CN-121717891-B - Peppermint lipid transport protein of affinity monoterpenes, and encoding gene and application thereof

CN121717891BCN 121717891 BCN121717891 BCN 121717891BCN-121717891-B

Abstract

The invention discloses a peppermint lipid transport protein of affinity monoterpenes, and a coding gene and application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the lipid transport protein provided by the invention is shown as SEQ ID NO. 1. The invention also provides a method for improving the tolerance of host cells to monoterpenes by using the protein and application of the protein in production of monoterpenes. Experiments prove that the protein can specifically bind with various monoterpenes such as menthol, menthone and the like, can obviously enhance the tolerance of cells to menthol after being expressed in yeast, and can promote the yeast cells to excrete and transfer menthol outwards. The lipid transport protein and the encoding gene thereof provided by the invention can be used for modifying and constructing a high-yield and high-tolerance monoterpene microbial cell factory (such as yeast engineering bacteria), and provide a new technical means and gene resources for improving the fermentation production efficiency and final yield of monoterpene compounds.

Inventors

LI LI
LIANG CHENGYUAN
WANG JINGTING
QI XIWU
BAI YANG
YU XU

Assignees

江苏省中国科学院植物研究所

Dates

Publication Date: 20260508
Application Date: 20260213

Claims (6)

1. An affinity monoterpene mint lipid transporter is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
2. A nucleic acid molecule encoding the mint lipid transporter of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence is set forth in SEQ ID No. 2.
4. A recombinant expression vector comprising the nucleic acid molecule of claim 2 or 3.
5. A recombinant host cell comprising the nucleic acid molecule of claim 2 or 3, or comprising the recombinant expression vector of claim 4.
6. A method of increasing host cell tolerance to monoterpene compounds, wherein the mint lipid transporter of claim 1 is expressed in the host cell, the host cell is saccharomyces cerevisiae, and the monoterpene compound is menthol.

Description

Peppermint lipid transport protein of affinity monoterpenes, and encoding gene and application thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to an affinity monoterpene mint lipid transport protein, and a coding gene and application thereof. Background Monoterpene compounds are terpenes formed by condensing two isoprene units (C5H 8), and are widely existing in gland, oil chamber, resin tract and other secretion tissues of higher plants, and are mainly one of volatile oil components, such as menthol, geraniol, limonene, camphor, carvacrol and the like. Monoterpene oxygen-containing derivatives such as alcohols, aldehydes and ketones generally have high biological activity and are widely applied to the industries of medicines, foods, spices, cosmetics, agriculture, energy sources and the like. The main modes for obtaining monoterpene compounds in the market at present are a plant extraction method, a chemical synthesis method and a biological synthesis method. Plant extraction relies on the supply of high quality plant material. Chemical synthesis is achieved by means of corresponding precursors and organocatalytic reactions, but it is difficult to achieve single structure terpenoid synthesis. Biosynthesis laws rely on microbial cell factory fermentation, such as Saccharomyces cerevisiae, as a model strain, which has been widely used to produce various monoterpenes. The main factors affecting the synthesis of monoterpenes by yeast are insufficient supply of precursors, low enzyme activity and expression level, cytotoxicity of monoterpenes, etc. Most monoterpenes have strong toxicity to microbial cells, such as menthol, alpha-pinene and limonene, and when a certain concentration is reached, the monoterpenes can be permanently damaged and even die, so that the alleviation of the cytotoxicity of the monoterpenes is an important direction for constructing a monoterpene efficient production strain. Recognition and promotion of extracellular excretion of monoterpene compounds from yeast cells by efflux proteins is one of the main strategies. In yeast cells, no native efflux proteins of the monoterpenes have been found, and therefore the introduction of heterologous transporters to transport the monoterpenes is beneficial to yeast cells. For example, in the construction and optimization of high-yield sabinene saccharomyces cerevisiae, the foreign protein GcABCG1 is expressed in a heterologous manner on the basis of the expression of sabinene synthase t34SabS1, so that sabinene yield is improved by 1.04 times. Transport proteins associated with D-limonene, β -pinene, geraniol, carene transport have been found, while other monoterpene related transport proteins are rarely evident. Therefore, there is a need to study more and more efficient monoterpene transporters to increase the tolerance of the cells to monoterpenes and the fermentation efficiency, thereby increasing the yield of monoterpenes. Provides technical support for efficiently biosynthesizing monoterpenes by utilizing a microbial cell factory. Disclosure of Invention Aiming at the problem of limited yield caused by toxicity of the monoterpene compounds to cells when the monoterpene compounds are produced by a microbial cell factory in the prior art, the application provides the lipid transport protein capable of improving tolerance of host cells to the monoterpene compounds and the coding gene thereof. The application further aims to provide specific application of the lipid transport protein and the encoding gene thereof. In order to solve the technical problems, the invention adopts the following technical scheme: a peppermint lipid transport protein has an amino acid sequence shown in SEQ ID NO. 1. A nucleic acid molecule encoding said mint lipid transporter. According to the nucleic acid molecule, the nucleotide sequence is shown as SEQ ID NO. 2. A recombinant expression vector comprising said nucleic acid molecule. A recombinant host cell comprising said nucleic acid molecule, or comprising said recombinant expression vector. A method of increasing host cell tolerance to monoterpene compounds, the method comprising the step of expressing the mint lipid transporter in the host cell. In some embodiments, the mint lipid transporter is capable of enhancing extracellular transport of monoterpene compounds. In some embodiments, the host cell is a yeast. In some embodiments, the yeast is saccharomyces cerevisiae. In some embodiments, the monoterpene is selected from at least one of menthol, menthone, pulegone, linalool, eucalyptol. The use of said mint lipid transporter, said recombinant expression vector or said recombinant host cell in the production of monoterpene compounds. In some embodiments, the monoterpene is selected from at least one of menthol, menthone, pulegone, linalool, eucalyptol. Compared with the prior art, the invention has the following beneficial effects: 1) The applicatio