CN103725716A - Method for biosynthesis of cis-form-3-hexenol employing neutral fat as substrate - Google Patents
Method for biosynthesis of cis-form-3-hexenol employing neutral fat as substrate Download PDFInfo
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- CN103725716A CN103725716A CN201310722845.7A CN201310722845A CN103725716A CN 103725716 A CN103725716 A CN 103725716A CN 201310722845 A CN201310722845 A CN 201310722845A CN 103725716 A CN103725716 A CN 103725716A
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- linolenic acid
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for the biosynthesis of cis-form-3-hexenol employing neutral fat as a substrate, and belongs to the field of bioengineering. A lipase gene which can be secreted out of a cell is expressed in a yeast cell or business-like lipase is directly added in the process of producing C3H employing the neutral fat as the substrate, the production of the C3H employing the neutral fat as a raw material is firstly achieved. By adopting the technical scheme disclosed by the invention, the problems that linolenic acid is poisonous to a host cell, and high in price are overcome, and the method has an industrial application prospect.
Description
Technical field
The present invention relates to a kind of method take neutral fat as substrate biosynthesizing cis-3-hexenol, belong to bioengineering field.
Background technology
Cis-3-hexenol (C3H, cis-3-hexen-1-ol), has another name called cis-blatter alcohol, leaf-alcohol, is a kind of colourless oil liquid, has the strong green meadow of just having been crossed by mowing and the smell of leaf.Most plants on a small quantity synthesizing cis-3-hexenol is used for luring many predatory insects.When plant meets with the predation of physical abuse or herbivore, can produce cis-3-hexenol and lure as a kind of " calling for help " signaling molecule natural enemy-predatory insect of herbivore.Cis-3-hexenol is applied to fruits and vegetables spices and perfume as a kind of important aromatic compound.But because the content of cis-3-hexenol in plant is low, correspondingly, the price of natural cis-3-hexenol is very expensive, the annual production of cis-3-hexenol is about 30 tons.
In plant, cis-3-hexenol is through these the 3 kinds of enzyme effects of lipoxygenase (LOX), fat hydroperoxide lyase (HPL) and ethanol dehydrogenase (ADH), to generate after elder generation take alpha-linolenic acid as substrate.Since the nineties in 20th century, the gene of encoding such enzymes is in succession cloned and is obtained from various plants, for these genes of heterogenous expression in microorganism (as intestinal bacteria and yeast) produce C3H, has created possibility.
One of key enzyme of synthetic C3H is lipoxygenase.Lipoxygenase is a class iron enzyme, can catalysis contain cis, and two oxygenations occur the polybasic unsaturated fatty acid in the lipid of cis-Isosorbide-5-Nitrae-pentadiene structure (as alpha-linolenic acid).The reaction of its catalysis is as follows: lipid acid+O
2=fatty acid peroxidase hydrogen thing.Fatty acid peroxidase hydrogen thing is the precursor of many important hormone (as prostanoid, lipoxin, jasmonic, traumatin) and spices/aroma substance mentioned above (as C3H, 1-OCOL).
Second enzyme in C3H building-up process is fat hydroperoxide lyase.When there is linolenic acid hydroperoxide substrate, fat hydroperoxide lyase can generate cis-3-hexenoic aldehyde by catalysis linolenic acid hydroperoxide, then by ethanol dehydrogenase, cis-3-hexenoic aldehyde is converted into C3H.
Alpha-linolenic acid (ALA) is necessary omega-3 fatty acid and the organic compound of seed (as salvia hispanica L, linseed oil, nut (it should be noted that walnut) and multiple Vegetable Oils).From the angle of structure, alpha-linolenic acid is named as entirely-18:3OMEGA-3.In physiology document, alpha-linolenic acid is named as 18:3(n-3).Alpha-linolenic acid is a kind of carboxylic acid with 18 carbon carbochains and 3 cis-double bondss.Wherein, first double bond position is in the 3rd carbon from methyl end (being n end), and therefore, alpha-linolenic acid is a kind of polynary unsaturated n-3(Ω-3) lipid acid, be polynary unsaturated n-6(Ω-6) lipid acid, the i.e. isomer of gamma-linolenic acid.
