CN107815424A - A kind of Yarrowia lipolytica gene engineering bacteria for producing limonene and its application - Google Patents
A kind of Yarrowia lipolytica gene engineering bacteria for producing limonene and its application Download PDFInfo
- Publication number
- CN107815424A CN107815424A CN201610817882.XA CN201610817882A CN107815424A CN 107815424 A CN107815424 A CN 107815424A CN 201610817882 A CN201610817882 A CN 201610817882A CN 107815424 A CN107815424 A CN 107815424A
- Authority
- CN
- China
- Prior art keywords
- gene
- yarrowia lipolytica
- plasmid
- limonene
- optimization
- 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
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
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- 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
- C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
-
- 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)
- C12Y402/0302—(R)-Limonene synthase (4.2.3.20)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of Yarrowia lipolytica gene engineering bacteria for producing limonene and its application.The genetic engineering bacterium is the gene LS containing optimization and gene NDPS1 recombinant vector to be converted into uracil and leucine auxotroph Yarrowia lipolytica (Yarrowia lipolytica) is built-up, and the gene LS and NDPS1 of optimization nucleotide sequence is respectively as shown in sequence table SEQ ID No.1~2.Gene HMG1 and gene ERG12 are overexpressed simultaneously on this basis, the very high throughput that limonene can be made to reach 0.67mg/g DCW.In addition, the pyruvic acid and the concentration of dodecane that add during by being fermented to the genetic engineering bacterium two-phase select, the yield of limonene is further improved.The genetic engineering bacterium can be used for large-scale commercial production, have good prospects.
Description
Technical field
The invention belongs to genetic engineering field, more particularly to a kind of Yarrowia lipolytica gene engineering bacteria for producing limonene
And its application.
Background technology
Limonene is prevalent in natural plants, is obtained because of the citrus aromes with pleasant in food essence
It is widely applied.In perfume industry, limonene can be used as preparing artificial flores aurantii, sweet tea flower, lemon, the original of bergamot oil in itself
Material, can act also as fresh head perfume (or spice) spices.Because limonene possesses pharmacological activity, it also has in field of medicaments widely should
With.Limonene is also a kind of substitute of jet fuel simultaneously.Itself or many important aromatic substances and medicine are such as purple in addition
The precursor of Soviet Union's alcohol, carvol and menthol etc..
The production method of limonene mainly has two kinds of natural extraction method and microbe fermentation method.Due to from trade waste mandarin orange
The limonene extracted in orange peel has residues of pesticides unavoidably, is not suitable as food additives, therefore microbial fermentation production lemon
Lemon alkene has obvious advantage.In particular with the fast development of biotechnology, using microbial fermentation produce limonene and its
Derivative turns into one of focus studied at present.
Limonene typically is produced by being exogenously introduced limonene synthase gene biological fermentation process, needs high yield lemon badly at present
The genetic engineering bacterium of alkene.
The content of the invention
The technical problem to be solved in the present invention is to overcome the genetic engineering bacterium of shortage high yield limonene in the prior art
Deficiency, there is provided it is a kind of produce limonene Yarrowia lipolytica gene engineering bacteria and its application.
Inventor is had found after paying creative work, and lemon is produced for Yarrowia lipolytica after introducing limonene synthetic gene
The metabolic pathway of lemon alkene, by two limonene synthesis related genes --- i.e. from tomato, can by isopentenyl pyrophosphate and
The condensation of methacrylic pyrophosphoric acid is the gene NDPS1 of neryl diphosphonic acid；With from wrinkled giant hyssop, can be different by neryl diphosphonic acid
After structure optimizes respectively for the gene LS of limonene, the genetic transformation Yarrowia lipolytica of the two optimizations can be made into its production
Limonene.HMG-CoA is reduced to first hydroxyl penta by inventor it has furthermore been found that being overexpressed on the basis of above-mentioned two gene is optimized
The gene HMG1 of acid, and be overexpressed the gene ERG12 that mevalonate is mevalonic acid -5- phosphoric acid simultaneously, can be greatly
Improve to amplitude the yield of limonene (referring to Fig. 1).In addition, inventor has found that the addition of specific dodecane can be in two-phase
Yarrowia lipolytica high yield limonene is advantageously promoted in fermentation.
