CN115044574B

CN115044574B - Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast

Info

Publication number: CN115044574B
Application number: CN202210571548.6A
Authority: CN
Inventors: 高蓓; 魏东芝; 王风清; 郭婧妍
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2023-07-14
Anticipated expiration: 2042-05-24
Also published as: CN115044574A

Abstract

The invention discloses a valencene synthase mutant and application thereof in synthesizing valencene in yeast, wherein the valencene synthase mutant comprises the following components: the valance-western-ene synthase mutant M560L is formed by site-directed mutagenesis by taking wild-type valance-western-ene synthase with the amino acid sequence shown in SEQ ID No.2 as a template. Compared with the wild type valencene synthase, the catalytic activity of the valencene synthase mutant M560L provided by the invention is greatly improved, the high-yield preparation of valencene is realized, the yield of valencene is improved by 30%, and the activity of a heterologous enzyme catalytic substrate FPP is improved by constructing the valencene synthase mutant, so that a foundation is laid for constructing a yeast high-yield valencene cell factory.

Description

Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a valance cemetery synthase mutant and application thereof in synthesizing valance cemetery in yeast.

Background

Valencene is a sesquiterpene compound with the chemical name (3R, 4aS, 5R) -4 o, 5-dimethyl-3-isopropenyl-1, 2,3, 4a,5,6, 7-octahydronaphthalene with the formula C ₁₅ H ₂₄ Molecular weight 204.35. Is colorless liquid at normal temperature, has the characteristic fragrance of sweet orange oil, is difficult to dissolve in water and is easy to dissolve in organic solvent.

The valance-grapefruit juice contains pleasant smell, and is commercially available as an essence and flavor to be added to foods and cosmetics. In addition, valance is also widely used in the pharmaceutical industry because of its specific odor activity and its safety to humans and mammals as evidenced by the FDA's view of GRAS materials. In recent years, the study of the related physiological effects of the valance cemetery is increasingly paid attention to, and the study finds that the valance cemetery has antibacterial, anti-inflammatory, antiproliferative and pro-apoptotic activities and has the function of inhibiting the growth of retinoblastoma cells. In addition, valance siemens has been found to have potential therapeutic effects on neuroinflammation and alzheimer's disease. The current industrial method for obtaining the valance cemetery mainly uses the citrus essential oil as a raw material and is separated through processes such as distillation, extraction and the like, but the concentration of the valance cemetery in the citrus fruits is low (0.2% -0.5%), and the plant cultivation period is long, the efficiency is low, the cost is high due to limited sources, and the valance cemetery cannot be used as a sustainable development obtaining means. In addition, chemical synthesis can also realize the synthesis of valance cemetery, but a large amount of toxic organic reagents are involved, so that the environment is polluted, the organic reagents are harmful to human bodies, and the edible grade cannot be achieved. With the development of synthetic biology, the construction of heterologous expression of microbial cell factories and related enzymes is a promising approach to the production of valencene in yeasts and E.coli.

To date, only a few valencene synthetases have been identified and tested for (+) -valencene biosynthesis, including VvVal of Vitis vinifera, cstps1 of Citrus sinesis, GFTpsD of Citrus paradis, and CnVS of Callitropsis nootkatensis. Among these, cnVS has been demonstrated to be one of the most stable at catalytic pH and temperature conditions, which is an ideal property for application in different hosts or under a variety of physiological conditions. The identified valencene synthase activity is too low compared to other sesquiterpene synthases, and overexpression of CnVS in yeast strain WAT11 only produces 1.36mg/L valencene, as a result of the challenge of reduced catalytic efficiency of microbial heterologous anabolites compared to native endogenous synthesis. Therefore, it is expected that the enhancement of the activity of the valencene synthase plays an important role in promoting the production of valencene.

Disclosure of Invention

The invention aims to provide a valance western-style alkene synthase mutant and application thereof in synthesizing valance western-style alkene in yeast, thereby solving the problems of lower activity of the valance western-style alkene synthase and lower yield of the valance western-style alkene in the prior art.

In order to solve the problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention there is provided a valencene synthase mutant of: the valance western-style alkene synthase mutant M560L is formed by taking wild valance western-style alkene synthase with an amino acid sequence shown as SEQ ID No.2 as a template and mutating methionine at position 560 into leucine.