But, free fatty acids, as alpha-linolenic acid, not only costliness also has toxicity to microorganism, current biotechnology is synthesized and from economy and biology angle, become infeasible.And on the other hand, low price and microorganism is not had to toxic action of triglyceride level or other neutral lipids.Triglyceride level is by glycerine and 3 synthetic obtaining of lipid acid, and triglyceride level is diversified, and according to oily source, some triglyceride level are highly undersaturated, and some degrees of unsaturation are low.Saturated compound refers to that hydrogen is saturated, used in carbon atom can be in conjunction with the position of hydrogen atom all in conjunction with upper hydrogen atom.
But bread yeast can not discharge free C18:3 owing to lacking extracellular lipase from triglyceride level, thereby can not directly utilize neutral lipid to generate C3H.Therefore, develop a kind of new C3H biosynthetic means that does not affect microorganism cells (as bacterium and yeast) survival in the process of synthetic C3H very necessary.
Summary of the invention
The present invention wants technical solution problem to be to provide a kind of method take neutral fat as substrate biosynthesizing cis-3-hexenol, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 with the auxiliary overexpression lipoxygenase of lipase and fat hydroperoxide lyase, take neutral fat as raw material production C3H.
Described neutral fat is C18:3 ester or oleum lini.C18:3 ester is one or more the combination in alpha-linolenic acid methyl esters, Alpha-ethyl linolenate, alpha-linolenic acid triglycerides.Oleum lini can be one or more the combination deriving from salvia hispanica L, Chinese gooseberry seed, purple perilla, flax, cowberry, shepherd's purse, purslane, sea-buckthorn, hemp, Semen Brassicae campestris or soybean.
Described lipase is derived from fold candida.It can also be one or more the combination being derived from pig pancreas, fold candida, Fructus Hordei Germinatus or snow-white head mold.The gene of described lipase is the lipase gene from pig pancreas, Fructus Hordei Germinatus or snow-white head mold.
The gene order of the described lipoxygenase of encoding (LoxD-TP), as shown in SEQ ID NO.5, is used LoxD-TP gene amplification product
the processing of TA clone test kit, imports gene
recombinant cloning vector platform.
Encode the gene order of described fat hydroperoxide lyase (HPL) as shown in SEQ ID NO.3.
Described LoxD-TP and HPL gene react clone by LR respectively and enter Yeast expression carrier pAG424GPD-ccdB and pAG425GPD-ccdB(Life Technologies) and for transforming S.cerevisiae WAT11.
The construction process reference literature Urban P of described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P
450reductases with P
450cYP
73A5.J Biol Chem272 (31): the construction process of this cell in 19176-19186.
Describedly with lipase supplement production C3H, can realize by two kinds of modes, take neutral fat as substrate, external source is added the gene of lipase or overexpression coding lipase:
(1) external source is added lipase
Cultivate the S.cerevisiae WAT11 that collects overexpression LoxD-TP and HPL gene, cultivate the S.cerevisiae WAT11 that collects overexpression lipoxygenase and hydroperoxide lyase gene, cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mM pH6.8, then, add neutral fat and lipase reaction generation C3H.
Described cell suspending liquid OD
600=10.
Described lipase derives from fold candida (Candida rugosa), purchased from Sigma company, and production number L1754, addition is 3mg/10mL.
Described neutral fat is oleum lini or 0.1% methyl esters linolenic acid or 0.1% the ethyl ester linolenic acid of final concentration (v/v) oleum lini that is 0.5% or 5%.
(2) overexpression lipase gene LIP2
Be that the lipase gene as shown in SEQ ID NO.4 clone enters Yeast expression carrier pAG423GPD-ccdB by sequence, and transform in the S.cerevisiae WAT11 that enters overexpression lipoxygenase and hydroperoxide lyase gene; Cultivate recombinant bacterium, cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mM pH6.8, then, add neutral fat to reaction system, reaction generates C3H.
Described cell suspending liquid OD
600=10.
Described neutral fat is methyl esters linolenic acid or the ethyl ester linolenic acid of 0.1% methyl esters linolenic acid or 0.05% or 0.1% the ethyl ester linolenic acid of final concentration (v/v) oleum lini that is 2% or 0.05%.
Second technical problem that the present invention will solve is to provide a strain yeast gene engineering bacteria, take yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 as host, lipoxygenase and the gene order fat hydroperoxide lyase as SEQ ID NO.3 as shown in of expressing gene sequence as shown in SEQ ID NO.5.More specifically, be the gene of the described lipoxygenase of coding and fat hydroperoxide lyase to be reacted to clone by LR respectively enter Yeast expression carrier pAG424GPD-ccdB and pAG425GPD-ccdB, and transform S.cerevisiae WAT11.