Technical scheme provided by the invention is as follows：
One of technical scheme is：It is a kind of produce limonene Yarrowia lipolytica gene engineering bacteria, its be by containing
There are the gene LS of optimization and the gene NDPS1 of optimization recombinant vector conversion uracil and leucine auxotroph solution fat Ye Shi
Yeast (Yarrowia lipolytica) is built-up, the gene LS of optimization nucleotide sequence such as sequence table SEQ ID
Shown in No.1；The gene NDPS1 of optimization nucleotide sequence is as shown in sequence table SEQ ID No.2.
In the present invention, uracil and the leucine auxotroph Yarrowia lipolytica (Yarrowialipolytica)
For this area conventional uracil and leucine auxotroph Yarrowia lipolytica, i.e., itself it is difficult to synthesize uracil and bright ammonia
The Yarrowia lipolytica of acid.It is preferred that the uracil and leucine auxotroph Yarrowia lipolytica are solution fat Ye Shi ferment
Female Po1f, it is according to Madzak C, Tr é ton B and Roland SB.Strong hybrid promoters and
integrative expression/secretion vectors for quasi-constitutive expression of
heterologous proteins in the yeast Yarrowia lipolytica.J Mol Microbiol
Biotechnol.2000,2(2):Preparation method described in 207-216 is made.
The gene LS of described optimization will be by that will derive from wrinkled giant hyssop (Agastache rugosa) LS genes (GB:
AY055214 nucleotide sequence) obtains after optimizing；The gene NDPS1 of the optimization will be by that will derive from tomato
NDPS1 genes (the GB of (Solanum lycopersicum):NM_001247704 nucleotide sequence) obtains after optimizing.
In the present invention, it is preferred that described recombinant vector contains PmlI restriction enzyme site.It is preferred that the recombinant vector
Also contain promoter and/or terminator sequence.More preferably, the recombinant vector also contains strong promoter hp4d.
Recombinant vector of the present invention is the conventional recombinant vector in this area, and it can convert uracil and leucine battalion
Support deficiency Yarrowia lipolytica, and gene LS and gene NDPS1 containing above-mentioned optimization.
It is preferred that described recombinant vector is that the gene LS of above-mentioned optimization and the gene NDPS1 of optimization are imported into plasmid
Plasmid pINA1312LN obtained by pINA1312, the plasmid pINA1312LN nucleotide sequence such as sequence table SEQ ID
Shown in No.5.
In the present invention, above-mentioned plasmid pINA1312LN is converted to above-mentioned Yarrowia lipolytica (Yarrowia
Lipolytica) Yarrowia lipolytica gene engineering bacteria built-up Po1f can produce more limonene, be named as
Yarrowia lipolytica Po1f-LN-000.
More preferably, the Yarrowia lipolytica gene engineering bacteria of production limonene of the present invention is by plasmid pINA1269-
The above-mentioned Yarrowia lipolytica Po1f-LN-000 of HMG1 conversions is built-up, the nucleotides sequence of the plasmid pINA1269-HMG1
Row are as shown in sequence table SEQ ID No.3.
Wherein, the gene HMG1 from Yarrowia lipolytica is contained in the plasmid pINA1269-HMG1 (in NCBI
Accession number is GB:NC_006071).
In the present invention, above-mentioned plasmid pINA1269-HMG1 is converted to above-mentioned Yarrowia lipolytica Po1f-LN-000 structures
The Yarrowia lipolytica gene engineering bacteria built is named as Yarrowia lipolytica Po1f-LN-004, and it can produce more lemons
Lemon alkene.
Most preferably, the Yarrowia lipolytica gene engineering bacteria of production limonene of the present invention is by plasmid pINA1269-
The above-mentioned Yarrowia lipolytica Po1f-LN-000 of HMG1-ERG12 conversions is built-up, the plasmid pINA1269-HMG1-
ERG12 nucleotide sequence is as shown in sequence table SEQ ID No.4.
Wherein, the gene from Yarrowia lipolytica is contained in the plasmid pINA1269-HMG1-ERG12 simultaneously
(accession number is GB to HMG1 in NCBI:NC_006071) and ERG12 (accession number is GB in NCBI:NC_006068).
In the present invention, above-mentioned plasmid pINA1269-HMG1-ERG12 is converted to above-mentioned Yarrowia lipolytica Po1f-
Yarrowia lipolytica gene engineering bacteria built-up LN-000 is named as Yarrowia lipolytica Po1f-LN-051, and it being capable of pole
Significantly high yield limonene.