According to a second aspect of the present invention there is provided a coding gene encoding said valencene synthase mutant.

According to a third aspect of the present invention there is provided a recombinant expression vector comprising a gene encoding said valencene synthase mutant.

According to a preferred embodiment of the invention, the vector plasmid in the recombinant expression vector is pESC-URA and the promoter is Gal1 inducible promoter. It will be appreciated that the invention is not limited to this vector plasmid and promoter but may be any other suitable vector plasmid and promoter.

According to a fourth aspect of the present invention, there is provided a recombinant genetically engineered bacterium comprising a gene encoding the valencene synthase mutant.

As a preferred embodiment of the present invention, a Saccharomyces cerevisiae strain BY4741 is randomly selected as the host in the present invention, but it is to be understood that the present invention is not limited to Saccharomyces cerevisiae, and virtually all yeast strains may be used, such as Saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica, etc. It should also be understood that the s.cerevisiae strains include, by way of example and not limitation: saccharomyces cerevisiae BY4741, saccharomyces cerevisiae CENPK2-1C, saccharomyces cerevisiae CENPK2-1D, saccharomyces cerevisiae BY4742, saccharomyces cerevisiae C800, saccharomyces cerevisiae CP08, etc. can also be used.

According to a fifth aspect of the present invention there is provided the use of said valencene synthase mutant in the synthesis of valencene in yeast.

The application comprises: transferring a recombinant expression vector containing the coding gene of the valance cemetery synthase mutant into yeast host bacteria, constructing a recombinant genetic engineering bacteria, and inoculating the recombinant genetic engineering bacteria into YPD liquid culture medium for fermentation for 6-8 days to realize the synthesis of the valance cemetery.

In the application, 2-3 mL of n-dodecane is preferably added for bi-directional fermentation at 10-12 h after the start of fermentation to reduce the volatilization loss of the valencene.

In this application, the valencene synthase mutant is M560L.

As described in the background of the invention section, only a few valencene synthetases have been identified and tested to date for (+) -valencene biosynthesis, but these valencene synthetases are relatively low in activity and do not allow for high-yield production of valencene. No literature has reported rational engineering of the sesquiterpene synthases CnVS, and the prior art generally optimizes CnVS expression patterns based solely on metabolic considerations, such as selecting promoter-terminator pairs, or increasing the copy number of CnVS in the genome. The key invention point of the invention is that the expression effect of the heterologous enzyme is optimized and the CnVS activity is improved by carrying out rational transformation on the Vanilla carbanilla synthase CnVS.

The invention obtains 13 valance western ene synthase mutants after the rational transformation, which comprises the following steps: a valance-western-ene synthase mutant G435V formed by taking a wild-type valance-western-ene synthase of an amino acid sequence shown in SEQ ID No.2 as a template and mutating 435 th glycine (Gly) into valine (Val); glycine 435 (Gly) to asparagine (Asn) to form a valance-western ene synthase mutant G435N; a valance-western ene synthase mutant G435S formed by mutation of glycine (Gly) at position 435 to serine (Ser); a valance-western-ene synthase mutant T479G formed by mutation of threonine (Thr) at position 479 to glycine (Gly); a valance-western ene synthase mutant F488H formed by mutation of phenylalanine (Phe) at position 488 to histidine (His); a valance-western-ene synthase mutant F488Q formed by mutation of phenylalanine (Phe) at position 488 to glutamine (Gln); a valance-western-ene synthase mutant F488Y formed by mutation of phenylalanine (Phe) at position 488 to tyrosine (Tyr); a valance-sienna alkene synthase mutant E489K formed by mutating glutamic acid (Glu) at position 489 to lysine (Lys); a valance-western ene synthase mutant E489M formed by mutation of glutamic acid (Glu) at position 489 to methionine; a valance-sienna alkene synthase mutant E489C formed by mutation of glutamic acid (Glu) at position 489 to cysteine (Cys); a valance-sienna alkene synthase mutant E489I formed by mutation of glutamic acid (Glu) at position 489 to isoleucine (IIe); a valance-western ene synthase mutant M560L formed by mutation of methionine (Met) at position 560 to leucine (Leu); a valance-sagitta synthase mutant M560A formed by the mutation of methionine (Met) at position 560 to alanine (Ala).

