CN116904383A

CN116904383A - Recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, and preparation method and application thereof

Info

Publication number: CN116904383A
Application number: CN202311105655.0A
Authority: CN
Inventors: 崔凤杰; 杨雨蒙; 昝新艺; 孙雷; 孙文敬; 孟利娟
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2023-08-30
Filing date: 2023-08-30
Publication date: 2023-10-20

Abstract

The invention discloses a recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, and a preparation method and application thereof, belonging to the technical fields of metabolic engineering and food fermentation. The recombinant corynebacterium glutamicum is constructed by introducing lipoxygenase genes, hydroperoxide lyase genes, alcohol oxidoreductase genes and a triple promoter into the corynebacterium glutamicum, wherein the nucleotide sequence of the triple promoter is shown as SEQ ID NO:1, wherein the nucleotide sequence of the lipoxygenase gene is shown as SEQ ID NO:2, the hydroperoxide lyase is shown as SEQ ID NO:3, the alcohol oxidoreductase is shown as SEQ ID NO: 4. Compared with the common recombinant bacteria, the recombinant corynebacterium glutamicum has 364% higher yield of 1-octene-3-alcohol, 22mg/L higher fermentation level than other species related genetically engineered bacteria, and good industrialized application prospect.

Description

Recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, and preparation method and application thereof

Technical Field

The invention relates to the technical fields of metabolic engineering and food fermentation, in particular to recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, and a preparation method and application thereof.

Background

The edible fungi contain components such as aroma, delicate flavor and the like, and are good seasonings, and the aroma can lead people to enjoy the sense and the mind, promote secretion of digestive juice and facilitate digestion and absorption of nutrient components by human bodies. Edible fungi have special flavor, and different kinds of edible fungi often show different flavors. The flavor of grifola frondosa is like shredded chicken, and the aroma is attractive. The volatile flavor components in the edible fungi mainly comprise eight-carbon volatile compounds, sulfur-containing compounds, some acid, ketone, aldehyde, ester compounds and the like. In recent years, researches show that the eight-carbon volatile compound is a main contributor to the fragrance of edible fungi in 110 or more volatile compounds, and even has various functions of affecting the growth, development, behavior and the like of fungi as a signal molecule oxyresinol and a quorum sensing molecule. At present, 1-octene-3-ol (namely 'mushroom alcohol') is the most important volatile compound in edible mushrooms, almost exists in all edible mushroom types, and is rich in content. In addition, 1-octen-3-ol also affects the growth, development and behavior of fungi, plants and insects as a signal molecule, oxylipoproteins and quorum sensing molecules. In particular, 1-octen-3-ol is considered a self-inhibitor of mushroom primordium formation, which results in an "overcrowding" effect, thereby limiting germination of spores at dense concentrations. The olfactory threshold of 1-octen-3-ol is very low, so that it is authorized by the U.S. food and drug administration to be used as a food additive (ASP 1154,Regnum 172.515), and can be used for daily chemicals and preparing edible essences such as mushrooms and earthy tastes, and artificial essential oils, recombinant essential oils or ester flavors, and can also be used as a chemical synthesis raw material for the pharmaceutical field; as a mosquito attractant, the mosquito attractant has higher economic value when applied to mosquito-killing products. These volatile compounds are the main source of the "fresh green" smell characteristic of mushrooms. For example, they are widely used as the taste of foods to restore the freshness of the foods after the sterilization process. Since these compounds are low in content in nature and are required in large quantities, it is necessary to synthesize them on a large scale. Lipoxygenase, hydroperoxide lyase and alcohol oxidoreductase are suitable biocatalysts for the production of the taste of "natural" foods.