The two of technical scheme are：A kind of method for producing limonene, it comprises the following steps：Cultivate above-mentioned
The Yarrowia lipolytica gene engineering bacteria of limonene is produced, addition dodecane carries out two-phase and fermented to obtain zymotic fluid, extractive fermentation liquid
Dodecane phase.
In the present invention, the culture medium of the culture is the conventional culture medium in this area, preferably YPD culture mediums.More preferably
Ground, described YPD culture mediums are made up of 2% glucose, 2% peptone and 1% yeast extract, and surplus is water, the percentage
Than for mass percent.
It is preferred that the culture medium also contains auxiliary carbon source.The auxiliary carbon source is the conventional auxiliary carbon source in this area, compared with
It is pyruvic acid goodly.The concentration of the pyruvic acid is the conventional concentration in this area, preferably 2~8g/L, is more preferably 4g/L,
The concentration is the ratio between volume of system of the culture before the quality and the addition pyruvic acid of the pyruvic acid.
In the present invention, because limonene is the monoterpene of high volatility, it is necessary to additionally add 1mL dodecanes in the medium
Two-phase fermentation is carried out as extractant, so as to which extraction is secreted into extracellular limonene at any time.It is preferred that add the dodecane
Time is the initial time of two-phase fermentation.The concentration of the dodecane is added as the conventional concentration in this area, preferably 2
~10%, it is more preferably 6~8%, is most preferably 8%, the percentage is the dodecane and institute before the addition dodecane
State the percent by volume of zymotic fluid.
The three of technical scheme are：Above-mentioned production limonene Yarrowia lipolytica gene engineering bacteria is preparing lemon
Application in alkene.
The four of technical scheme are：It is a kind of to prepare above-mentioned production limonene Yarrowia lipolytica gene engineering bacteria
Method, it comprises the following steps：
(1) the structure gene LS containing optimization and gene NDPS1 recombinant vector, the gene LS of optimization nucleotides
Sequence is as shown in sequence table SEQ ID No.1；The gene NDPS1 of optimization nucleotide sequence such as sequence table SEQ ID
Shown in No.2；
(2) recombinant vector for preparing step (1) converts uracil and leucine auxotroph Yarrowia lipolytica
(Yarrowia lipolytica), obtains transformant.
It is preferred that also comprise the following steps：
(3) plasmid pINA1269-HMG1 and plasmid pINA1269-HMG1-ERG12 is built, by the plasmid pINA1269-
HMG1 or the transformant obtained by plasmid pINA1269-HMG1-ERG12 step of converting (2), wherein, the plasmid pINA1269-
HMG1 nucleotide sequence is as shown in sequence table SEQ ID No.3；The nucleotides sequence of the plasmid pINA1269-HMG1-ERG12
Row are as shown in sequence table SEQ ID No.4.
The five of technical scheme are：A kind of restructuring for being used to prepare above-mentioned Yarrowia lipolytica gene engineering bacteria
Carrier, it contains the gene LS of optimization and gene NDPS1, the gene LS of optimization nucleotide sequence such as sequence table SEQ ID
Shown in No.1；The gene NDPS1 of optimization nucleotide sequence is as shown in sequence table SEQ ID No.2.
It is preferred that described recombinant vector is that the gene LS of above-mentioned optimization and the gene NDPS1 of optimization are imported into plasmid
Plasmid pINA1312LN obtained by pINA1312, the plasmid pINA1312LN nucleotide sequence such as sequence table SEQ ID
Shown in No.3.
It on the basis of common sense in the field is met, above-mentioned each optimum condition, can be combined, it is each preferably real to produce the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available.
The positive effect of the present invention is：The present invention is by by from the gene NDPS1 of tomato and from the leaves of pulse plants
Yarrowia lipolytica is imported after fragrant gene LS nucleotide sequence optimization, is overexpressed gene HMG1 on this basis, and more enter
One step is overexpressed gene HMG1 and gene ERG12 simultaneously, obtains the production limonene for the yield that can more greatly improve limonene
Yarrowia lipolytica gene engineering bacteria.The Yarrowia lipolytica gene engineering bacteria of the production limonene can reach limonene
0.67mg/g DCW very high throughput, more than the yield 111 that only quiding gene NDPS1 and gene LS bacterial strain produces limonene
Times.
Meanwhile the pyruvic acid and the concentration of dodecane added during by being fermented to Yarrowia lipolytica gene engineering bacteria two-phase
Selected, further improve the yield of limonene.In addition, produced using Yarrowia lipolytica gene engineering bacteria provided by the invention
The method of limonene, easy to operate, stable reaction is reliable, can be used in and commercially produce on a large scale, the limonene of gained
The preparation of food additives can be can be safely used for, is had good prospects.
