CN115044574A - Valencienene synthase mutant and application thereof in synthesizing Valencienene in yeast - Google Patents
Valencienene synthase mutant and application thereof in synthesizing Valencienene in yeast Download PDFInfo
- Publication number
- CN115044574A CN115044574A CN202210571548.6A CN202210571548A CN115044574A CN 115044574 A CN115044574 A CN 115044574A CN 202210571548 A CN202210571548 A CN 202210571548A CN 115044574 A CN115044574 A CN 115044574A
- Authority
- CN
- China
- Prior art keywords
- valencene
- synthase mutant
- yeast
- valencene synthase
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03073—Valencene synthase (4.2.3.73)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a valencene synthase mutant and application thereof in synthesizing valencene in yeast, wherein the valencene synthase mutant is as follows: the valencene synthase mutant M560L is formed by site-directed mutagenesis by using a wild valencene synthase of an amino acid sequence shown in SEQ ID No.2 as a template. Compared with wild valencene synthase, the valencene synthase mutant M560L provided by the invention has greatly improved catalytic activity, realizes high-yield preparation of valencene, improves yield of valencene by 30%, improves activity of heterologous enzyme catalysis substrate FPP by constructing valencene synthase mutant, and lays a foundation for constructing a yeast high-yield valencene cell factory.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a valencene synthase mutant and application thereof in synthesizing valencene in yeast.
Background
Valencene (valencene) is a sesquiterpene compound with the chemical name of (3R, 4aS, 5R) -4 o, 5-dimethyl-3-isopropenyl-1, 2, 3, 4, 4a, 5, 6, 7-octahydronaphthalene and the molecular formula C 15 H 24 Molecular weight 204.35. Is colorless liquid at normal temperature, has sweet orange oil fragrance, is insoluble in water and is easily soluble in organic solvent.
Valencene has a pleasant odor in orange and grapefruit juice and is commercially added as a flavor and fragrance in foods, cosmetics. Furthermore, valencene is also widely used in the pharmaceutical industry because of its specific odor activity and its recognition by the FDA as a GRAS substance, which proves safe for humans and mammals. In recent years, the research on related physiological effects of valencene is increasingly gaining attention, and the valencene is found to have antibacterial, anti-inflammatory, antiproliferative and apoptosis-promoting activities and has the function of inhibiting the growth of retinoblastoma cells. Furthermore, valencene has been found to have potential therapeutic effects on neuroinflammation and alzheimer's disease. At present, the method for industrially obtaining valencene mainly uses orange essential oil as a raw material and separates the valencene by processes of distillation, extraction and the like, but the valencene concentration in orange fruits is low (0.2% -0.5%), and the method has the defects of limited sources, long plant culture period, low efficiency and high cost, and cannot be used as an acquisition means for sustainable development. In addition, the chemical synthesis method can also realize the synthesis of valencene, but a large amount of toxic organic reagents are involved, so that the chemical synthesis method not only pollutes the environment, but also is harmful to human bodies, and can not reach the edible level. With the development of synthetic biology, the construction of microbial cell factories and the heterologous expression of related enzymes has become a promising approach for the production of valencene in yeast and E.coli.
To date, only a few valencene synthases have been identified and tested for (+) -valencene biosynthesis, including VvVal from vitas vinifera, Cstps1 from Citrus sinensis, GFTpsD from Citrus paradis, and CnVS from Callitropsis nootkatensis. Among these, CnVS has been shown to be the most stable one under catalytic pH and temperature conditions, which are desirable characteristics for applications in different hosts or under a variety of physiological conditions. The identified valencene synthase activity was too low compared to other sesquiterpene synthases, and overexpression of CnVS in yeast strain WAT11 produced only 1.36mg/L of valencene, which is a challenge due to microbial heterologous synthesis of metabolites with reduced catalytic efficiency compared to natural endogenous synthesis. Therefore, the improvement of the activity of valencene synthase is expected to play an important role in promoting the production of valencene.
Disclosure of Invention
The invention aims to provide a valencene synthase mutant and application of the valencene synthase mutant in yeast synthesis, so as to solve the problems of low valencene synthase activity and low valencene yield 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 which is: the valencene synthase mutant M560L is formed by mutating methionine 560 to leucine by using wild valencene synthase of amino acid sequence shown in SEQ ID No.2 as template.
According to a second aspect of the present invention, there is provided a coding gene encoding the valencene synthase mutant.