Apply described yeast gene engineering bacteria and produce the method for C3H, to cultivate the S.cerevisiae WAT11 that collects overexpression LoxD-TP and HPL gene, cultivate the S.cerevisiae WAT11 that collects overexpression lipoxygenase and hydroperoxide lyase gene, cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mM pH6.8, then, add neutral fat and lipase reaction generation C3H.
Described cell suspending liquid OD
600=10.Described lipase derives from fold candida (Candida rugosa), purchased from Sigma company, and production number L1754, addition is 3mg/10mL.Described neutral fat is oleum lini or 0.1% methyl esters linolenic acid or 0.1% the ethyl ester linolenic acid of final concentration (v/v) oleum lini that is 0.5% or 5%.
The 3rd technical problem that the present invention will solve is to provide a strain yeast gene engineering bacteria, take yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 as host, lipoxygenase, gene order fat hydroperoxide lyase and the gene order lipase as SEQ ID NO.4 as shown in as SEQ ID NO.3 as shown in of expressing gene sequence as shown in SEQ ID NO.5.More specifically, be the gene of coding described lipoxygenase, fat hydroperoxide lyase and lipase to be reacted to clone by LR respectively enter Yeast expression carrier pAG424GPD-ccdB, pAG425GPD-ccdB and pAG423GPD-ccdB, and transform S.cerevisiae WAT11.
Applying described yeast gene engineering bacteria and produce the method for C3H, is to cultivate recombinant bacterium, and cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mMpH6.8, then, adds neutral fat to reaction system, and reaction generates C3H.
Described cell suspending liquid OD
600=10.Described neutral fat is methyl esters linolenic acid or the ethyl ester linolenic acid of 0.1% methyl esters linolenic acid or 0.05% or 0.1% the ethyl ester linolenic acid of final concentration (v/v) oleum lini that is 2% or 0.05%.
The suitability for industrialized production of current C3H is take the linolenic acid that dissociates as raw material, and linolenic acid is poisonous to host cell, and expensive.The present invention, by expressing and can be secreted into the lipase gene outside born of the same parents or directly add business-like lipase in reaction system in yeast cell, first realizes take neutral fat as raw material production C3H, has industrial applications prospect.
Accompanying drawing explanation
Fig. 1 is the structural formula of cis-3-hexenol.
Fig. 2 is linolenic structural formula.
Fig. 3 is the biosynthetic process of C3H.
Fig. 4 is that the free linolenic acid take 0.1% or 2% oleum lini are produced C3H as substrate through the S.cerevisiae Wat11 of overexpression LoxD-TP, the HPL of genetic modification, Lip2 cell.
Fig. 5 is through the S.cerevisiae Wat11 of overexpression LoxD-TP, the HPL of genetic modification cells produce C3H; CK: the C18:3 take 0.2% is substrate production C3H; P0.5: produce C3H take 0.5% oleum lini as substrate and from the lipase of pig pancreas; C0.5: produce C3H take 0.5% oleum lini as substrate and from the lipase of fold candida; W0.5: produce C3H take 0.5% oleum lini as substrate and from the lipase of Fructus Hordei Germinatus; R0.5: produce C3H take 0.5% oleum lini as substrate and from the lipase of Fructus Hordei Germinatus; P5: produce C3H take 5% oleum lini as substrate and from the lipase of pig pancreas; C5: produce C3H take 5% oleum lini as substrate and from the lipase of fold candida; W5: produce C3H take 5% oleum lini as substrate and from the lipase of Fructus Hordei Germinatus; R5: produce C3H take 5% oleum lini as substrate and from the lipase of Fructus Hordei Germinatus.
Fig. 6 produces C3H take C18:3, methyl esters linolenic acid or ethyl ester linolenic acid as substrate through the S.cerevisiae Wat11 of overexpression LoxD-TP, the HPL of genetic modification, Lip2 cell; The C18:3 of CK:0.1%; The methyl esters linolenic acid of ME-0.05:0.05%; The methyl esters linolenic acid of ME-0.1:0.1%; The ethyl ester linolenic acid of ET-0.05:0.05%; The ethyl ester linolenic acid of ET-0.1:0.1%.
Fig. 7 is the output through the S.cerevisiae Wat11 of the overexpression LoxD-TP of genetic modification and HPL cells produce C3H; CK-0.1: represent the 0.1% free linolenic acid of feeding; ME-0.1 and ET-0.1: represent respectively feed 0.1% methyl esters linolenic acid or ethyl ester linolenic acid; ME+LP and ET+LP, representative is added from the lipase of fold candida feed 0.1% methyl esters linolenic acid or ethyl ester linolenic acid simultaneously respectively.