Based on the above, the inventor further screens out three key residue sites through rational design modification, and finds that the three key residue sites have promotion effect on synthesizing valance cetylene by CnVS catalytic substrate FPP. And by means of force analysis and combination of binding free energy values, the mutant M560L with the best fermentation result improves the binding force of CnVS and a substrate.

According to the invention, 13 valance western ene synthase mutants G435V, G435N, G435S, T479G, F488H, F488 5226 488Y, E4848 489M, E489C, E489I, M560L, M560A are initially obtained through rational design modification, and a gene recombination yeast strain is further constructed through a genetic engineering technology, finally, compared with a wild strain, the yield of valance western ene produced by the M560L mutant strain is improved by 30 percent through experiments.

In summary, the invention provides a valance cemetery synthase mutant and application of synthesizing valance cemetery in yeast by utilizing means of genetic engineering and enzyme engineering, and provides conditions for industrial production of valance cemetery by providing the valance cemetery synthase mutant with improved catalytic activity.

Drawings

FIG. 1 shows a plasmid schematic of the gene expression vector for Van-Sesamene synthase CnVS;

FIG. 2 shows a graph of the yield of valencene in a genetically recombinant yeast strain;

Detailed Description

The invention is further described below with reference to examples and figures, but embodiments of the invention are not limited thereto.

The following examples are examples of experimental methods that are used to identify specific experimental conditions, typically according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are those commercially available.

The preparation method of the solution provided by the invention comprises the following steps:

LB (Luria-Bertani) medium: 10g/L Peptone (Peptone), 5g/L Yeast extract (Yeast extract), 10g/L NaCl, and 2% (2 g/100 mL) agar powder added to the solid medium, and sterilizing at 121deg.C under high temperature and high pressure for 20min.

YPD (Yeast Extract Peptone Dextrose): 20g/L glucose (Dextrose), 10g/L Yeast extract (Yeast extract), 20g/L Peptone (Peptone), 2% (2 g/100 mL) agar powder was added to the solid medium, and the mixture was sterilized at 115℃under high temperature and high pressure for 20 minutes.

SD-URA auxotroph solid Medium: weighing 8g of corresponding auxotroph powder, adding 950mL of deionized water, uniformly mixing, adjusting the pH value to 6.0-6.5 by using 5M sodium hydroxide solution, subpackaging into 95 mL-250 mL conical flasks, adding 2% (2 g/100 mL) of agar powder, sterilizing at high temperature and high pressure at 115 ℃ for 20min, melting, cooling to about 50 ℃ and adding 5mL of 40% sterile glucose solution.

2 XPimeSTAR MAX used for PCR was purchased from Takara; hieff CloneTM One Step Cloning Kit from YEASEN; rTaq enzyme was purchased from Thermo Scientific; frozen-EZ Yeast Transformation II Kit is available from Epigenetics.

The yeast fermentation and product extraction method comprises the following steps: the monoclonal yeast colony is picked up on a flat plate and cultured for 24 hours in a test tube containing 5mL of YPD liquid culture medium at a temperature of 30 ℃ by a shaking table of 220rpm, then the colony is transferred to a 250mL conical flask filled with 20mL of YPD culture medium at a temperature of 30 ℃ by a shaking table of 220rpm for 12 hours, the bacterial OD600 is sampled and detected, the inoculum size is calculated, the initial OD600 is adjusted to 0.1, three parallel samples of each strain are fermented in a shaking flask, the sample is sampled every 48 hours for 7 days, and 2% n-dodecane is added to the shaking flask for covering the 60 th hour of fermentation. After fermentation, standing and shaking the bottle, sucking out the organic phase into an EP tube by using a gun head until the organic phase is layered with the water phase, centrifuging at1,2000 rpm for 10min, taking out the upper organic phase, adding a proper amount of anhydrous sodium sulfate, fully shaking and mixing, centrifuging at1,2000 rpm for 10min, sucking the upper organic phase, filtering into a gas chromatographic bottle by using a membrane, and taking the gas chromatographic bottle as a GC sample to be detected.