According to a third aspect of the present invention, there is provided a recombinant expression vector comprising a gene encoding the valencia ene synthase mutant.
According to a preferred embodiment of the present invention, the vector plasmid in the recombinant expression vector is pESC-URA, and the promoter is Gal1 inducible promoter. It is to be understood, however, that the present 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, the present invention randomly selects a Saccharomyces cerevisiae strain BY4741 as a host, but it should be understood that the present invention is not limited to Saccharomyces cerevisiae, and virtually all yeast strains such as Saccharomyces cerevisiae, Pichia pastoris, yarrowia lipolytica, and the like are possible. It should also be understood that, by way of example and not limitation, strains of Saccharomyces cerevisiae include: saccharomyces cerevisiae BY4741, Saccharomyces cerevisiae CENPK2-1C, Saccharomyces cerevisiae CENPK2-1D, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae C800 or Saccharomyces cerevisiae CP08 can also be selected.
According to a fifth aspect of the invention, there is provided a use of the valencene synthase mutant in the synthesis of valencene in yeast.
The application comprises the following steps: transferring a recombinant expression vector containing the coding gene of the valencene synthase mutant into yeast host bacteria to construct a recombinant genetic engineering bacterium, inoculating the recombinant genetic engineering bacterium into a YPD liquid culture medium, and fermenting for 6-8 days to realize the synthesis of valencene.
In the application, 2-3 mL of n-dodecane is preferably added for bidirectional fermentation in 10-12 h after fermentation is started, so that volatilization loss of valencene is reduced.
In the application, the valencene synthase mutant is M560L.
As described in the background section of the present invention, only a few valencene synthases have been identified and tested for (+) -valencene biosynthesis, but these valencene synthases have low enzymatic activity and do not allow the production of high yields of valencene. There is no report in the literature on rational modification of the sesquiterpene synthase CnVS, and the prior art usually optimizes the expression mode of CnVS only from the perspective of metabolism, such as screening promoter-terminator pairs, or increasing the copy number of CnVS in the genome. The key point of the invention is that the expression effect of heterologous enzymes is optimized and the CnVS activity is improved by rationally modifying the valencene synthase CnVS.
The invention obtains 13 valencia ene synthase mutants through the rational transformation, which comprises the following steps: a valencene synthase mutant G435V formed by mutating glycine (Gly) at position 435 to valine (Val) by using a wild valencene synthase of an amino acid sequence shown in SEQ ID No.2 as a template; a valencene synthase mutant G435N formed by mutating glycine (Gly) at position 435 to asparagine (Asn); a valencene synthase mutant G435S formed by mutating glycine (Gly) at position 435 to serine (Ser); a valencene synthase mutant T479G formed by mutating threonine (Thr) at the 479 th position into glycine (Gly); valencene synthase mutant F488H, which is formed by mutation of phenylalanine (Phe) at position 488 to histidine (His); valencene synthase mutant F488Q, which was mutated to phenylalanine (Phe) at position 488 to glutamine (Gln); valencene synthase mutant F488Y, which is formed by mutating phenylalanine (Phe) at position 488 to tyrosine (Tyr); a valency-senia alkene synthase mutant E489K formed by mutating glutamic acid (Glu) at the 489 th position into lysine (Lys); a valencene synthase mutant E489M formed by mutating glutamic acid (Glu) at the 489 th position into methionine; a valencene synthase mutant E489C formed by mutating glutamic acid (Glu) at position 489 to cysteine (Cys); a valencene synthase mutant E489I formed by mutating glutamic acid (Glu) at position 489 into isoleucine (IIe); a valencene synthase mutant M560L formed by mutating methionine (Met) at position 560 to leucine (Leu); a valencene synthase mutant M560A formed by mutating methionine (Met) at position 560 to alanine (Ala).
On the basis, the inventors further screen three key residue sites through rational design and modification, and find that the three key residue sites have a promoting effect on synthesis of valencene by a CnVS catalytic substrate FPP. And the mutant M560L with the best fermentation result improves the binding force of CnVS and the substrate through the analysis of acting force and the combination of binding free energy values.
According to the invention, 13 valencene synthase mutants G435V, G435N, G435S, T479G, F488H, F488Q, F488Y, E489K, E489M, E489C, E489I, M560L and M560A are obtained through rational design and transformation, and genetic engineering technology is used for further constructing the gene recombinant yeast strains, and finally experiments prove that compared with wild strains, the yield of valencene produced by the M560L mutant strains is improved by 30%.
