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

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...

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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, this method also has obvious defects. For example, using When microorganisms catalyze the corresponding substrates to obtain CLA, most of the strains involved are strict anaerobic bacteria such as bifidobacteria. Strict anaerobic bacteria are difficult to cultivate in industry or laboratories and have low yields, so it is difficult to be widely used in food, medicine, and cosmetics. Medium; When using enzymes to catalyze corresponding substrates to obtain CLA, multiple enzymes need to be used at the same time, and the multi-enzyme catalysis has many intermediate products and complex pathways, which is not suitable for the industrial production of CLA

[0007] In addition, although the types of CLA isomers produced by biosynthesis are relatively single, there is still a long way to go to obtain high-purity CLA monomers, especially high-purity cis9, trans11-CLA and trans10, cis12-CLA monomers. a certain distance

[0008] The above-mentioned defects make the existing biosynthesis method unable to realize the large-scale industrial production of high-purity CLA monomers, especially high-purity cis9, trans11-CLA and trans10, cis12-CLA monomers. Therefore, it is urgent to find a high-yield A method for producing high-purity CLA monomers, especially high-purity cis9, trans11-CLA and trans10, cis12-CLA monomers to overcome the shortcomings of existing biosynthetic methods

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