Embodiment
Embodiment 1 produces C3H with the bread yeast WAT11 that expresses LoxD, HLP gene
(1) structure of expression vector
Lox gene (Lycopersicon esculentum, GenBank:U37840) sequence is as shown in SEQ ID NO.1, after being optimized based on Yeast genome codon, Lox gene obtains LoxD gene, sequence is as shown in SEQ ID NO.2, this gene is connected to pUC57 carrier (GenScript USA Inc., Piscataway, NJ, USA), obtain recombinant vectors pUC57-LoxD.
LoxD-TP gene order, as shown in SEQ ID NO.5, is to obtain from the amplification of pUC57-LoxD carrier by PCR, and the primer is LoxD-TPF:ATGATTTCAGAAAACTTGGTTAA and LoxD-R:CTAGATGGAAACACTATTAGGA.174 bases of LoxD-TP genetically deficient 5 ' end, the chloroplast(id) positioning sequence of 58 amino acid compositions of coding, by LoxD-TP gene amplification product clone into
tA cloning vector (Life Technologies).
The sequence of HPL gene is as shown in SEQ ID NO.3, based on GenBank:AF230372 design primer, HPL-F:atgaattctgctcctctatcaa and HPL-R:tcaactagcctttttcacagatgtga, extract tomato blade RNA(Solanum lycopersicum Moneymaker), by RT-PCR, obtain HPL gene.
LoxD-TP and HPL gene are reacted to clone by LR respectively and enter Yeast expression carrier pAG424GPD-ccdB and pAG425GPD-ccdB(Life Technologies) and for transforming S.cerevisiae WAT11.Transform forwarding method and see document (Chen DC, Yang BC, Kuo TT.One-step transformation of yeast in stationary phase.Current Genetics.1992,21:83-84).
The construction process reference literature Urban P of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P
450reductases with P
450cYP
73A5.J Biol Chem272 (31): 19176-19186.
(2) screening of recombinant bacterium and the Synthesis of C3H
By the S.cerevisiaeWAT11 of gained while overexpression LoxD-TP and HPL gene seed selection and growth in the synthetic medium (SD-Trp-Leu, U.S. Clontech company produces) of disappearance Trp and Leu.At the shaking tables of 30 ℃, 225 revs/min, cultivate two days later, 3000 leave 5 minutes collecting cells of the heart, by cell Eddy diffusion in the phosphoric acid buffer of 100mM pH6.8 to concentration OD
600=10, then, add the production for C3H to reaction system of 0.1% free linolenic acid (being dissolved in 95% alcohol), in 30 ℃, the shaking table reaction of 225rpm, generate C3H.In different time points, get 2ml reaction solution and add 2ml MTBE(Methyl tert-butyl ether) oscillation extraction C3H.Organic phase is measured C3H content for GC/MS, and GC/MS analyzes and adopts Shimadzu GC-2010 system, and this system is with GCMS-QP2010S detector, and analytical column is: Rtx-5MS(Thickness0.25u; Length30m; Diameter0.25mm), 265 ℃ of sample introduction temperature, sample introduction pattern is split, 50 ℃ of furnace temperature, thermograde: 0-4min50 ℃, 4-7.3min50 ℃-100 ℃, gradient 15,7.3-15.3min100 ℃-260 ℃, gradient 20, the retention time of C3H is approximately 5.1min.The results are shown in the interpretation of result of embodiment 2.
Because embodiment 1 gained recombinant bacterial strain can not utilize neutral fat, we continue to import lipase gene LIP2(GenBank:DQ831123 on this basis).The sequence of described lipase gene LIP2 is as shown in SEQ ID NO.4, it is the genomic dna (YeaStar Genomic DNA reagent closes) that extracts Yarrowia lipolytica ATCC90811, by pcr amplification, obtain, the primer is Lip2_F:ATGAAGCTTTCCACCATCCTTTTCA and Lip2_R:TTAGATACCACAGACACCCTCGGTGA, and PCR product connects
carrier, LIP2 gene reacts clone by LR and enters Yeast expression carrier pAG423GPD-ccdB and for transformed yeast.