Brief description of the drawings
Fig. 1 is the metabolic pathway of Yarrowia lipolytica generation limonene after introducing limonene synthetic gene.
Fig. 2 is the plasmid construct figure of the plasmid pINA1312LN containing two limonene synthetic genes.
Fig. 3 is the plasmid construct containing two plasmid pINA1269-HMG1-ERG12 for being overexpressed gene HMG1 and ERG12
Figure.
Fig. 4 is the result that bacterial strain Po1f-LN-000, Po1f-LN-004 and Po1f-LN-051 shake flask fermentation produce limonene
Figure.
Fig. 5 is the result figure that bacterial strain Po1f-LN-051 shake flask fermentations after fermentation condition optimization produce limonene.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification selects.
The embodiment of the present invention although with bacterial strain Po1f as starting strain, but conventional uracil and leucine nutrition
Deficiency Yarrowia lipolytica (Yarrowia lipolytica) can be used as starting strain, to convert, to be implemented
Experiment in example.
The starting strain Po1f of Yarrowia lipolytica (Yarrowia lipolytica) in embodiment is according to Madzak
C,Tréton B and Roland SB.Strong hybrid promoters and integrative expression/
secretion vectors for quasi-constitutive expression of heterologous proteins
in the yeast Yarrowia lipolytica.J Mol Microbiol Biotechnol.(2000)2(2):207-
Preparation method described in 216 is made.
Plasmid pINA1312 preparation method referring to Nicaud, J.M., Madzak, C., Broek, P., Gysler, C.,
Duboc,P.,Niederberger,P.,Gaillardin,C.,2002,Protein expression and secretion
in the yeast Yarrowia lipolytica.FEMS yeast research 2,371-379。
Plasmid pINA1269 preparation method is referring to Madzak C, Tr é ton B and Roland SB.Strong
hybrid promoters and integrative expression/secretion vectors for quasi-
constitutive expression of heterologous proteins in the yeast Yarrowia
lipolytica.J Mol Microbiol Biotechnol.(2000)2(2):207-216。
Embodiment 1 builds bacterial strain Po1f-LN-000
(1) the gene LS of wrinkled giant hyssop (Agastache rugosa) optimization (its nucleotide sequence will be derived from respectively
As shown in sequence table SEQ ID NO.1) and gene NDPS from tomato (Solanum lycopersicum) optimization sequence
Row (its nucleotide sequence is as shown in sequence table SEQ ID NO.2) are building up to plasmid pINA1312 by restriction enzyme site PmlI, obtain
To plasmid pINA1312-LS and pINA1312-NDPS1.
(2) expression cassette of NDPS1 genes in the plasmid pINA1312-NDPS1 obtained by step (1) is passed through into restriction enzyme site
StuI is connected on the plasmid pINA1312-LS obtained by step (1) and obtains the plasmid containing gene LS and NDPS1
pINA1312LN.Wherein plasmid pINA1312LN nucleotide sequence is as shown in sequence table SEQ ID NO.5.Plasmid
PINA1312LN plasmid construct is referring to Fig. 2.
(3) the plasmid pINA1312LN linearisations obtained by step (2) are transformed into solution fat using the method for homologous recombination
In family name's yeast Po1f, the initial strains Po1f-LN-000 that can generate limonene is obtained.Wherein, conversion uses kit
Frozen EZ Yeast Transformation IITM(being purchased from Zymo Research), recorded according to the kit specification
Method operated.
Embodiment 2 builds bacterial strain Po1f-LN-004 and Po1f-LN-051
(1) by from the gene HMG1 of Yarrowia lipolytica, (accession number is GB in NCBI:NC_006071) and
(accession number is GB to ERG12 in NCBI:NC_006068) it is connected respectively to by restriction enzyme site PmlI on plasmid pINA1269,
Obtain plasmid pINA1269-HMG1 and pINA1269-ERG12.Such as sequence table of plasmid pINA1269-HMG1 nucleotide sequence
Shown in SEQ ID NO.3.
(2) expression cassette of ERG12 genes will be passed through into restriction enzyme site in the plasmid pINA1269-ERG12 obtained by step (1)
SpeI is connected on the plasmid pINA1269-HMG1 obtained by step (1) and obtains the plasmid containing gene HMG1 and ERG12
pINA1269-HMG1-ERG12.Wherein plasmid pINA1269-HMG1-ERG12 nucleotide sequence such as sequence table SEQ ID
Shown in NO.4.Plasmid pINA1269-HMG1-ERG12 plasmid construct is referring to Fig. 3.