It is generally believed that the process of synthesizing the volatile compound 1-octen-3-ol in edible mushrooms may involve the following major steps: (1) The substrate linoleic acid was stereoscopically oxidized by Lipoxygenase (LOX) to hydroperoxide 10-HPOD; (2) A relatively stable hydroperoxide lyase (Hydroperoxide lyase, HPL) and alcohol oxidoreductase (Alcohol oxidoreductase, AOR) together catalyze cleavage of the C-C bond in polyunsaturated fatty acid hydroperoxides 10-HPOD and form volatile 1-octen-3-ol and 10-oxo-trans-8-decenoic acid. The reaction process for synthesizing the 1-octen-3-ol with volatile products by catalytically cracking the low hydroperoxide isomer 10-HPOD comprises the following steps: CH (CH) ₃ (CH ₂ ) ₄ CHCHCH ₂ CHOOHCHCH(CH ₂ ) ₆ COOH→CHOCHCH(CH ₂ ) ₆ COOH+CH ₂ ＝CHCH(OH)CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ +HOCHCH(CH ₂ ) ₅ CH ₃ . Because the formed hydrogen peroxide fatty acid intermediate has high risk to human body, accurately elucidating the catalytic property of key enzyme system and the contribution of the key enzyme system to the fragrance of edible fungi is a key step for understanding the synthesis of 1-octene-3-ol.

Currently, the problem of accumulation of polyunsaturated fatty acid hydroperoxide 10-HPOD is a major bottleneck for sustainable biosynthesis of 1-octen-3-ol from linoleic acid. Lipoxygenase, hydroperoxide lyase and alcohol oxidoreductase for 1-octene-3-alcohol production are deficient in activity, and are extremely prone to accumulation of toxic 10-HPOD, damaging cell growth. And the mechanism of cleavage of the hydroperoxide 10-HPOD by the hydroperoxide lyase in Grifola frondosa is still unclear, and no report has been found of fermentative production of 1-octen-3-ol by recombinant bacteria.

Disclosure of Invention

The invention aims to provide a recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, a preparation method and application thereof, so as to solve the problems in the prior art, and the recombinant corynebacterium glutamicum realizes the purpose of mass production of 1-octene-3-alcohol by a microbiological method and provides an effective solving strategy and path for safely and efficiently producing aromatic octanene.

In order to achieve the above object, the present invention provides the following solutions:

the present invention provides a recombinant corynebacterium glutamicum producing 1-octen-3-ol, which is constructed by introducing lipoxygenase gene, hydroperoxide lyase gene, alcohol oxidoreductase gene and triple promoter into corynebacterium glutamicum, wherein the triple promoter (P _tac -P _tuf -P _gro ) The nucleotide sequence of (2) is shown as SEQ ID NO:1, wherein the nucleotide sequence of the lipoxygenase (lox) gene is shown in SEQ ID NO:2, the hydroperoxide lyase (hplc) is shown in SEQ ID NO:3, the alcohol oxidoreductase (aor) is shown in SEQ ID NO: 4.

The invention also provides a construction method of the recombinant corynebacterium glutamicum for producing 1-octene-3-alcohol, which comprises the following steps:

introducing nucleotide sequences of lipoxygenase genes, hydroperoxide lyase genes, alcohol oxidoreductase genes and triple promoters into a plasmid vector to construct a recombinant plasmid;

and electrotransferring the recombinant plasmid into competent cells of corynebacterium glutamicum, and screening positive clones to obtain recombinant engineering bacteria.

Preferably, the primers for amplifying the hydroperoxide lyase are:

gfhpl-F：5’-ttgtatgaatggcgtccgcactcagaga-3’，SEQ ID NO：6；

gfhpl-R：5’-atcccttgatcatgcacgctgacgcgga-3’，SEQ ID NO：7；

the primers for amplifying the alcohol oxidoreductase gene are:

gfaor-F：5’-gtgcatgatcaagggattatcacagccctgaac-3’，SEQ ID NO：8；

gfaor-R：5’-caaaacagccaagctgatgtggatgattgtgcccatcg-3’，SEQ ID NO：9；

the primers for amplifying the lipoxygenase gene are as follows:

lox-F：5’-ttcaagtaaa tggctccctt caagggg-3’，SEQ ID NO：10；

lox-R：5’-ggacgccatt catacaaggt ttaatgagag aaggtcaatgt-3’，SEQ ID NO：11；

the primers for amplifying the triple promoter are as follows:

P _tac -P _tuf -P _gro -F：5’-gtatcccact accgagatat gcctatcttc aagaagacgc tca-3’，SEQ ID NO：12；

P _tac -P _tuf -P _gro -R：5’-caaaacagcc aagctgttac ttgaagatcg ttaccttctt acgtgtacc-3’，SEQ ID NO：13。

preferably, the electrical conversion conditions are: the volume ratio of the recombinant plasmid to the competent cells of the corynebacterium glutamicum is 1:50, and the electrotransformation voltage is 2.5kV.

The invention also provides a method for producing 1-octene-3-alcohol by utilizing the recombinant corynebacterium glutamicum, which comprises the steps of obtaining fermentation liquor by fermenting and culturing the recombinant corynebacterium glutamicum, and separating and purifying the fermentation liquor to obtain the 1-octene-3-alcohol.

Preferably, the fermentation medium of the fermentation culture comprises the following components: 10g of bean flour, 2.5g of soybean oil, 0.2g of diammonium phosphate and KH ₂ PO ₄ 0.068g, 8.0mL of corn steep liquor, 2.941g of citrate buffer and 100mL of mineral solution, and fixing the volume to 1L;

the conditions of the fermentation culture are as follows: the inoculation amount is 1-10%, the rotating speed is 200r/min, and the culture time is 24-48 h.

Preferably, the separation is purified as: centrifuging the fermentation liquor, taking supernatant I, adding sodium phosphate buffer solution and linoleic acid into the supernatant I, uniformly mixing, and incubating;

after the incubation, the pH value of the mixture is adjusted, the mixture is centrifuged to obtain a supernatant II, the supernatant II is extracted by diethyl ether, the diethyl ether part is collected, and the diethyl ether is volatilized to obtain the 1-octen-3-ol.

Preferably, the concentration of the sodium phosphate buffer is 0.1M and the pH value is 6.5; the concentration of the sub-oleic acid is 130 mu M;

the conditions of the incubation are: incubate at 25℃for 60min.

Preferably, the pH of the mixture is adjusted to 9.0.

The invention also provides application of the recombinant corynebacterium glutamicum in producing 1-octene-3-alcohol.

The invention discloses the following technical effects:

according to the invention, the gene related to the synthesis path of 1-octene-3-ol is introduced into the food-grade safety strain Corynebacterium glutamicum, so that the heterologous biosynthesis of 1-octene-3-ol is successfully realized, the Corynebacterium glutamicum is used as an important industrial fermentation engineering bacterium, the culture cost is relatively low, and the production intensity is high. Compared with other strains synthesized by 1-octene-3-alcohol, the product completely meets the food safety requirement and has great application advantages. Meanwhile, the invention also introduces a triple promoter, which is compared with a common single-promoter control group, so that the transcription efficiency of related genes is effectively improved, the 1-octene-3-alcohol yield is improved by 364%, 22mg/L is achieved, and the method is far higher than the fermentation level of other species related genetic engineering bacteria, and has good industrialized application prospect.

The invention is helpful for understanding the synthesis mechanism of the volatile compound 1-octen-3-ol in the edible fungi from the molecular level, and the development of the biotechnology process provides important technical support and reference for producing the 1-octen-3-ol product with stable quality by efficient fermentation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the pathway for the synthesis of 1-octen-3-ol (1-octen-3-ol) from the head of Corynebacterium glutamicum;

FIG. 2 is an agarose gel electrophoresis of EcoRI and EcoRV double digested plasmid pXMJ 19; 1, representing a plasmid pXMJ19 sample; m represents a standard DNA molecule;

FIG. 3 shows plasmid pX _lox-hpl-aor Is a map of (2);

FIG. 4 is a GC/MS spectrum of a 1-octen-3-ol standard;