In conclusion, the invention provides a valencene synthase mutant and application of valencene in yeast synthesis by means of genetic engineering and enzyme engineering, improves the yield of valencene by providing the valencene synthase mutant with improved catalytic activity, and creates conditions for industrial production of valencene.
Drawings
FIG. 1 shows a plasmid schematic of a gene expression vector for valencene synthase CnVS;
FIG. 2 shows a plot of the Valencia yield of a genetically recombinant yeast strain;
Detailed Description
The invention is further described below with reference to examples and figures, but the embodiments of the invention are not limited thereto.
The experimental procedures for specifying specific experimental conditions in the following examples are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are commercially available reagents and materials.
The preparation method of the solution comprises the following steps:
LB (Luria-Bertani) medium: 10g/L Peptone (Peptone), 5g/L Yeast extract (Yeast extract), 10g/L NaCl, 2% (2g/100mL) agar powder added in the solid medium, and high temperature and high pressure sterilization at 121 ℃ for 20 min.
Ypd (Yeast Extract Peptone Dextrose): 20g/L glucose (Dextrose), 10g/L Yeast extract (Yeast extract), 20g/L Peptone (Peptone), 2% (2g/100mL) agar powder added to the solid medium, and sterilized at 115 ℃ under high pressure and high temperature for 20 min.
SD-URA auxotrophic 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 a 5M sodium hydroxide solution, subpackaging 95mL to 250mL conical bottles, adding 2% (2g/100mL) of agar powder, sterilizing at the high temperature and the high pressure of 115 ℃ for 20min, melting when in use, cooling to about 50 ℃, and adding 5mL of 40% sterile glucose solution.
2 × PrimeSTAR MAX for PCR was purchased from Takara; hieff clone One Step Cloning Kit from YEASEN; rTaq enzyme was purchased from Thermo Scientific; the Frozen-EZ Yeast Transformation II Kit was purchased from Epigentics.
The yeast fermentation and product extraction method comprises the following steps: selecting a monoclonal yeast colony on a plate, culturing for 24h in a test tube containing 5mL of YPD liquid culture medium at 220rpm of a shaking table at 30 ℃, then transferring the colony into a 250mL conical flask containing 20mL of YPD culture medium at 1-2% of inoculum size, culturing for 12h at 220rpm of the shaking table at 30 ℃, sampling and detecting the OD600 of thalli, calculating the inoculum size, adjusting the initial OD600 to be 0.1, fermenting three parallel samples of each strain in a shaking flask, fermenting for 7 days, sampling once every 48h, and adding 2% n-dodecane to cover in the shaking flask at 60h of fermentation. And after the fermentation is finished, standing and shaking the flask, sucking out the organic phase into an EP (EP) tube by using a gun head after the organic phase and the water phase are layered, centrifuging for 10min at1,2000 rpm, taking out the upper organic phase, adding a proper amount of anhydrous sodium sulfate, fully oscillating and uniformly mixing, centrifuging for 10min at1,2000 rpm, sucking the upper organic phase, filtering through a membrane into a gas chromatography bottle, and detecting as a GC sample.
Valencia alkene gas phase detection method
Valencene standard bottles were purchased from Sigma, valencene standard solutions: weighing 10mg of valencene standard substance, dissolving in n-dodecane solution, and fixing the volume to 1mL, wherein the concentration of the mother solution of the standard substance is 10 mg/mL.
The method adopts gas chromatography to carry out qualitative and quantitative analysis on valencene, and the detection conditions are as follows:
a chromatographic column: HP-5(30m x 0.32mm, 0.25 μm, Agilent, USA); the temperature of a sample inlet is 250 ℃; temperature programming: the column temperature was started at 100 ℃ for 4min, then increased to 250 ℃ at a rate of 10 ℃/min for 5 min.
Hydrogen Flame Ionization Detector (FID) temperature: 280 ℃; the split ratio is 5:1, and the split flow is 6 mL/min; air flow rate: 300 mL/min; hydrogen flow rate: 30 mL/min; flow rate of tail gas blowing(N 2 ): 15 mL/min; average linear velocity: 24.802 cm/s; pressure: 7.1413 psi; sample introduction amount: 1 μ L.