(3) by the plasmid pINA1269-ERG12 obtained by step (1) and the plasmid pINA1269-HMG1- obtained by step (2)
ERG12 is linearized, and is transformed into respectively using the method for homologous recombination in the initial strains Po1f-LN-000 of the gained of embodiment 1, according to
It is secondary to obtain bacterial strain Po1f-LN-004 and Po1f-LN-051.Wherein, the method for conversion is identical with the method converted in embodiment 1.
Embodiment 3 determines the yield of bacterial strain production limonene
It is prepared by initial strains Po1f-LN-000 prepared by bacterial strain Yarrowia lipolytica Po1f, embodiment 1, embodiment 2
Bacterial strain Po1f-LN-004 and Po1f-LN-051 be inoculated in respectively 2mL YPD culture mediums (the YPD culture mediums by 2% glucose,
2% peptone and 1% yeast extract composition, surplus is water, and the percentage is mass percent), culture 24 hours, then
New 50mL YPD culture mediums are inoculated in as 0.01 inoculum concentration to be cultivated, and add 2% dodecane using initial OD, described hundred
Divide than the percent by volume for the dodecane and the zymotic fluid before the addition dodecane.After two-phase fermented and cultured 3 days, take
Dodecane mutually detects limonene with GC-MS.The method of detection is referring to Jongedijk, E., Cankar, K., Ranzijn, J., van
der Krol,S.,Bouwmeester,H.,Beekwilder,J.,2015.Capturing of themonoterpene
olef in limonene produced in Saccharomyces cerevisiae.Yeast.32,159-171。
The result of detection is referring to Fig. 4 and table 1.The result explanation of table 1, bacterial strain Po1f-LN-004 and Po1f-LN-051 production lemon
The ability of lemon alkene is significantly lifted.
The limonene yield of the different strains of table 1
Embodiment 4 improves the optimization fermentation process of the yield of bacterial strain production limonene
A, using the pyruvic acid of more excellent concentration
Bacterial strain Po1f-LN-051 prepared by embodiment 2 is inoculated in 2mL YPD medium cultures 24 hours, then with first
The inoculum concentration that beginning OD is 0.01 is inoculated in new 50mL YPD culture mediums and cultivated.The pyruvic acid of various concentrations is added as auxiliary
Carbon source is helped to be cultivated.The dodecane of extra addition 2% is as extractant progress two-phase fermentation in the medium simultaneously, and described hundred
Divide than the percent by volume for the dodecane and the zymotic fluid before the addition dodecane.
After two-phase fermented and cultured 3 days, dodecane is taken mutually to detect the content of limonene with GC-MS, as a result such as Fig. 5 and the institute of table 2
Show.The result explanation of table 2, when the concentration of the pyruvic acid added is 4g/L, the yield highest of limonene, reaches 0.985mg/g
DCW, wherein, the unit (g/L) is the body of the quality and the system of the culture before the addition pyruvic acid of the pyruvic acid
The ratio between product.
Limonene yield -1 after the fermentation condition optimization of table 2
B, using the dodecane of more excellent concentration
Bacterial strain Po1f-LN-051 prepared by embodiment 2 is inoculated in 2mL YPD medium cultures 24 hours, then with first
The inoculum concentration that beginning OD is 0.01 is inoculated in new 50mL YPD culture mediums and cultivated.4g/L pyruvic acid is added as auxiliary carbon
Source is cultivated, the concentration for the pyruvic acid quality with add the system of the culture before the pyruvic acid volume it
Than.The dodecane for adding various concentrations in the medium simultaneously carries out two-phase fermentation as extractant.
After two-phase fermented and cultured 3 days, dodecane is taken mutually to detect the content of limonene with GC-MS, as a result such as Fig. 5 and the institute of table 3
Show.The result explanation of table 3, when 4mL dodecanes are added in 50mL culture mediums, the yield highest of limonene, reaches 1.36mg/g
DCW.Wherein, the percentage is the percent by volume of the dodecane and the zymotic fluid before the addition dodecane.
Limonene yield -2 after the fermentation condition optimization of table 3
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above of the present invention has been read, those skilled in the art can be right
The present invention makes various changes or modifications, and these equivalent form of values equally fall within the application appended claims limited range.