The S.cerevisiaeWAT11 of overexpression LoxD-TP, HPL and LIP2 gene seed selection and growth (SD-Trp-Leu in the synthetic medium of disappearance Trp, Leu and His simultaneously, U.S. Clontech company produces), at the shaking tables of 30 ℃, 225 revs/min, cultivate two days later, 3000 leave 5 minutes collecting cells of the heart, by cell Eddy diffusion in the phosphoric acid buffer of 100mM pH6.8 to concentration OD
600=10, then, methyl esters linolenic acid (being dissolved in 95% alcohol) production for C3H to reaction system that to add final concentration be 0.1%, generates C3H in 30 ℃, the shaking table reaction of 225rpm.In different time points, get 2ml reaction solution and add 2ml MTBE(Methyl tert-butyl ether) oscillation extraction C3H.Organic phase is measured C3H content with GC/MS, and GC/MS analyzes and adopts Shimadzu GC-2010 system, and this system is with GCMS-QP2010S detector, and analytical column is: Rtx-5MS(Thickness0.25u; Length30m; Diameter0.25mm), 265 ℃ of sample introduction temperature, sample introduction pattern is split, 50 ℃ of furnace temperature, thermograde: 0-4min50 ℃, 4-7.3min50 ℃-100 ℃, gradient 15,7.3-15.3min100 ℃-260 ℃, gradient 20, the retention time of C3H is approximately 5.1min.
Simultaneously with not expressing the bacterial strain of LIP2 gene and adding methyl esters linolenic acid and do negative contrast, with not expressing the bacterial strain of LIP2 gene and adding free linolenic acid and make positive control.As shown in Figure 4, also express LoxD-TP and HPL when expressing Lip2 gene, S.cerevisiaeWAT11 can be directly take oleum lini as substrate synthetic C3H on a small quantity.
For only overexpression LoxD-TP and HPL, do not express the S.cerevisiaeWAT11 of Lip2, add the lipase from fold candida, can synthesize and obtain C3H(Fig. 5).Only the S.cerevisiaeWAT11 of overexpression LoxD-TP and HPL can not be with C18:3 ester, and C18:3 methyl esters (C18:3-ME) or C18:3 ethyl ester (C18:3-ET) obtain C3H for substrate.The endogenous lipase that this means S.cerevisiaeWAT11 can not be hydrolyzed C18:3 ester.On the contrary, when express again Lip2 in S.cerevisiaeWAT11, or while adding commercialization candida rugosa lipase, can synthesize C3H.
During by lipase, the effect of producing C3H take methyl esters linolenic acid as substrate is better than produces C3H take oleum lini as substrate with take free fatty acids as substrate.
The gene of coding lipase is from pig pancreas, fold candida, Fructus Hordei Germinatus, snow-white head mold, and selects in their combination.C18:3 ester is to select from alpha-linolenic acid methyl esters, Alpha-ethyl linolenate, alpha-linolenic acid triglycerides and their combination.C18:3 ester derives from oleum lini.C18:3 ester derives from the oil of selecting from salvia hispanica L, Chinese gooseberry seed, purple perilla, flax, cowberry, shepherd's purse, purslane, sea-buckthorn, hemp, Semen Brassicae campestris, soybean and their combination.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.In addition,, except special instruction, scientific terminology involved in the present invention has the general implication of understanding of those skilled in the art.Although have much, all can use the preferred above-mentioned embodiment of the present invention to material method similar, that be equal to of the present invention.
Other aspects, object of the present invention and advantage can obtain from the accompanying drawing of the present invention's record, claim.
Claims (26)
1. the method take neutral fat as substrate biosynthesizing cis-3-hexenol, be with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 of the auxiliary overexpression lipoxygenase of lipase and fat hydroperoxide lyase, take neutral fat as substrate, produce C3H.
2. method according to claim 1, is characterized in that, described neutral fat is C18:3 ester or oleum lini.
3. method according to claim 1, is characterized in that, described lipase is derived from fold candida.
4. method according to claim 1, is characterized in that, the gene order of the described lipoxygenase of encoding is as shown in SEQ ID NO.5.
5. method according to claim 1, is characterized in that, the gene order of the described fat hydroperoxide lyase of encoding is as shown in SEQ ID NO.3.
6. method according to claim 1, it is characterized in that, encode described lipoxygenase and hydroperoxide lyase gene reacts clone by LR respectively and enters Yeast expression carrier pAG424GPD-ccdB and pAG425GPD-ccdB, and for transforming S.cerevisiae WAT11.