FIG. 5 shows the results of fermentation production of 1-octen-3-ol from recombinant Corynebacterium glutamicum.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The invention provides a technical scheme for efficiently expressing lipoxygenase gene lox, hydroperoxide lyase gene hplc and alcohol oxidoreductase gene aor in edible fungi grifola frondosa by integrating a triple promoter in a corynebacterium glutamicum expression system (the specific construction way is shown in figure 1). Most corynebacterium glutamicum is nonpathogenic, and the U.S. Food and Drug Administration (FDA) has listed the corynebacterium glutamicum as a microorganism (GRAS) which is regarded as safe in principle and is regarded as an ideal microorganism chassis for biological production, and the recombinant corynebacterium glutamicum for efficiently synthesizing the volatile compound 1-octen-3-ol and the method for producing 1-octen-3-ol are specifically constructed, so that an effective solving strategy and approach are provided for safely and efficiently producing aromatic octans. The constructed corynebacterium glutamicum can synthesize 1-octen-3-ol by taking linoleic acid and the like as substrates. The above technical idea will be further described with specific examples.

EXAMPLE 1 construction of recombinant Corynebacterium glutamicum Cg/pX by introduction of genes involved in biosynthesis of 1-octen-3-ol _lox-hpl-aor

1. Expression plasmid pX _lox-hpl-aor Comprises the following steps:

(1) PCR amplification of the hplc gene (SEQ ID NO. 3) and aor gene (SEQ ID NO. 4) fragments using primers gfhplc-F, gfhpl-R and gfacor-F, gfaor-R, respectively, using genomic DNA of Grifola frondosa Grifola frondosa strain GF9801 as a template, and recovery and purification;

the primer sequences were as follows:

gfhpl-F：5’-ttgtatgaatggcgtccgcactcagaga-3’；

gfhpl-R：5’-atcccttgatcatgcacgctgacgcgga-3’；

gfaor-F：5’-gtgcatgatcaagggattatcacagccctgaac-3’；

gfaor-R：5’-caaaacagccaagctgatgtggatgattgtgcccatcg-3’。

the PCR reaction system is as follows:

TABLE 1

Reagent(s)	Dosage of
		Primer F	1μL
Primer R	1μL
		10×Buffer	5μL
DNA	5μL
		ExTag	0.25μL
dNTP	4μL
		ddH ₂ O	33.75μL

The PCR reaction conditions were:

TABLE 2

Temperature (temperature)	Time
		98℃	30s
98℃	10s
		55℃	30s
72℃	2min
		32	cycles

(2) EcoRI and EcoRV double digested plasmid pXMJ19 was digested and the 5.3Kb fragment was recovered and purified. See fig. 2.

Double cleavage reaction system:

TABLE 3 Table 3

Reagent(s)	Dosage of
		Plasmid pXMJ19	5μL
EcoRⅠ,EcoRⅤ	1μL+1μL
		Buffer	3μL
ddH ₂ O	20μL

The double enzyme cutting condition is 37 ℃ and the enzyme cutting is carried out for 4 hours.

(3) Synthesis of HR1-P _tac -P _tuf -P _gro A lox-HR2 gene element (SEQ ID NO: 5) comprising 20bp pXMJ19 terminal homology sequence, triplet codon sequence P _tac -P _tuf -P _gro (SEQ ID NO: 1), lox gene sequence and hplc terminal homologous sequence. The gene sequence was delegated to Jiangsu Style Biotechnology Co.

(4) Connecting the Hpl, aor gene fragment purified and recovered in the step (1) and the nucleotide fragment synthesized in the step (3) to a plasmid vector pXMJ19 recovered by double digestion in the step (2) through Gibson assembly, thus obtaining a recombinant plasmid pX _lox-hpl-aor (see FIG. 3 for plasmid map).

The Gibson assembly system is 1.5 mu L of each of the hplc gene and aor gene fragment, P _tac -P _tuf -P _gro 1 mu L of lox fragment, 1 mu L of restriction enzyme linearized plasmid vector pXMJ, 2X Gibson assembly Mix mu L, 1h of reaction condition at 50 ℃, transforming E.coli DH5 alpha competent cells, screening with 12.5 mu g/mL chloramphenicol, picking single colony for inoculation, extracting plasmid, ecoRI restriction enzyme single restriction enzyme verification, and positive recombinant sequencing verification.

