Genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris

A conjugated linoleic acid and Pichia pastoris technology, which is applied in the fields of genetic engineering and microbial engineering, can solve the problems of multiple types of conjugated linoleic acid isomers, single type of CLA isomers, poisoning and the like

Active Publication Date: 2021-07-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Among them, the chemical synthesis method is mainly to obtain CLA through alkali isomerization and transition metal catalysis of the corresponding substrate. However, the process of obtaining CLA by chemical synthesis will produce many toxic by-products, which are harmful to the environment and the human body. There are toxic effects, and there are many types of conjugated linoleic acid isomers prepared by chemical synthesis, which are difficult to effectively separate (see references for details: Philippaerts, A., VanAelst, J., Sels, B. , 2013. Conjugated linoleic acids and conjugated vegetable oils: from nutraceutical to bio-polymer. Eur. J. Lipid Sci. Technol. 115, 717–720. and Nicod, N., Parker, R.S., Giordano, E., Maestro, V., Davalos, A., Visioli, F., 2015. Isomer specific effects of conjugated linoleic acid on HDL functionality associated with reverse cholesterol transport. J. Nutr. Biochem. 26, 165-172.), therefore, chemical synthesis can not really achieve high-purity CLA monomer, Especially the large-scale industrial production of high-purity cis9, trans11-CLA and trans10, cis12-CLA monomers
[0006] The biosynthesis method is mainly to obtain CLA by using specific enzymes or microorganisms to catalyze corresponding substrates, which has the advantages of less pollution and relatively single types of synthetic CLA isomers. However, thi...

Method used

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  • Genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris
  • Genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris
  • Genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Screening of genes encoding linoleic acid isomerase

[0082] 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 protein 1" increased 68 times at 8h compared wi...

Embodiment 2

[0083] Example 2: Cloning of the gene encoding linoleic acid isomerase

[0084] 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 genomic DNA extraction The kit extracts the genomic DNA from the wet...

Embodiment 3

[0089] Example 3: Optimization of the gene encoding linoleic acid isomerase

[0090] According to the codon preference of yeast, the bbi gene was optimized using Genscript OptimumGene TM software, and the optimized gene was named obbi gene. The obbi gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the vector pUC57 , to obtain the recombinant plasmid pUC57-obbi; wherein, the nucleotide sequence of the bbi gene is shown in SEQ ID No.2, and the nucleotide sequence of the obbi gene is shown in SEQ ID No.5.

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Abstract

The invention discloses genetically-engineered pichia pastoris capable of producing conjugated linoleic acid and application of genetically-engineered pichia pastoris, belonging to the technical fields of genetic engineering and microbial engineering. The genetically-engineered pichia pastoris can be used for producing conjugated linoleic acid; linoleate isomerase-containing cell disruption supernatant obtained by fermentation of the genetically-engineered pichia pastoris is added into a reaction system containing 0.9 g/L of free linoleic acid for a reaction; and only after the reaction is carried out for 3 h, the yield of conjugated linoleic acid in reaction liquid can reach 0.855 g/L, a conversion rate reaches 95%, all the conjugated linoleic acid in the reaction is cis9,trans11-CLA, and no other conjugated linoleic acid isomers exist.

Description

technical field [0001] The invention relates to a Pichia pastoris engineering bacterium capable of producing conjugated linoleic acid and an application thereof, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Conjugated linoleic acid (CLA) is a general term for a group of octadecadienoic acids containing conjugated double bonds, and is a stereogeometric isomer of linoleic acid (LA). Due to the different positions of the conjugated double bonds and the two geometric structures of the double bonds, cis and trans, the types of conjugated linoleic acid are very rich. [0003] Among the many isomers of conjugated linoleic acid, cis9, trans11-CLA, trans10, cis12-CLA have been proved to have important physiological activities. For example, studies have shown that cis9, trans11-CLA and trans10, cis12-CLA have Anti-cancer physiological activity, among which, cis9 and trans11-CLA have stronger anti-cancer ability (see...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P7/64C12N9/90C12R1/84
CPCC12N9/90C12Y502/01005C12P7/6427
Inventor 陈海琴杨波赵建新李秀清高鹤张灏陈卫
Owner JIANGNAN UNIV
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