Claims (10)
1. a kind of Yarrowia lipolytica gene engineering bacteria for producing limonene, it is characterised in that it is by the gene LS containing optimization
Uracil and leucine auxotroph Yarrowia lipolytica (Yarrowia are converted with the gene NDPS1 of optimization recombinant vector
Lipolytica) built-up, the gene LS of optimization nucleotide sequence is as shown in sequence table SEQ ID No.1；It is described
The gene NDPS1 of optimization nucleotide sequence is as shown in sequence table SEQ ID No.2.
2. the Yarrowia lipolytica gene engineering bacteria of production limonene as claimed in claim 1, it is characterised in that the restructuring carries
Body is plasmid pINA1312LN, the plasmid pINA1312LN nucleotide sequence as shown in sequence table SEQ ID No.5；With/
Or, the uracil and leucine auxotroph Yarrowia lipolytica are Yarrowia lipolytica Po1f, the solution fat Ye Shi ferment
Female Po1f is according to Madzak C, Tr é ton B and Roland SB.Strong hybrid promoters and
integrative expression/secretion vectors for quasi-constitutive expression of
heterologous proteins in the yeast Yarrowia lipolytica.J Mol Microbiol
Biotechnol.2000,2(2):Preparation method in 207-216 is made.
3. the Yarrowia lipolytica gene engineering bacteria of production limonene as claimed in claim 2, it is characterised in that it is by plasmid
Built-up, the nucleotide sequence such as sequence table of the plasmid pINA1269-HMG1 after pINA1269-HMG1 conversion transformant A
Shown in SEQ ID No.3；The transformant A is that the plasmid pINA1312LN is converted into uracil and leucine auxotroph
Yarrowia lipolytica (Yarrowia lipolytica) is built-up.
4. the Yarrowia lipolytica gene engineering bacteria of production limonene as claimed in claim 2, it is characterised in that it is by plasmid
Built-up, the nucleotides of the plasmid pINA1269-HMG1-ERG12 after pINA1269-HMG1-ERG12 conversion transformant A
Sequence is as shown in sequence table SEQ ID No.4；The transformant A is that the plasmid pINA1312LN is converted into uracil and bright ammonia
Sour auxotroph Yarrowia lipolytica (Yarrowia lipolytica) is built-up.
A kind of 5. method for producing limonene, it is characterised in that it comprises the following steps：Culture such as any one of Claims 1 to 4
The Yarrowia lipolytica gene engineering bacteria of described production limonene, addition dodecane carry out two-phase and fermented to obtain zymotic fluid, extraction hair
The dodecane phase of zymotic fluid.
6. method as claimed in claim 5, it is characterised in that pyruvic acid is also added in described culture, it is preferred that the acetone
The concentration of acid is 2~8g/L, preferably 4g/L, and the concentration is institute before the quality and the addition pyruvic acid of the pyruvic acid
State the ratio between volume of system of culture；And/or the concentration of the dodecane is 2~10%, preferably 6~8%, described hundred
Divide than the percent by volume for the dodecane and the zymotic fluid before the addition dodecane.
7. the Yarrowia lipolytica gene engineering bacteria of the production limonene as described in any one of Claims 1 to 4 is preparing limonene
In application.
A kind of 8. method for the Yarrowia lipolytica gene engineering bacteria for preparing production limonene, it is characterised in that it includes following
Step：
(1) gene LS of the structure containing optimization and the gene NDPS1 of optimization recombinant vector, the gene LS of optimization nucleosides
Acid sequence is as shown in sequence table SEQ ID No.1；The gene NDPS1 of optimization nucleotide sequence such as sequence table SEQ ID
Shown in No.2；
(2) recombinant vector for preparing step (1) converts uracil and leucine auxotroph Yarrowia lipolytica
(Yarrowia lipolytica), obtains transformant.
9. method as claimed in claim 8, it is characterised in that it also comprises the following steps：
(3) plasmid pINA1269-HMG1 and plasmid pINA1269-HMG1-ERG12 is built, by the plasmid pINA1269-HMG1
Or the transformant obtained by plasmid pINA1269-HMG1-ERG12 step of converting (2), wherein, the plasmid pINA1269-HMG1's
Nucleotide sequence is as shown in sequence table SEQ ID No.3；The nucleotide sequence such as sequence of the plasmid pINA1269-HMG1-ERG12
Shown in list SEQ ID No.4.
