids. (B) Comparison of cell viability following electric shock. The present invention will be described in detail below. The present invention relates to a Lactobacillus reuteri transformant having a deletion of the gene encoding prophage Φ3 consisting of the sequence of SEQ ID NO. 1. The above transformant may have at least a portion of the gene encoding prophage Φ4, which consists of the sequence of SEQ ID NO. 2, additionally deleted. That is, all or part of the prophage Φ4 may be deleted, for example, 50%, 60%, or 70% or more of the continuous or discontinuous sequence of SEQ ID NO. 2 may be deleted, and the upper limit thereof may be indicated as 60%, 70%, 80%, 90%, or 100% or less. The above-mentioned Lactobacillus reuteri is a microorganism used to produce 1,3-propanediol from glycerol. For example, by culturing the microorganism in the prese