7. according to the arbitrary described method of claim 1-6, it is characterized in that the described gene realization that can add by external source lipase or overexpression coding lipase with lipase supplement production C3H.
8. method according to claim 7, it is characterized in that, describedly by external source, add lipase and generate C3H and cultivate the S.cerevisiae WAT11 that collects overexpression lipoxygenase and hydroperoxide lyase gene, cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mM pH6.8, then, add neutral fat and lipase reaction generation C3H.
9. method according to claim 8, is characterized in that, described cell suspending liquid OD
600=10.
10. method according to claim 8, is characterized in that, the addition of described lipase is 3mg/10mL.
11. methods according to claim 8, is characterized in that, described neutral fat is oleum lini, addition 0.5%.
12. methods according to claim 8, is characterized in that, described neutral fat is oleum lini, addition 5%.
13. methods according to claim 8, is characterized in that, described neutral fat is methyl esters linolenic acid, addition 0.1%.
14. methods according to claim 8, is characterized in that, described neutral fat is ethyl ester linolenic acid, addition 0.1%.
15. methods according to claim 7, it is characterized in that, it is that the clone of the lipase gene as shown in SEQ ID NO.4 enters Yeast expression carrier pAG423GPD-ccdB by sequence that described overexpression lipase gene generates C3H, and transforms in the S.cerevisiae WAT11 that enters overexpression lipoxygenase and hydroperoxide lyase gene; Cultivate recombinant bacterium, cell Eddy diffusion is obtained to cell suspending liquid in the phosphoric acid buffer of 100mM pH6.8, then, add neutral fat to reaction system, reaction generates C3H.
16. methods according to claim 15, is characterized in that, described cell suspending liquid OD
600=10.
17. methods according to claim 15, is characterized in that, described neutral fat is oleum lini, addition 2%.
18. methods according to claim 15, is characterized in that, described neutral fat is methyl esters linolenic acid, addition 0.05%.
19. methods according to claim 15, is characterized in that, described neutral fat is methyl esters linolenic acid, addition 0.1%.
20. methods according to claim 15, is characterized in that, described neutral fat is ethyl ester linolenic acid, addition 0.05%.
21. methods according to claim 15, is characterized in that, described neutral fat is ethyl ester linolenic acid, addition 0.1%.
22. 1 Accharomyces cerevisiae genetic engineering bacteriums, it is characterized in that, take yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 as host, lipoxygenase and the gene order fat hydroperoxide lyase as SEQ ID NO.3 as shown in of expressing gene sequence as shown in SEQ ID NO.5.
23. according to the yeast gene engineering bacteria shown in claim 22, it is characterized in that, the gene of the described lipoxygenase of encoding and fat hydroperoxide lyase reacts clone by LR respectively and enters Yeast expression carrier pAG424GPD-ccdB and pAG425GPD-ccdB, and transforms S.cerevisiae WAT11.
24. 1 Accharomyces cerevisiae genetic engineering bacteriums, it is characterized in that, take yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) WAT11 as host, lipoxygenase, gene order fat hydroperoxide lyase and the gene order lipase as SEQ ID NO.4 as shown in as SEQ ID NO.3 as shown in of expressing gene sequence as shown in SEQ ID NO.5.
25. according to the yeast gene engineering bacteria shown in claim 24, it is characterized in that, the gene of described lipoxygenase, fat hydroperoxide lyase and lipase of encoding reacts clone by LR respectively and enters Yeast expression carrier pAG424GPD-ccdB, pAG425GPD-ccdB and pAG423GPD-ccdB, and transforms S.cerevisiae WAT11.
The arbitrary described yeast gene engineering bacteria of 26. claim 22-25 is applied to production cis-3-hexenol.
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| CN108719230A (en) * | 2018-04-26 | 2018-11-02 | 华南农业大学 | A kind of combination trapping combined method of diaphorina citri |
| CN110724696A (en) * | 2019-11-19 | 2020-01-24 | 安徽农业大学 | A kind of lipid hydroperoxide lyase and its gene and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108719230A (en) * | 2018-04-26 | 2018-11-02 | 华南农业大学 | A kind of combination trapping combined method of diaphorina citri |
| CN110724696A (en) * | 2019-11-19 | 2020-01-24 | 安徽农业大学 | A kind of lipid hydroperoxide lyase and its gene and application |
| CN110724696B (en) * | 2019-11-19 | 2021-01-12 | 安徽农业大学 | Lipid hydroperoxide lyase and gene and application thereof |
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