A kind of 10. Yarrowia lipolytica gene engineering bacteria for being used to prepare the production limonene as described in any one of Claims 1 to 4
Recombinant vector, it is characterised in that its contain the gene LS of optimization and optimization gene NDPS1, the gene LS's of the optimization
Nucleotide sequence is as shown in sequence table SEQ ID No.1；The gene NDPS1 of optimization nucleotide sequence such as sequence table SEQ
Shown in ID No.2；It is preferred that described recombinant vector is to import the gene LS of described optimization and the gene NDPS1 of optimization
Plasmid pINA1312LN obtained by plasmid pINA1312, the plasmid pINA1312LN nucleotide sequence such as sequence table SEQ ID
Shown in No.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610817882.XA CN107815424B (en) | 2016-09-12 | 2016-09-12 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610817882.XA CN107815424B (en) | 2016-09-12 | 2016-09-12 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107815424A true CN107815424A (en) | 2018-03-20 |
| CN107815424B CN107815424B (en) | 2021-03-05 |
Family
ID=61600491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610817882.XA Active CN107815424B (en) | 2016-09-12 | 2016-09-12 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107815424B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108587934A (en) * | 2018-05-10 | 2018-09-28 | 天津科技大学 | A kind of Yarrowia lipolytica of production limonene and its construction method and application |
| CN110499259A (en) * | 2019-07-22 | 2019-11-26 | 浙江工业大学 | A kind of solution ester Ye Shi yeast YW100-1 and its application |
| CN110669713A (en) * | 2019-10-18 | 2020-01-10 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium for synthesizing D-limonene and construction method and application thereof |
| CN111979133A (en) * | 2020-08-14 | 2020-11-24 | 华东理工大学 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
| CN112011471A (en) * | 2019-05-31 | 2020-12-01 | 深圳华大生命科学研究院 | Yeast strain for brewing lemon-flavored beer, preparation method thereof and beer brewing method |
| CN112626103A (en) * | 2020-10-21 | 2021-04-09 | 天津科技大学 | Yarrowia lipolytica engineering bacterium for producing limonene and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010031062A1 (en) * | 2008-09-15 | 2010-03-18 | Danisco Us Inc. | Increased isoprene production using the archaeal lower mevalonate pathway |
| CN102071153A (en) * | 2010-12-08 | 2011-05-25 | 江南大学 | Yeast engineering bacterium for producing d-limonene and construction method thereof |
| WO2013052914A2 (en) * | 2011-10-07 | 2013-04-11 | Danisco Us Inc. | Methods for increasing microbial production of isoprene, isoprenoids, and isoprenoid precursor molecules using glucose and acetate co-metabolism |
| CN104039844A (en) * | 2011-08-04 | 2014-09-10 | 丹尼斯科美国公司 | Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase |
| CN104053765A (en) * | 2011-10-07 | 2014-09-17 | 丹尼斯科美国公司 | Utilization of phosphoketolase in production of mevalonate, isoprenoid precursors, and isoprene |
| EP2899263A4 (en) * | 2012-09-21 | 2016-04-13 | Sekisui Chemical Co Ltd | Recombinant cell, and method for producing beta-phellandrene |
-
2016
- 2016-09-12 CN CN201610817882.XA patent/CN107815424B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010031062A1 (en) * | 2008-09-15 | 2010-03-18 | Danisco Us Inc. | Increased isoprene production using the archaeal lower mevalonate pathway |
| CN102071153A (en) * | 2010-12-08 | 2011-05-25 | 江南大学 | Yeast engineering bacterium for producing d-limonene and construction method thereof |
| CN104039844A (en) * | 2011-08-04 | 2014-09-10 | 丹尼斯科美国公司 | Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase |
| WO2013052914A2 (en) * | 2011-10-07 | 2013-04-11 | Danisco Us Inc. | Methods for increasing microbial production of isoprene, isoprenoids, and isoprenoid precursor molecules using glucose and acetate co-metabolism |
| CN104053765A (en) * | 2011-10-07 | 2014-09-17 | 丹尼斯科美国公司 | Utilization of phosphoketolase in production of mevalonate, isoprenoid precursors, and isoprene |
| EP2899263A4 (en) * | 2012-09-21 | 2016-04-13 | Sekisui Chemical Co Ltd | Recombinant cell, and method for producing beta-phellandrene |
Non-Patent Citations (6)
| Title |
|---|
| ESMER JONGEDIJK等: "Biotechnological production of limonene in microorganisms", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| ESMER JONGEDIJK等: "Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae", 《YEAST》 * |
| FU-LIANG DU等: "Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli", 《BIORESOURCES AND BIOPROCESSING》 * |