Vanilencia alkene gas phase detection method

The valance carbapenem standard bottle is purchased from Sigma company and prepared from valance carbarene standard solution: 10mg of a valance cemetery standard substance is weighed and dissolved in n-dodecane solution, the volume is fixed to 1mL, and the concentration of mother liquor of the standard substance is 10mg/mL.

The invention adopts gas chromatography to carry out qualitative and quantitative analysis of the valencene, and the detection conditions are as follows:

chromatographic column: HP-5 (30m x 0.32mm,0.25 μm, agilent, USA); the temperature of the sample inlet is 250 ℃; programming temperature: the column temperature was maintained at 100℃for 4min at the initial temperature, and then raised to 250℃at a rate of 10℃per minute for 5min.

Hydrogen Flame Ionization Detector (FID) temperature: 280 ℃; split ratio = 5:1, split flow 6mL/min; air flow rate: 300mL/min; hydrogen flow rate: 30mL/min; tail blow flow (N) ₂ ): 15mL/min; average linear velocity: 24.802cm/s; pressure: 7.1413psi; sample injection amount: 1 mul.

Example 1: construction of valance Sesamene Gene expression cassettes

First, the GenScript company was entrusted to synthesize a codon-optimized CnVS derived from Callitropsis nootkatensis (the nucleotide sequence of which is shown as SEQ ID No. 1), an expression vector pESC-URA empty plasmid was selected, cnVs-BamHI-F/CnVs-XhoI-R amplified CnVs genes were selected, and the CnVs genes were recovered and purified after agarose gel electrophoresis verification. And then carrying out double enzyme digestion on empty carrier plasmid pESC-URA and CnVS gene fragments by using restriction endonucleases BamHI and XhoI respectively, then connecting by using T4 ligase, introducing DH5 alpha competence through escherichia coli transformation, carrying out PCR verification by using pGal1-cx-F/pGal1-cx-R primers, and sending the strain corresponding to the positive strip to DNA sequencing to obtain a positive recombinant plasmid pESC-URA-CnVS, thus completing the construction of the pESC-URA-CnVS plasmid.

TABLE 1 primer sequence listing

Example 2: preparation of valance-western ene synthase mutants

The recombinant plasmid pESC-URA-CnVS constructed according to example 1 was designed and synthesized, respectively, using PCR techniques, to site-directed mutate the valencene synthase gene with primers that introduce mutations of G435V, G435N, G435S, T479G, T A, F488H, F488Q, F488Y, E489K, E489M, E489 4825 549I, M560L, M a.

TABLE 2 primer sequence listing

The PCR reaction system is as follows: prime STAR MAX (25. Mu.L), double distilled water (21. Mu.L), forward primer (1. Mu.L), reverse primer (1. Mu.L), template DNA (1. Mu.L), and the total system was 50. Mu.L.

The PCR amplification conditions were: pre-denaturation at 95℃for 3min; followed by 30 cycles (95 ℃ 15s,53 ℃ 5s;72 ℃ 15 s); and (5) continuing to extend at 72 ℃ for 5min, and finally preserving at 4 ℃.

The PCR product is purified after agarose gel electrophoresis verification, then the E.coli DH5 alpha competent cells are transformed after Dpn I digestion, competent cells are cultured overnight in LB solid medium (containing 50mg/L kana), plasmids are extracted after being selected and cloned in LB liquid medium containing 50mg/L kana, and positive mutant plasmids are obtained after sequencing correctly.

Example 3: construction of recombinant Saccharomyces cerevisiae expressing mutants

The wild-type and mutant plasmids constructed in example 1 and example 2, respectively, were introduced into a strain of Saccharomyces cerevisiae as Vanilla-Xieya-synthase expression vectors. The plasmid was added in an amount of 100 to 200ng BY using the Saccharomyces cerevisiae transformation kit Frozen-EZ Yeast Transformation II to Saccharomyces cerevisiae BY47419 (the genotype of the strain is listed in Table 3) as an example. The recombinant Saccharomyces cerevisiae cells transferred with plasmids are coated on SD-URA auxotroph screening solid culture medium plates, and cultured for 2-3 days in a 30 ℃ incubator, so as to obtain Saccharomyces cerevisiae valiene synthase mutant expression strains G435V, G435N, G435S, T4743 488H, F488 5226 488Y, E489K, E489 48M, E489C, E489I, M560L, M560A and valiene synthase wild type expression strains respectively.