2. Plasmid pX _lox-hpl-aor Electrotransfer into competent cells of corynebacterium glutamicum, and screening chloramphenicol with 5 mug/mL to obtain recombinant corynebacterium glutamicum Cg/pX _lox-hpl-aor 。

The preparation method of the electrotransformation competent cells of the corynebacterium glutamicum comprises the following steps:

(1) The Corynebacterium glutamicum CICC 10186 glycerol bacteria stored in a refrigerator at-80℃were cultured overnight at 30℃with 20. Mu.L of the empty LB plates. Selecting single bacteria on a flat plate, and culturing for 16 hours at 30 ℃ and 200r/min in an LB liquid culture medium to obtain corynebacterium glutamicum seed liquid.

(2) Inoculating the seed solution to LB medium containing 3% glycine and 0.1% Tween at 10% inoculating ratio, culturing at 30deg.C and 200r/min until OD ₆₀₀ 1-1.2.

(3) The pre-cultured bacterial liquid is transferred into a 50mL centrifuge tube ice bath for 30min, the temperature is 4 ℃,5000g is centrifuged for 10min, and the supernatant is removed.

(4) The cells were resuspended in pre-chilled sterile water, centrifuged at 5000g for 10min at 4℃and the supernatant removed.

(5) Repeating the step (4) for 2 times.

(6) The cells were resuspended in pre-chilled 10% glycerol and centrifuged at 5000g for 10min at 4℃to remove the supernatant.

(7) Repeating the step (6) for 2 times.

(8) The thalli are resuspended with pre-chilled 10% glycerol, and the thalli are respectively packed into pre-chilled 1.5mL centrifuge tubes according to 100 mu L of each tube, rapidly cooled in liquid nitrogen for 5-10min, and stored in a refrigerator at-80 ℃.

The specific operation of the electric conversion is as follows:

and (3) inoculating 2 mu L of plasmids into 100 mu L of electric transfer corynebacterium glutamicum which is split into a centrifuge tube, transferring to a precooled 2mm electric transfer cup, carrying out ice bath for 5min, regulating the voltage of an electroporation device to 2.5kV, sucking the outer surface of a sample tank by using paper towels, loading the electric transfer cup into the electric transfer device, and pressing an electric shock key. After the electric shock is finished, 1mL of recovery culture medium (LB culture medium is added with 0.5M sorbitol and 0.38M mannitol) is immediately added into the electric rotating cup, and the thallus is resuspended and transferred into a 1.5mL centrifuge tube together, and recovered for 3 hours at 37 ℃ and 200 r/min.

Construction of recombinant Corynebacterium glutamicum Cg/pXMJ19-lox

1. Construction of the expression plasmid pXMJ19-lox, comprising the steps of:

(1) HR1-P synthesized in example 1 _tac -P _tuf -P _gro And (3) taking lox-HR2 as a template, amplifying a lox gene fragment by using a primer lox-F and lox-R, and recovering and purifying. Above-mentionedThe primer sequences were as follows:

lox-F：5’-ttcaagtaaa tggctccctt caagggg-3’；

lox-R：5’-ggacgccatt catacaaggt ttaatgagag aaggtcaatgt-3’

the PCR reaction system is as follows:

TABLE 4 Table 4

Reagent(s)	Volume of
		Primer F	1μL
Primer R	1μL
		10×Buffer	5μL
HR1-P _tac -P _tuf -P _gro -lox-HR2	2μL
		ExTag	0.25μL
dNTP	4μL
		ddH ₂ O	36.75μL

The PCR reaction conditions were:

TABLE 5

(2) EcoRI and EcoRV double digested plasmid pXMJ19 was digested and the 5.3Kb fragment was recovered and purified.