| MADZAK C等: "Strong Hybrid Promoters and Integrative Expression/Secretion Vectors for Quasi-Constitutive Expression of Heterologous Proteins in the Yeast Yarrowia lipolytica", 《J.MOL.MICROBIOL.BIOTECHNOL》 * |
| XUAN CAO等: "Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction", 《BIOTECHNOLOGY FOR BIOFUELS》 * |
| 刘继栋: "代谢工程改造酿酒酵母生产单萜的关键问题解析", 《中国博士学位论文全文数据库基础科学辑》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108587934A (en) * | 2018-05-10 | 2018-09-28 | 天津科技大学 | A kind of Yarrowia lipolytica of production limonene and its construction method and application |
| CN112011471A (en) * | 2019-05-31 | 2020-12-01 | 深圳华大生命科学研究院 | Yeast strain for brewing lemon-flavored beer, preparation method thereof and beer brewing method |
| CN112011471B (en) * | 2019-05-31 | 2022-07-22 | 深圳华大生命科学研究院 | Yeast strain for brewing lemon-flavored beer, preparation method thereof and beer brewing method |
| CN110499259A (en) * | 2019-07-22 | 2019-11-26 | 浙江工业大学 | A kind of solution ester Ye Shi yeast YW100-1 and its application |
| CN110669713A (en) * | 2019-10-18 | 2020-01-10 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium for synthesizing D-limonene and construction method and application thereof |
| CN111979133A (en) * | 2020-08-14 | 2020-11-24 | 华东理工大学 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
| CN111979133B (en) * | 2020-08-14 | 2023-02-28 | 华东理工大学 | Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof |
| CN112626103A (en) * | 2020-10-21 | 2021-04-09 | 天津科技大学 | Yarrowia lipolytica engineering bacterium for producing limonene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107815424B (en) | 2021-03-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | Elevating limonene production in oleaginous yeast Yarrowia lipolytica via genetic engineering of limonene biosynthesis pathway and optimization of medium composition | |
| CN107815424A (en) | A kind of Yarrowia lipolytica gene engineering bacteria for producing limonene and its application | |
| Zhang et al. | Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica | |
| Tang et al. | Metabolic engineering of Yarrowia lipolytica for improving squalene production | |
| Mirończuk et al. | A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol | |
| EP2657344B1 (en) | Method for producing butanol using two-phase extractive fermentation | |
| CN105420134B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN106566779B (en) | A kind of restructuring yeast strains and its construction method and application | |
| CN104962488A (en) | Recombinant yeast strain, and construction method and application thereof | |
| CN113234610B (en) | Saccharomyces cerevisiae strain for synthesizing squalene and application thereof | |
| CN104651287A (en) | Engineering bacterium for synthesizing glycosylglycerol and application thereof | |
| CN107723252A (en) | Produce the restructuring Yarrowia lipolytica and construction method of valencia orange alkene and nootkatone | |
| CN114507613B (en) | Yeast engineering bacteria for producing alpha-santalene by fermentation and application thereof | |
| CN106350476A (en) | Genetic engineering strain capable of coproducing isoprene and 1,3-propylene glycol and establishment method and application thereof | |
| CN108998383B (en) | Yarrowia lipolytica genetically engineered bacterium for producing linalool and application thereof | |
| CN104017853A (en) | Method for producing gamma-aminobutyric acid by fermentation | |
| CN111088175A (en) | Yarrowia lipolytica for producing bisabolene and construction method and application thereof | |
| CN111334459B (en) | Construction method and application of Klebsiella engineering bacteria for improving yield of 1, 3-propylene glycol | |
| CN106434772B (en) | One plant of genetic engineering bacterium for producing L MALIC ACID and its construction method and application | |
| CN106867922A (en) | The method that Klebsiella pneumoniae produces KIV and isobutanol | |
| CN101130782B (en) | Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate | |
| CN106244615B (en) | Engineering bacterium, construction method thereof and application of engineering bacterium in preparation of geraniol | |
| CN104789487A (en) | Bacterial strain capable of respectively producing butyric acid and n-butanol and method for producing n-butanol | |
| CN106148255B (en) | Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol | |
| CN107815425A (en) | A kind of Yarrowia lipolytica gene engineering bacteria for producing fatty-acid ethyl ester and its application |
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 |