Example 1: construction of Valencia ene Gene expression cassette
Firstly, GenScript company is entrusted to synthesize codon-optimized CnVS (the codon-optimized nucleotide sequence is shown as a sequence SEQ ID No. 1) from Callitropsis nootkatensis, an expression vector pESC-URA unloaded plasmid is selected, CnVs-BamHI-F/CnVs-XhoI-R is selected to amplify CnVs gene, and the CnVs gene is recovered and purified after agarose gel electrophoresis verification. And carrying out double enzyme digestion on empty plasmid pESC-URA and CnVS gene fragments by using restriction endonucleases BamHI and XhoI respectively, then adopting T4 ligase to connect, introducing DH5 alpha competence through escherichia coli transformation, using pGal1-cx-F/pGal1-cx-R primer PCR verification, sending a strain corresponding to a positive band to DNA sequencing to obtain a positive recombinant plasmid pESC-URA-CnVS, and completing the construction of the pESC-URA-CnVS plasmid.
TABLE 1 primer sequence Listing
Example 2: preparation of valencene synthase mutants
Using PCR technology, the recombinant plasmid pESC-URA-CnVS constructed according to example 1 was designed and synthesized to introduce primers for mutations of G435V, G435N, G435S, T479G, T479A, F488H, F488N, F488Q, F488Y, E489K, E489M, E489C, E489I, M560L, M560A, respectively, to perform site-directed mutagenesis of the valencene synthase gene.
TABLE 2 primer sequence Listing
The PCR reaction systems are 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), total 50. mu.L.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; followed by 30 cycles (95 ℃ for 15s, 53 ℃ for 5 s; 72 ℃ for 15 s); extension was continued for 5min at 72 ℃ and finally stored at 4 ℃.
And (3) purifying the PCR product after agarose gel electrophoresis verification, then transforming Escherichia coli DH5 alpha competence after digestion by Dpn I, culturing competent cells in an LB solid culture medium (containing 50mg/L kana) overnight, selecting and cloning in an LB liquid culture medium containing 50mg/L kana to culture, extracting plasmids, and obtaining positive mutant plasmids after correct sequencing.
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 s.cerevisiae strain as valencia ene synthase expression vectors. For example, the strain was transformed into Saccharomyces cerevisiae BY47419 (the strain genotype is shown in Table 3) using Saccharomyces cerevisiae Transformation kit Frozen-EZ Yeast Transformation II, and the amount of plasmid added was 100 to 200 ng. And (3) coating the recombinant saccharomyces cerevisiae cells transferred with the plasmid on an SD-URA auxotrophic screening solid medium plate, and culturing for 2-3 days at the temperature of 30 ℃ in an incubator to respectively obtain saccharomyces cerevisiae valencene synthase mutant expression strains G435V, G435N, G435S, T479G, F488H, F488Q, F488Y, E489K, E489M, E489C, E489I, M560L, M560A and valencene synthase wild-type expression strains.
TABLE 3 strains to which the present invention relates
Example 4: production of valencene by recombinant saccharomyces cerevisiae shake flask fermentation
Picking the recombinant Saccharomyces cerevisiae strain constructed in example 3 for shake flask fermentationThe varenidene synthase wild-type expression strain served as a control. Single colony of each strain is picked up and cultured in a test tube containing 5mLYPD medium for 24h, and then 1mL of bacterial liquid is transferred to a seed culture medium containing 20mLYPD medium for 12 h. Adjusting the initial OD of the seed culture solution 600nm 0.1, the fermentation was transferred to a 250mL conical flask containing 50mLYPD liquid medium, and since valencene is volatile and insoluble in water, the volatilization loss of valencene was reduced by adding 2mL of n-dodecane to the 12h of the initial fermentation for bidirectional fermentation, and the yield of valencene was measured at the 7 th day of fermentation.
As shown in FIG. 2, the M560L mutant strain has improved catalytic activity, and the yield of valencene is obviously improved compared with that of a wild-type expression strain, the yield is 5.01mg/L, and the improvement ratio is 30.1%.
The above 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 may be made to the above-described embodiment of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in the conventional technical content.
