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Lactobacillus plantarum engineering bacterium capable of producing conjugated linoleic acid at high yield

A technology of Lactobacillus plantarum and conjugated linoleic acid, which is applied in the fields of genetic engineering and microbial engineering, can solve the problems of low production efficiency, difficult cultivation, and safety issues, and achieve the effect of high safety, easy cultivation and more cultivation

Active Publication Date: 2021-04-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] First, most microorganisms that can produce high-yielding conjugated linoleic acid are pathogenic bacteria, which have great safety problems and cannot be directly used as conjugated linoleic acid production strains for industrial production. For example, Vibrio fibrinolyticus, Propionibacterium and Clostridium sporogenes, etc.;
[0007] Second, most of the microorganisms that can produce high-yielding conjugated linoleic acid are strictly anaerobic bacteria, which are difficult to cultivate in industry or laboratories and have low yields, so it is difficult to be widely used in food and medicine, for example, Vibrio fibrinolyticus and bifidobacteria, etc.;
[0008] Third, the yield of some microorganisms that can produce conjugated linoleic acid is not high. If it is used as a conjugated linoleic acid production strain for industrial production, the production efficiency is too low, for example, Lactobacillus plantarum ZS2058 (see references for details: Qi Hui, Yang Bo et al. Research on the mechanism of biotransformation of conjugated linoleic acid by Lactobacillus plantarum ZS2058 [D], Jiangnan University, 2017),
[0009] The above defects make the existing microbial synthesis method unable to realize the large-scale industrial production of CLA. Therefore, it is urgent to find a CLA production strain with high safety, non-strict anaerobic and high yield to overcome above defects

Method used

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  • Lactobacillus plantarum engineering bacterium capable of producing conjugated linoleic acid at high yield
  • Lactobacillus plantarum engineering bacterium capable of producing conjugated linoleic acid at high yield
  • Lactobacillus plantarum engineering bacterium capable of producing conjugated linoleic acid at high yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Screening of genes encoding linoleic acid isomerase

[0047] Specific steps are as follows:

[0048] The transcriptomics data of Bifidobacterium breve (Bifidobacterium breve) CGMCCNo.11828 (recorded in the patent application text with publication number CN105925514A) under linoleic acid stress was collected through the PacBio sequencing platform, and the sampling time points were 3h, 8h, respectively. 15h. After bioinformatics analysis, it was found that in Bifidobacterium breve (Bifidobacterium breve) CGMCCNo.11828, there were 8 genes with increased gene transcription levels at the three time points, and these 8 genes were respectively annotated as coding " Unknown protein 1", "mellibiose carrier protein", "ribokinase", linoleate hydratase, "unknown protein 2", "transcriptional regulatory protein", "ribose-binding ABC channel protein 1" and "ribose-binding ABC channel Protein 2" gene, among them, the transcription level of the gene encoding "unknown protei...

Embodiment 2

[0049] Example 2: Cloning of the gene encoding linoleic acid isomerase

[0050] Specific steps are as follows:

[0051] Pick the bacteria solution of Bifidobacterium breve (Bifidobacterium breve) CGMCC No.11828 from the bacteria preservation tube, streak it on the MRS solid medium, cultivate it in a constant temperature anaerobic workstation at 37°C for 48 hours, and obtain a single colony; pick a single colony for inoculation In the MRS liquid medium, continue to stand still for 24 hours in a constant temperature anaerobic workstation at 37°C, and activate continuously for 3 generations to obtain an activated bacterial liquid; inoculate the activated bacterial liquid at an inoculum of 1% (v / v) Inoculate into MRS liquid medium, culture in a constant temperature anaerobic workstation at 37°C for 24 hours to obtain a bacterial suspension; centrifuge the obtained bacterial suspension at 25°C and 12000g for 10 minutes to obtain wet bacterial cells; use bacterial genomic DNA The e...

Embodiment 3

[0056] Example 3: Optimization of the gene encoding linoleic acid isomerase

[0057] Specific steps are as follows:

[0058] Without affecting the precursor of the expressed protein, reduce the content of GC in the bbi obtained in Example 2, and make the corresponding codon more suitable for the biological utilization of Lactobacillus, the optimization of the codon and the synthesis of the gene sequence are provided by General Bio Completed by System (Anhui) Co., Ltd. The enzyme cutting sites at both ends of the sequence are Kpn I and Xba I respectively, and the sequence is ligated in the pU57 plasmid, which is stored in E. coli E.coli DH5α, and the recombinant E. coli DH5α / pU57-bbi(U); wherein, the nucleotide sequence of the unoptimized bbi sequence is shown in SEQ ID No.2, and the nucleotide sequence of the optimized bbi sequence is shown in SEQ ID No.5.

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Abstract

The invention discloses a lactobacillus plantarum engineering bacterium capable of producing conjugated linoleic acid at high yield, and belongs to the technical field of gene engineering and microbial engineering. The lactobacillus plantarum engineering bacterium can produce the conjugated linoleic acid at high yield, most of produced conjugated linoleic acid isomers are cis9 and trans11-CLA, the lactobacillus plantarum engineering bacterium is added into a culture medium containing linoleic acid to be cultured for 72 hours, the conversion rate of conjugated linoleic acid can reach 89.9%, and the content of the cis9 and the trans11-CLA in the conjugated linoleic acid can reach 100%. The lactobacillus plantarum is one of probiotics and is already brought into 'strain list capable of being used for foods' issued by Ministry of Health at present, so that the conjugated linoleic acid produced by the lactobacillus plantarum engineering bacterium is higher in safety for a human body.

Description

technical field [0001] The invention relates to a plant lactobacillus engineering bacterium capable of high-yielding conjugated linoleic acid, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Since the discovery of conjugated linoleic acid by Michael W.Pariza of Wisconsin State University in 1978, research on conjugated linoleic acid has attracted much attention. Studies have confirmed that conjugated linoleic acid has many important biological functions such as anti-tumor, anti-oxidation, lowering animal and human cholesterol, anti-atherosclerosis, improving immunity and increasing bone marrow density. In the United States, CLA has been marketed as a food supplement, and it is one of the popular supplements recommended by the American Food Institute. my country has approved conjugated linoleic acid to be used in the production of health food with weight loss, blood lipid regulation and immune regulation funct...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/61C12N9/90C12N15/74C12P7/64C12R1/25
CPCC12N9/90C12P7/6472C12Y502/01005
Inventor 杨波陈海琴高鹤赵建新张灏陈卫
Owner JIANGNAN UNIV
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