TABLE 3 strains according to the invention

Example 4: vanilla production by shake flask fermentation of recombinant saccharomyces cerevisiae

The recombinant Saccharomyces cerevisiae strain constructed in example 3 was selected for shake flask fermentation, and a Vanilla alkene synthase wild type expression strain was used as a control. Single colonies of each strain were picked and cultured in a test tube containing 5mLYPD medium for 24 hours, and then 1mL of the bacterial solution was inoculated in a seed medium containing 20mLYPD medium for 12 hours. Adjusting the initial OD of the seed culture solution _600nm For 0.1, transfer and containing 50mLYPD liquid medium of 250mL conical flask fermentation, because the valance is volatile and insoluble in water, at 12h of the beginning of fermentation, adding 2mL n-dodecane for two-way fermentation reduces the valance loss, at 7 days of fermentation, detection of valance yield.

As shown in FIG. 2, the catalytic activity of the M560L mutant strain is improved, the yield of the valance cemeterol is obviously improved compared with that of the wild type expression strain, the yield is 5.01mg/L, and the improvement ratio is 30.1%.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.

SEQUENCE LISTING

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<211> 36

<212> DNA

<213> artificial sequence

<400> 23

tgtcaaggac tttatggacg aacaagctca cggtga 36

<210> 24

<211> 32

<212> DNA

<213> artificial sequence

<400> 24

gttcgtccat aaagtccttg acatcgtcga cc 32

<210> 25

<211> 36

<212> DNA

<213> artificial sequence

<400> 25

tgtcaaggac ttttgtgacg aacaagctca cggtga 36

<210> 26

<211> 32

<212> DNA

<213> artificial sequence

<400> 26

gttcgtcaca aaagtccttg acatcgtcga cc 32

<210> 27

<211> 29

<212> DNA

<213> artificial sequence

<400> 27

actttattga cgaacaagct cacggtgaa 29

<210> 28

<211> 41

<212> DNA

<213> artificial sequence

<400> 28

cgtgagcttg ttcgtcaata aagtccttga catcgtcgac c 41

<210> 29

<211> 41

<212> DNA

<213> artificial sequence

<400> 29

agagttatat ttttcttgtt gaacgacggt gacttgttca c 41

<210> 30

<211> 41

<212> DNA

<213> artificial sequence

<400> 30

gttcaacaag aaaaatataa ctctcatacc aacgttcaag t 41

<210> 31

<211> 41

<212> DNA

<213> artificial sequence

<400> 31

agagttatat ttttcgcttt gaacgacggt gacttgttca c 41

<210> 32

<211> 39

<212> DNA

<213> artificial sequence

<400> 32

tcaaagcgaa aaatataact ctcataccaa cgttcaagt 39

Claims

1. A valencene synthase mutant, characterized in that the valencene synthase mutant is: the valance western-style alkene synthase mutant M560L is formed by taking wild valance western-style alkene synthase with an amino acid sequence shown as SEQ ID No.2 as a template and mutating methionine at position 560 into leucine.

2. A coding gene encoding the valencene synthase mutant of claim 1.

3. A recombinant expression vector comprising a gene encoding the valencene synthase mutant of claim 2.

4. The recombinant expression vector according to claim 3, wherein the vector plasmid in the recombinant expression vector is pESC-URA and the promoter is Gal1 inducible promoter.

5. A recombinant genetically engineered bacterium comprising a gene encoding the valencene synthase mutant of claim 2.

6. The recombinant genetically engineered bacterium of claim 5, wherein the host bacterium employed is a yeast strain.

7. Use of a valencene synthase mutant according to claim 1, for the synthesis of valencene in yeast.

8. The application according to claim 7, characterized in that it comprises:

transferring a recombinant expression vector containing the coding gene of the valance cemetery synthase mutant of claim 2 into yeast host bacteria to construct a recombinant genetic engineering bacteria, and inoculating the recombinant genetic engineering bacteria into YPD liquid culture medium to ferment for 6-8 days to realize the synthesis of the valance cemetery.

9. Use according to claim 8, characterized in that 2-3 mL of n-dodecane is added for bi-directional fermentation at 10-12 h after the start of fermentation to reduce the volatilization losses of valencene.