SEQUENCE LISTING
<110> university of east China's college of science
<120> varenidene synthase mutant and application thereof in synthesizing varenidene in yeast
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 1770
<212> DNA
<213> Callitropsis nootkatensis
<400> 1
atggctgaaa tgtttaacgg caactcttcc aacgatggtt cctcatgtat gccagtcaag 60
gacgctttga gaagaactgg taaccaccac ccaaacttgt ggactgatga tttcattcaa 120
tccttgaact ctccatactc cgactcttct taccacaagc acagagaaat cttgattgat 180
gaaatcagag acatgttctc caatggtgaa ggtgacgaat tcggtgtctt ggaaaacatc 240
tggttcgtcg atgttgttca aagattgggt attgacagac acttccaaga agaaatcaag 300
actgctttgg actacatcta caagttctgg aaccacgatt ccatcttcgg tgacctcaac 360
atggttgctt taggtttccg tatcttgaga ttgaatagat acgttgcctc ttctgacgtt 420
ttcaagaaat tcaagggtga agaaggtcaa ttctctggtt tcgaatcttc agaccaagat 480
gccaagttgg aaatgatgct taacttgtac aaggctagcg aattggactt cccagacgaa 540
gacattttga aagaagccag agcttttgct tccatgtact tgaagcacgt catcaaggaa 600
tatggtgata tccaagaatc taagaaccca ttgttaatgg aaattgaata cactttcaag 660
tacccatgga gatgtcgttt gccaaggtta gaagcttgga acttcatcca catcatgaga 720
caacaagact gtaacatttc cttggctaac aacttgtaca agatcccaaa gatttacatg 780
aagaagattt tggagttagc catcttggat tttaatatcc tacaatctca acaccaacac 840
gaaatgaagt tgatttctac ctggtggaag aactcctctg ctattcaatt ggacttcttc 900
agacacagac atatcgaatc ctacttctgg tgggcttctc cattattcga accagaattt 960
agcacctgta gaatcaactg tactaaattg tccaccaaga tgttcttgtt ggatgacatt 1020
tacgacactt acggtaccgt tgaagaattg aagccattca ccactaccct aaccagatgg 1080
gatgtctcta ctgttgacaa tcatccagat tatatgaaga ttgctttcaa cttctcttac 1140
gaaatctaca aagaaattgc ttctgaagct gaaagaaagc atggtccttt cgtttacaag 1200
tatttacaat cttgttggaa atcttacatt gaagcttaca tgcaagaagc agaatggatt 1260
gcctctaacc atattccagg tttcgatgaa tacttgatga acggtgttaa gtcttctggt 1320
atgagaatct tgatgattca cgctttaatc ttgatggaca ccccattgtc tgatgaaatt 1380
ctagaacaac ttgacatacc ttcctccaag tcccaagctt tgttgtcttt gatcactcgt 1440
ttggtcgacg atgtcaagga ctttgaagac gaacaagctc acggtgaaat ggcctcctct 1500
atcgaatgtt acatgaaaga taaccatgga tcgacacgtg aagatgcttt gaactactta 1560
aagatcagaa ttgaatcctg cgtccaagaa ctaaacaagg aattgttgga accatctaac 1620
atgcacggtt ccttcagaaa cttatacttg aacgttggta tgagagttat atttttcatg 1680
ttgaacgacg gtgacttgtt cactcactct aacagaaagg aaatccaaga cgctattacc 1740
aagttctttg tcgaaccaat cattccatga 1770
<210> 2
<211> 589
<212> PRT
<213> Callitropsis nootkatensis
<400> 2
Met Ala Glu Met Phe Asn Gly Asn Ser Ser Asn Asp Gly Ser Ser Cys
1 5 10 15
Met Pro Val Lys Asp Ala Leu Arg Arg Thr Gly Asn His His Pro Asn
20 25 30
Leu Trp Thr Asp Asp Phe Ile Gln Ser Leu Asn Ser Pro Tyr Ser Asp
35 40 45
Ser Ser Tyr His Lys His Arg Glu Ile Leu Ile Asp Glu Ile Arg Asp
50 55 60
Met Phe Ser Asn Gly Glu Gly Asp Glu Phe Gly Val Leu Glu Asn Ile
65 70 75 80
Trp Phe Val Asp Val Val Gln Arg Leu Gly Ile Asp Arg His Phe Gln
85 90 95