The double enzyme digestion system is as follows:

reagent(s)	Dosage of
		Plasmid pXMJ19	5μL
EcoRⅠ，BamHⅠ	1μL+1μL
		Buffer	3μL
ddH ₂ O	20μL

(3) The lox gene fragment recovered by purification in the step (1) and the hplc and aor fragments recovered by purification in the example 1 were ligated to the plasmid vector pXMJ19 recovered by double digestion in the step (2) by Gibson assembly, thereby obtaining the recombinant plasmid pXMJ19-lox.

The Gibson assembly system is lox, hplc, aor gene fragments of 2 mu L, 1 mu L of plasmid vector linearized by enzyme digestion, 2X Gibson assembly Mix mu L, reaction condition of 50 ℃ for 1h, transforming E.coli DH5 alpha competent cells, chloramphenicol of 12.5 mu g/mL screening, picking single colony for inoculation, extracting plasmid, ecoRI single enzyme digestion verification, positive recombinant sequencing verification, and obtaining recombinant Corynebacterium glutamicum Cg/pXMJ19-lox.

EXAMPLE 2 use of recombinant Corynebacterium glutamicum Cg/pX _lox-hpl-aor Expression of volatile compound 1-octen-3-ol in edible fungi

1. Recombinant corynebacterium glutamicum fermentation

(1) Recombinant Corynebacterium glutamicum Cg/pX of example 1 _lox-hpl-aor And the recombinant corynebacterium glutamicum Cg/pXMJ19-lox of the comparative example are respectively inoculated into LB liquid culture medium for culture, so as to obtain pre-cultured engineering bacteria liquid.

(2) The pre-culture bacterial liquid is inoculated into a fermentation medium according to the volume ratio of 10 percent, and the fermentation liquid containing 1-octene-3-alcohol is obtained after the fermentation medium is inoculated at 30 ℃ and 200r/min for 48 hours. Fermentation medium composition (1000 ml, ph 5.0): 10g of bean flour, 2.5g of soybean oil, 0.2g of diammonium phosphate and KH ₂ PO ₄ 0.068g, corn steep liquor 8.0mL, lemon2.941g of acid salt buffer, 100mL of mineral solution, and the volume was set to 1L.

2. Separating 1-octen-3-ol from fermentation liquid

The separation method of the 1-octene-3-alcohol in the fermentation liquor comprises the following steps:

(1) Taking fermentation liquor, subpackaging in a centrifugal cup, centrifuging at 12000rpm for 10min, taking supernatant, adding 0.1M sodium phosphate buffer solution (pH 6.5) and 130 mu M linoleic acid, and fully and uniformly mixing;

(2) After incubation for 60min at 25 ℃, the reaction mixture was adjusted to pH 9.0 (dilute NaOH), centrifuged for 20min at 100000g, and the supernatant was taken;

(3) To fully isolate 1-octen-3-ol, the supernatant was extracted twice with 100mL diethyl ether.

3. Detecting 1-octene-3-alcohol content in fermentation liquor

The method for detecting the content of 1-octene-3-alcohol in the fermentation broth comprises the following steps:

(1) After the diethyl ether extraction is finished, the sample is placed in a centrifuge tube, centrifuged for 8min at 5000g, and the supernatant is carefully collected;

(2) Taking the supernatant after centrifugation, evaporating in a fume hood until the volume is concentrated to 1mL;

(3) Volatiles were enriched by using HS-SPME (50/30 μm DVB/CAR/PDMS SPME, supelco, bellefonte, pa., USA) and volatility analysis was performed using a GC Trace1300 gas chromatograph in combination with ISQ-Mass (Thermo Scientific, fair down, NJ, USA);

(4) After the volatile matter is enriched, the SPME component is extracted from the bottle and inserted into a GC injection port, and the volatile matter is kept at 220 ℃ for 20min for volatile analysis;

(5) The separation was done in a DB-5MS (30 m 0.25 mm) column with the carrier gas being high purity helium gas at a flow rate of 1.0mL/min in a split mode of 2:1, with the injector temperature at 250 ℃ (oven temperature program range 50-280 ℃), and the ramp rate at 10 ℃/min. The mass spectral data is received by a mass detector operating in a full scan in the range of 20-500 amu. The standard 1-octen-3-ol for detection has a GC/MS spectrum shown in figure 4.