Glu Glu Ile Lys Thr Ala Leu Asp Tyr Ile Tyr Lys Phe Trp Asn His
100 105 110
Asp Ser Ile Phe Gly Asp Leu Asn Met Val Ala Leu Gly Phe Arg Ile
115 120 125
Leu Arg Leu Asn Arg Tyr Val Ala Ser Ser Asp Val Phe Lys Lys Phe
130 135 140
Lys Gly Glu Glu Gly Gln Phe Ser Gly Phe Glu Ser Ser Asp Gln Asp
145 150 155 160
Ala Lys Leu Glu Met Met Leu Asn Leu Tyr Lys Ala Ser Glu Leu Asp
165 170 175
Phe Pro Asp Glu Asp Ile Leu Lys Glu Ala Arg Ala Phe Ala Ser Met
180 185 190
Tyr Leu Lys His Val Ile Lys Glu Tyr Gly Asp Ile Gln Glu Ser Lys
195 200 205
Asn Pro Leu Leu Met Glu Ile Glu Tyr Thr Phe Lys Tyr Pro Trp Arg
210 215 220
Cys Arg Leu Pro Arg Leu Glu Ala Trp Asn Phe Ile His Ile Met Arg
225 230 235 240
Gln Gln Asp Cys Asn Ile Ser Leu Ala Asn Asn Leu Tyr Lys Ile Pro
245 250 255
Lys Ile Tyr Met Lys Lys Ile Leu Glu Leu Ala Ile Leu Asp Phe Asn
260 265 270
Ile Leu Gln Ser Gln His Gln His Glu Met Lys Leu Ile Ser Thr Trp
275 280 285
Trp Lys Asn Ser Ser Ala Ile Gln Leu Asp Phe Phe Arg His Arg His
290 295 300
Ile Glu Ser Tyr Phe Trp Trp Ala Ser Pro Leu Phe Glu Pro Glu Phe
305 310 315 320
Ser Thr Cys Arg Ile Asn Cys Thr Lys Leu Ser Thr Lys Met Phe Leu
325 330 335
Leu Asp Asp Ile Tyr Asp Thr Tyr Gly Thr Val Glu Glu Leu Lys Pro
340 345 350
Phe Thr Thr Thr Leu Thr Arg Trp Asp Val Ser Thr Val Asp Asn His
355 360 365
Pro Asp Tyr Met Lys Ile Ala Phe Asn Phe Ser Tyr Glu Ile Tyr Lys
370 375 380
Glu Ile Ala Ser Glu Ala Glu Arg Lys His Gly Pro Phe Val Tyr Lys
385 390 395 400
Tyr Leu Gln Ser Cys Trp Lys Ser Tyr Ile Glu Ala Tyr Met Gln Glu
405 410 415
Ala Glu Trp Ile Ala Ser Asn His Ile Pro Gly Phe Asp Glu Tyr Leu
420 425 430
Met Asn Gly Val Lys Ser Ser Gly Met Arg Ile Leu Met Ile His Ala
435 440 445
Leu Ile Leu Met Asp Thr Pro Leu Ser Asp Glu Ile Leu Glu Gln Leu
450 455 460
Asp Ile Pro Ser Ser Lys Ser Gln Ala Leu Leu Ser Leu Ile Thr Arg
465 470 475 480
Leu Val Asp Asp Val Lys Asp Phe Glu Asp Glu Gln Ala His Gly Glu
485 490 495
Met Ala Ser Ser Ile Glu Cys Tyr Met Lys Asp Asn His Gly Ser Thr
500 505 510
Arg Glu Asp Ala Leu Asn Tyr Leu Lys Ile Arg Ile Glu Ser Cys Val
515 520 525
Gln Glu Leu Asn Lys Glu Leu Leu Glu Pro Ser Asn Met His Gly Ser
530 535 540
Phe Arg Asn Leu Tyr Leu Asn Val Gly Met Arg Val Ile Phe Phe Met
545 550 555 560
Leu Asn Asp Gly Asp Leu Phe Thr His Ser Asn Arg Lys Glu Ile Gln
565 570 575
Asp Ala Ile Thr Lys Phe Phe Val Glu Pro Ile Ile Pro
580 585
<210> 3
<211> 32
<212> DNA
<213> Artificial sequence
<400> 3
atggatccat ggctgaaatg tttaacggca ac 32
<210> 4
<211> 36
<212> DNA
<213> Artificial sequence
<400> 4
tactcgagtc atggaatgat tggttcgaca aagaac 36
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<400> 5
gccccacaaa ccttcaaatg a 21
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence
<400> 6
gggacctaga cttcaggttg t 21
<210> 7
<211> 41
<212> DNA
<213> Artificial sequence
<400> 7
atgaacgttg ttaagtcttc tggtatgaga atcttgatga t 41
<210> 8
<211> 42
<212> DNA
<213> Artificial sequence
<400> 8
agaagactta acaacgttca tcaagtattc atcgaaacct gg 42
<210> 9
<211> 43
<212> DNA
<213> Artificial sequence
<400> 9
tgatgaacaa cgttaagtct tctggtatga gaatcttgat gat 43
<210> 10
<211> 42
<212> DNA
<213> Artificial sequence
<400> 10
agaagactta acgttgttca tcaagtattc atcgaaacct gg 42
<210> 11
<211> 43
<212> DNA
<213> Artificial sequence
<400> 11
tgatgaactc tgttaagtct tctggtatga gaatcttgat gat 43
<210> 12
<211> 42
<212> DNA
<213> Artificial sequence
<400> 12
agaagactta acagagttca tcaagtattc atcgaaacct gg 42
<210> 13
<211> 34
<212> DNA
<213> Artificial sequence
<400> 13
gtctttgatc tgtcgtttgg tcgacgatgt caag 34
<210> 14
<211> 36
<212> DNA
<213> Artificial sequence
<400> 14
accaaacgac agatcaaaga caacaaagct tgggac 36
<210> 15
<211> 33
<212> DNA
<213> Artificial sequence
<400> 15
tcaaggacca cgaagacgaa caagctcacg gtg 33
<210> 16
<211> 35
<212> DNA
<213> Artificial sequence
<400> 16
ttcgtcttcg tggtccttga catcgtcgac caaac 35
<210> 17
<211> 33
<212> DNA
<213> Artificial sequence
<400> 17
tgtcaaggac caagaagacg aacaagctca cgg 33
<210> 18
<211> 31
<212> DNA
<213> Artificial sequence
<400> 18
tcgtcttctt ggtccttgac atcgtcgacc a 31
<210> 19
<211> 28
<212> DNA
<213> Artificial sequence
<400> 19
aggactatga agacgaacaa gctcacgg 28
<210> 20
<211> 36
<212> DNA
<213> Artificial sequence
<400> 20
cttgttcgtc ttcatagtcc ttgacatcgt cgacca 36
<210> 21
<211> 38
<212> DNA
<213> Artificial sequence
<400> 21
gatgtcaagg actttaagga cgaacaagct cacggtga 38
<210> 22
<211> 30
<212> DNA
<213> Artificial sequence
<400> 22
tcgtccttaa agtccttgac atcgtcgacc 30
<210> 23
<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 (9)
1. A valencene synthase mutant, wherein the valencene synthase mutant is: the Valencien synthase mutant M560L is formed by mutating the 560 th methionine to leucine by using the wild Valencien synthase of the amino acid sequence shown in SEQ ID No.2 as a template.
2. A coding gene encoding the valencene synthase mutant according to claim 1.
3. A recombinant expression vector comprising a gene encoding the valencia ene synthase mutant of claim 2.
4. The recombinant expression vector of 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 according to claim 2.
6. The recombinant genetically engineered bacterium of claim 5, wherein the host bacterium is a yeast strain.
7. Use of the valencene synthase mutant according to claim 1 for the synthesis of valencene in yeast.
8. The application according to claim 7, wherein the application comprises:
transferring a recombinant expression vector containing the coding gene of the valencene synthase mutant as claimed in claim 2 into yeast host bacteria to construct a recombinant genetic engineering bacterium, inoculating the recombinant genetic engineering bacterium into a YPD liquid culture medium, and fermenting for 6-8 days to realize the synthesis of valencene.