FIG. 5 shows a recombinant engineering bacterium Cg/pX _lox-hpl-aor Comparison of the yields of 1-octen-3-ol with Cg/pXMJ19-lox fermentation. As can be seen from the figure, in example 1Recombinant Corynebacterium glutamicum Cg/pX _lox-hpl-aor Can successfully ferment and synthesize volatile compounds with higher yield.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A recombinant corynebacterium glutamicum for producing 1-octen-3-ol, which is characterized in that the recombinant corynebacterium glutamicum is constructed by introducing lipoxygenase genes, hydroperoxide lyase genes, alcohol oxidoreductase genes and a triple promoter into the corynebacterium glutamicum, wherein the nucleotide sequence of the triple promoter is shown as SEQ ID NO:1, wherein the nucleotide sequence of the lipoxygenase gene is shown as SEQ ID NO:2, the hydroperoxide lyase is shown as SEQ ID NO:3, the alcohol oxidoreductase is shown as SEQ ID NO: 4.

2. A method for constructing a recombinant corynebacterium glutamicum producing 1-octen-3-ol according to claim 1, comprising the steps of:

3. The construction method according to claim 2, wherein the primers for amplifying the hydroperoxide lyase are:

gfhpl-F：5’-ttgtatgaatggcgtccgcactcagaga-3’；

gfhpl-R：5’-atcccttgatcatgcacgctgacgcgga-3’；

the primers for amplifying the alcohol oxidoreductase gene are:

gfaor-F：5’-gtgcatgatcaagggattatcacagccctgaac-3’；

gfaor-R：5’-caaaacagccaagctgatgtggatgattgtgcccatcg-3’；

the primers for amplifying the lipoxygenase gene are as follows:

lox-F：5’-ttcaagtaaa tggctccctt caagggg-3’；

lox-R：5’-ggacgccatt catacaaggt ttaatgagag aaggtcaatgt-3’；

the primers for amplifying the triple promoter are as follows:

P _tac -P _tuf -P _gro -F：5’-gtatcccact accgagatat gcctatcttc aagaagacgc tca-3’；

P _tac -P _tuf -P _gro -R：5’-caaaacagcc aagctgttac ttgaagatcg ttaccttctt acgtgtacc-3’。

4. the method of claim 2, wherein the electrotransformation conditions are: the volume ratio of the recombinant plasmid to the competent cells of the corynebacterium glutamicum is 1:50, and the electrotransformation voltage is 2.5kV.

5. The method for producing 1-octen-3-ol by using the recombinant corynebacterium glutamicum according to claim 1, which comprises the steps of obtaining a fermentation broth by fermenting and culturing the recombinant corynebacterium glutamicum, and separating and purifying the fermentation broth to obtain 1-octen-3-ol.

6. The method of claim 5, wherein the fermentation medium of the fermentation culture comprises the following components: 10g of bean flour, 2.5g of soybean oil, 0.2g of diammonium phosphate and KH ₂ PO ₄ 0.068g, 8.0mL of corn steep liquor, 2.941g of citrate buffer and 100mL of mineral solution, and fixing the volume to 1L;

7. The method of claim 5, wherein the separation purification is: centrifuging the fermentation liquor, taking supernatant I, adding sodium phosphate buffer solution and linoleic acid into the supernatant I, uniformly mixing, and incubating;

8. The method of claim 7, wherein the sodium phosphate buffer has a concentration of 0.1m and a ph of 6.5; the concentration of the sub-oleic acid is 130 mu M;

the conditions of the incubation are: incubate at 25℃for 60min.

9. The method of claim 7, wherein the pH of the mixture is adjusted to 9.0.

10. Use of the recombinant corynebacterium glutamicum according to claim 1 for the production of 1-octen-3-ol.