9. The use of claim 8, wherein 2-3 mL of n-dodecane is added 10-12 h after the start of fermentation for bidirectional fermentation to reduce volatilization loss of valencene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210571548.6A CN115044574B (en) | 2022-05-24 | 2022-05-24 | Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210571548.6A CN115044574B (en) | 2022-05-24 | 2022-05-24 | Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115044574A true CN115044574A (en) | 2022-09-13 |
| CN115044574B CN115044574B (en) | 2023-07-14 |
Family
ID=83158722
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210571548.6A Active CN115044574B (en) | 2022-05-24 | 2022-05-24 | Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115044574B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150007368A1 (en) * | 2013-03-14 | 2015-01-01 | Dayal Saran | Valencene Synthase Polypeptides, Encoding Nucleic Acid Molecules And Uses Thereof |
| CN110117551A (en) * | 2019-04-04 | 2019-08-13 | 华南理工大学 | The saccharomyces cerevisiae engineered yeast and its construction method of production Valencia alkene and application |
| CN114480512A (en) * | 2021-12-13 | 2022-05-13 | 华南理工大学 | Application of oxidoreductase and mutant thereof in biosynthesis of nootkatone |
-
2022
- 2022-05-24 CN CN202210571548.6A patent/CN115044574B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150007368A1 (en) * | 2013-03-14 | 2015-01-01 | Dayal Saran | Valencene Synthase Polypeptides, Encoding Nucleic Acid Molecules And Uses Thereof |
| CN110117551A (en) * | 2019-04-04 | 2019-08-13 | 华南理工大学 | The saccharomyces cerevisiae engineered yeast and its construction method of production Valencia alkene and application |
| CN114480512A (en) * | 2021-12-13 | 2022-05-13 | 华南理工大学 | Application of oxidoreductase and mutant thereof in biosynthesis of nootkatone |
Non-Patent Citations (3)
| Title |
|---|
| BEEKWILDER,J.等: "terpene synthase [Callitropsis nootkatensis]", NCBI GENBANK, pages 1 * |
| J GUO等: "Combination of protein engineering and metabolic engineering to enhance(+)-nootkatone production in saccharomyces cerevisiae", FOOD BIOENGINEERING, pages 192 - 202 * |
| 陈和锋;朱晁谊;李爽;: "产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化", 生物技术通报, no. 01, pages 209 - 219 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115044574B (en) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111051503B (en) | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol | |
| CN107723307A (en) | A kind of method and its application for efficiently preparing the epimerase of D psicoses 3 | |
| CN110607286B (en) | Application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in synthesis of ergothioneine | |
| CN112251427B (en) | Sesquiterpene synthase IlTPS1, coding nucleotide sequence and application thereof | |
| CN106350565A (en) | Production method of rare ginsenoside Rh2 | |
| CN114480512B (en) | Application of oxidoreductase and mutant thereof in biosynthesis of nootkatone | |
| CN112159769B (en) | Genetically engineered bacterium for producing L-menthol and construction method and application thereof | |
| CN113755354A (en) | Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application thereof | |
| CN113444654B (en) | Saccharomyces cerevisiae engineering bacterium for producing dihydroartemisinic acid and construction method and application thereof | |
| CN109913380B (en) | Recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and construction method and application thereof | |
| CN113897325B (en) | Recombinant escherichia coli for producing salidroside as well as construction method and application thereof | |
| CN111334522B (en) | Recombinant saccharomyces cerevisiae for producing ambergris alcohol and construction method | |
| CN112175848A (en) | Patchouli alcohol production yeast strain and construction method and application thereof | |
| CN115044574B (en) | Vanilla alkene synthase mutant and application thereof in synthesizing valansia alkene in yeast | |
| CN115786389A (en) | Saccharomyces cerevisiae engineering bacterium for high-yield santalol as well as construction method and application thereof | |
| CN105802926B (en) | 2 α of triterpene-hydroxylase MAA45 and its relevant biological material and they preparation hawthorn acid and Corosolic acid in application | |
| CN114277024B (en) | Novel triterpene synthase and application thereof | |
| CN110004099A (en) | A kind of fermentation method for producing of rhodioside | |
| CN113817757A (en) | Recombinant yeast engineering strain for producing cherry glycoside and application | |
| CN113604448B (en) | Polyketide synthase Preu3 and application thereof in preparation of 2,4-dihydroxy-3,6-dimethylbenzoic acid | |
| CN113801861B (en) | Flavone 4' -O-methyltransferase from Korean epimedium and application thereof | |
| KR101826927B1 (en) | A microorganism having enhanced levansucrase productivity and a method of producing levan using the microorganism | |
| CN114806910B (en) | Candida tropicalis engineering bacteria for producing L-limonene and construction method thereof | |
| CN114908067B (en) | Application of oxymethyl transferase fsr2 in catalyzing oxygenation methylation of kaempferol 4' hydroxyl | |
| CN113278597B (en) | Novel short side chain fatty acid CoA ligase and application thereof in preparation of patchoulenone |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |