CN107815424B - Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof - Google Patents
Yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof Download PDFInfo
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Abstract
The invention discloses a yarrowia lipolytica gene engineering bacterium for producing limonene and application thereof. The genetic engineering bacteria are constructed by transforming uracil and leucine auxotroph Yarrowia lipolytica (Yarrowia lipolytica) into a recombinant vector containing an optimized gene LS and a gene NDPS1, wherein the nucleotide sequences of the optimized gene LS and the optimized gene NDPS1 are respectively shown in sequence tables SEQ ID No. 1-2. On the basis, the gene HMG1 and the gene ERG12 are simultaneously over-expressed, so that the extremely high yield of 0.67mg/g DCW can be achieved. In addition, the concentrations of pyruvic acid and dodecane added during two-phase fermentation of the genetically engineered bacteria are selected, so that the yield of the limonene is further improved. The gene engineering bacterium can be used for large-scale commercial production and has good prospect.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a yarrowia lipolytica genetic engineering bacterium for producing limonene and application thereof.
Background
Limonene is ubiquitous in natural plants and is widely used in food flavors due to its pleasant citrus flavor. In the flavor industry, limonene itself can be used as a raw material for formulating artificial neroli, sweet flowers, lemons, bergamot oils, and also as a fresh top flavor. Limonene has pharmacological activity and thus has wide application in medicine. Limonene is also a substitute for jet fuel. It is also a precursor to many important aromatic substances and drugs such as perillyl alcohol, carvone, and menthol.
The production method of limonene mainly comprises two methods, namely a natural extraction method and a microbial fermentation method. The limonene extracted from industrial waste citrus peel is difficult to avoid pesticide residue and is not suitable for being used as a food additive, so that the limonene production by microbial fermentation has obvious advantages. Particularly, with the rapid development of biotechnology, the fermentation production of limonene and derivatives thereof by using microorganisms becomes one of the hot spots of the current research.
Generally, limonene is produced by introducing a limonene synthetase gene from an external source through a biological fermentation method, and a gene engineering bacterium with high limonene yield is needed at present.
Disclosure of Invention
The invention aims to overcome the defect that the genetic engineering bacteria for high-yield limonene production is lacked in the prior art, and provides a yarrowia lipolytica genetic engineering bacteria for limonene production and application thereof.
The inventor finds out after creative work that aiming at the metabolic pathway of producing limonene by yarrowia lipolytica after introducing the limonene synthesis gene, two genes related to limonene synthesis, namely, the gene NDPS1 which is derived from tomato and can condense isopentenyl pyrophosphate and methallyl pyrophosphate into neryl diphosphate; and genes LS which are derived from agastache rugosus and can isomerize neryl diphosphate into limonene are respectively optimized, and the optimized genes are transformed into yarrowia lipolytica to enable the yarrowia lipolytica to produce limonene. The inventors further found that overexpression of HMG1, which reduces HMG-CoA to mevalonate, and simultaneously overexpression of ERG12, which phosphorylates mevalonate to mevalonate-5-phosphate, based on optimization of the above two genes, greatly increased the yield of limonene (see FIG. 1). Furthermore, the inventors have found that a specific amount of dodecane added is better able to promote high yields of limonene by yarrowia lipolytica in a two-phase fermentation.
The technical scheme provided by the invention is as follows:
one of the technical schemes of the invention is as follows: a Yarrowia lipolytica genetic engineering bacterium for producing limonene is constructed by transforming a recombinant vector containing an optimized gene LS and an optimized gene NDPS1 into uracil and leucine auxotrophic Yarrowia lipolytica (Yarrowia lipolytica), wherein the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2.
In the present invention, the uracil and leucine auxotrophic yarrowia lipolytica (yarrowia lipolytica) is a uracil and leucine auxotrophic yarrowia lipolytica that is conventional in the art, i.e., yarrowia lipolytica that itself has difficulty in synthesizing uracil and leucine. Preferably, the uracil and leucine auxotrophic Yarrowia lipolytica is Yarrowia lipolytica Po1f, produced according to the preparation method described in Madzak C, Traton B and Roland SB.Strong hybrid promoters and integral expression/secretion vectors for quasi-dependent expression of heterologous proteins in the layer yeast below specific molecular biotech.2000, 2: (2): 207-.
The optimized gene LS is obtained by optimizing a nucleotide sequence derived from an ageratum (Agastache rugosa) LS gene (GB: AY 055214); the optimized gene NDPS1 is obtained by optimizing the nucleotide sequence of NDPS1 gene (GB: NM-001247704) derived from tomato (Solanum lycopersicum).
In the present invention, preferably, the recombinant vector contains the cleavage site of PmlI. Preferably, the recombinant vector further comprises a promoter and/or terminator sequence. More preferably, the recombinant vector further comprises a strong promoter hp4 d.
The recombinant vector is a conventional recombinant vector in the field, can be used for transforming uracil and leucine auxotrophic yarrowia lipolytica, and contains the optimized gene LS and the optimized gene NDPS 1.
Preferably, the recombinant vector is a plasmid pINA1312LN obtained by introducing the optimized gene LS and the optimized gene NDPS1 into a plasmid pINA1312, and the nucleotide sequence of the plasmid pINA1312LN is shown in a sequence table SEQ ID No. 5.
In the present invention, the above-mentioned genetically engineered Yarrowia lipolytica strain constructed by transforming the above-mentioned plasmid pINA1312LN into Yarrowia lipolytica (Yarrowia lipolytica) Po1f was named as Yarrowia lipolytica Po1f-LN-000, because it can produce much limonene.
More preferably, the yarrowia lipolytica genetically engineered bacterium for producing limonene is constructed by transforming the plasmid pINA1269-HMG1 into the yarrowia lipolytica Po1f-LN-000, and the nucleotide sequence of the plasmid pINA1269-HMG1 is shown in the sequence table SEQ ID No. 3.
Wherein the plasmid pINA1269-HMG1 contains gene HMG1 (accession number GB: NC-006071 in NCBI) derived from yarrowia lipolytica.
In the present invention, yarrowia lipolytica genetically engineered bacterium constructed by transforming the above-mentioned yarrowia lipolytica Po1f-LN-000 with the above-mentioned plasmid pINA1269-HMG1 was named yarrowia lipolytica Po1f-LN-004, and it was possible to produce more limonene.
Preferably, the genetically engineered yarrowia lipolytica for producing limonene is constructed by transforming the plasmid pINA1269-HMG1-ERG12 into the yarrowia lipolytica Po1f-LN-000, and the nucleotide sequence of the plasmid pINA1269-HMG1-ERG12 is shown in a sequence table SEQ ID No. 4.
Wherein the plasmid pINA1269-HMG1-ERG12 contains genes HMG1 (accession number GB: NC-006071 in NCBI) and ERG12 (accession number GB: NC-006068 in NCBI) which are derived from yarrowia lipolytica.
In the present invention, yarrowia lipolytica genetically engineered bacterium constructed by transforming the above-mentioned yarrowia lipolytica Po1f-LN-000 with the above-mentioned plasmid pINA1269-HMG1-ERG12 is named yarrowia lipolytica Po1f-LN-051, and it is possible to produce limonene with a very high yield.
The second technical scheme of the invention is as follows: a method of producing limonene, comprising the steps of: culturing the yarrowia lipolytica gene engineering bacteria producing limonene, adding dodecane to perform two-phase fermentation to obtain fermentation liquor, and extracting the dodecane phase of the fermentation liquor.
In the present invention, the culture medium is a medium conventional in the art, preferably a YPD medium. More preferably, the YPD medium consists of 2% of glucose, 2% of peptone and 1% of yeast extract, and the balance of water, wherein the percentages are mass percentages.
Preferably, the medium further comprises an auxiliary carbon source. The auxiliary carbon source is an auxiliary carbon source conventional in the art, preferably pyruvate. The concentration of pyruvic acid is a concentration conventional in the art, preferably 2-8 g/L, more preferably 4g/L, and the concentration is a ratio of the mass of pyruvic acid to the volume of the cultured system before addition of pyruvic acid.
In the present invention, since limonene is a highly volatile monoterpene, 1mL of dodecane needs to be additionally added to the culture medium as an extractant to perform two-phase fermentation, so that limonene secreted to the extracellular space is extracted at any time. Preferably, the dodecane is added for the start time of the two-phase fermentation. The dodecane is added in a concentration conventional in the art, preferably 2 to 10%, more preferably 6 to 8%, and most preferably 8%, wherein the percentage is the volume percentage of the dodecane and the fermentation broth before adding the dodecane.
The third technical scheme of the invention is as follows: the application of the yarrowia lipolytica gene engineering bacteria for producing limonene in the preparation of limonene.
The fourth technical scheme of the invention is as follows: a method for preparing the yarrowia lipolytica genetically engineered bacterium for producing limonene comprises the following steps:
(1) constructing a recombinant vector containing an optimized gene LS and a gene NDPS1, wherein the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2;
(2) transforming the recombinant vector prepared in the step (1) into uracil and leucine auxotrophic Yarrowia lipolytica (Yarrowia lipolytica) to obtain a transformant.
Preferably, the method further comprises the following steps:
(3) constructing a plasmid pINA1269-HMG1 and a plasmid pINA1269-HMG1-ERG12, and transforming the plasmid pINA1269-HMG1 or the plasmid pINA1269-HMG1-ERG12 into the transformant obtained in the step (2), wherein the nucleotide sequence of the plasmid pINA1269-HMG1 is shown in a sequence table SEQ ID No. 3; the nucleotide sequence of the plasmid pINA1269-HMG1-ERG12 is shown in a sequence table SEQ ID No. 4.
The fifth technical scheme of the invention is as follows: a recombinant vector for preparing the yarrowia lipolytica genetically engineered bacterium contains an optimized gene LS and a gene NDPS1, wherein the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2.
Preferably, the recombinant vector is a plasmid pINA1312LN obtained by introducing the optimized gene LS and the optimized gene NDPS1 into a plasmid pINA1312, and the nucleotide sequence of the plasmid pINA1312LN is shown in a sequence table SEQ ID No. 3.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the present invention obtains gene engineering bacterium of yarrowia lipolytica capable of greatly raising the yield of limonene by optimizing the nucleotide sequence of tomato-derived gene NDPS1 and agastache rugosus-derived gene LS and introducing it into yarrowia lipolytica, on the basis of said nucleotide sequence the gene HMG1 can be over-expressed, and further the gene HMG1 and gene ERG12 can be over-expressed simultaneously. The yarrowia lipolytica gene engineering bacterium for producing limonene can enable limonene to reach extremely high yield of 0.67mg/g DCW, and the yield of limonene is 111 times higher than that of a strain only introduced with the gene NDPS1 and the gene LS.
Meanwhile, the concentrations of pyruvic acid and dodecane added during two-phase fermentation of the yarrowia lipolytica genetic engineering bacteria are selected, so that the yield of the limonene is further improved. In addition, the method for producing the limonene by utilizing the yarrowia lipolytica genetic engineering bacteria provided by the invention is simple and convenient to operate, stable and reliable in reaction, capable of being used for large-scale commercial production, and good in prospect, and the obtained limonene can be safely used for preparing food additives.
Drawings
FIG. 1 is a diagram showing the metabolic pathways for the production of limonene by yarrowia lipolytica after the introduction of the limonene synthesizing gene.
FIG. 2 is a plasmid structure diagram of plasmid pINA1312LN containing two limonene synthesizing genes.
FIG. 3 is a plasmid structure diagram of plasmid pINA1269-HMG1-ERG12 containing two overexpressed genes HMG1 and ERG 12.
FIG. 4 is a graph showing the results of shake flask fermentation for producing limonene by strains Po1f-LN-000, Po1f-LN-004 and Po1 f-LN-051.
FIG. 5 is a graph showing the results of the fermentation of strain Po1f-LN-051 in a shake flask for producing limonene after the fermentation conditions are optimized.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Although the strain Po1f was used as the starting strain in the examples of the present invention, conventional uracil and leucine auxotrophic Yarrowia lipolytica (Yarrowia lipolytica) could be used as the starting strain for transformation to perform the tests in the examples.
The starting strain Po1f of Yarrowia lipolytica (Yarrowia lipolytica) in the examples was prepared according to the preparation method described in Madzak C, Traton B and Roland SB.Strong primers and integral expression/genetic vectors for the quantitative-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.J. Mol Microbiol Biotechnology (2000)2(2) 207-.
Plasmid pINA1312 was prepared as described in Nicaud, J.M., Madzak, C., Broek, P., Gysler, C., Duboc, P., Niederberger, P., Gallardin, C.,2002, Protein expression and differentiation in the term Yarrowia lipolytica FEMS layer research 2, 371-.
Plasmid pINA1269 can be prepared as described in Madzak C, Traton B and road SB.Strong hybrid promoters and integral expression/secretion vectors for the quantitative-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.J.mol Microbiol Biotechnology (2000)2(2): 207-.
EXAMPLE 1 construction of the Strain Po1f-LN-000
(1) Respectively constructing an optimized sequence of a gene LS (nucleotide sequence is shown in a sequence table SEQ ID NO. 1) derived from Agastache rugosa (Agastache rugosa) and an optimized sequence of a gene NDPS (nucleotide sequence is shown in a sequence table SEQ ID NO. 2) derived from tomato (Solanum lycopersicum) to a plasmid pINA1312 through a restriction enzyme cutting site PmlI to obtain plasmids pINA1312-LS and pINA1312-NDPS 1.
(2) And (2) connecting the expression cassette of the NDPS1 gene in the plasmid pINA1312-NDPS1 obtained in the step (1) to the plasmid pINA1312-LS obtained in the step (1) through a restriction enzyme site StuI to obtain a plasmid pINA1312LN containing genes LS and NDPS 1. Wherein the nucleotide sequence of the plasmid pINA1312LN is shown in a sequence table SEQ ID NO. 5. The plasmid structure of plasmid pINA1312LN is shown in FIG. 2.
(3) Linearizing the plasmid pINA1312LN obtained in the step (2), and transforming the plasmid pINA1312LN into yarrowia lipolytica Po1f by using a homologous recombination method to obtain an initial strain Po1f-LN-000 capable of producing limonene. Wherein the Transformation kit Frozen EZ Yeast Transformation II is used for TransformationTM(purchased from Zymo Research) according to the kitThe method described in the specification was performed.
Example 2 construction of strains Po1f-LN-004 and Po1f-LN-051
(1) Genes HMG1 (accession number GB: NC-006071 in NCBI) and ERG12 (accession number GB: NC-006068 in NCBI) derived from yarrowia lipolytica were ligated to plasmid pINA1269 via cleavage site PmlI, respectively, to give plasmids pINA1269-HMG1 and pINA1269-ERG 12. The nucleotide sequence of the plasmid pINA1269-HMG1 is shown in a sequence table SEQ ID NO. 3.
(2) The expression cassette of ERG12 gene in the plasmid pINA1269-ERG12 obtained in the step (1) is connected to the plasmid pINA1269-HMG1 obtained in the step (1) through a cleavage site SpeI to obtain the plasmid pINA1269-HMG1-ERG12 containing genes HMG1 and ERG 12. Wherein the nucleotide sequence of the plasmid pINA1269-HMG1-ERG12 is shown in a sequence table SEQ ID NO. 4. The plasmid structure of plasmid pINA1269-HMG1-ERG12 is shown in FIG. 3.
(3) The plasmid pINA1269-ERG12 obtained in step (1) and the plasmid pINA1269-HMG1-ERG12 obtained in step (2) were linearized and transformed into the initial strain Po1f-LN-000 obtained in example 1 by homologous recombination, respectively, to obtain strains Po1f-LN-004 and Po1f-LN-051 in this order. The transformation method was the same as that of example 1.
Example 3 determination of the yield of limonene produced by a Strain
Yarrowia lipolytica Po1f, the initial strain Po1f-LN-000 prepared in example 1, the strains Po1f-LN-004 and Po1f-LN-051 prepared in example 2 were inoculated, respectively, into 2mL of YPD medium consisting of 2% glucose, 2% peptone and 1% yeast extract, the balance being water, said percentages being percentages by mass), cultured for 24 hours, then inoculated into a new 50mL of YPD medium at an initial OD of 0.01, cultured, and 2% dodecane was added, said percentages being percentages by volume of the dodecane and the fermentation broth before addition of the dodecane. After 3 days of two-phase fermentation culture, taking dodecane phase and detecting limonene by GC-MS. Methods of detection are described in Jongedijk, e., Cankar, k., Ranzijn, j., van der Krol, s., Bouwmeester, h., beekwelder, j.,2015, Capturing of the monoterpene ole in limonene produced in Saccharomyces cerevisiae.yeasts.32, 159-171.
The results of the test are shown in FIG. 4 and Table 1. The results in Table 1 show that the strains Po1f-LN-004 and Po1f-LN-051 have greatly improved limonene-producing capability.
TABLE 1 limonene production by different strains
Example 4 optimized fermentation method for increasing yield of limonene produced by strain
A. Using a preferred concentration of pyruvic acid
The strain Po1f-LN-051 prepared in example 2 was inoculated into 2mL of YPD medium and cultured for 24 hours, and then inoculated into a new 50mL of YPD medium at an initial OD of 0.01 for culture. Adding pyruvic acid with different concentrations as auxiliary carbon source for culturing. Meanwhile, 2% of dodecane is additionally added into the culture medium as an extractant for two-phase fermentation, wherein the percentage is the volume percentage of the dodecane to the fermentation liquor before the dodecane is added.
After 3 days of two-phase fermentation, the dodecane phase was taken and the limonene content was measured by GC-MS, and the results are shown in FIG. 5 and Table 2. The results in Table 2 show that the highest yield of limonene reached 0.985mg/g DCW at a concentration of 4g/L of pyruvic acid added, where the unit (g/L) is the ratio of the mass of pyruvic acid to the volume of the cultured system before addition of pyruvic acid.
TABLE 2 limonene production after fermentation conditions optimization-1
B. Using dodecane in relatively optimum concentrations
The strain Po1f-LN-051 prepared in example 2 was inoculated into 2mL of YPD medium and cultured for 24 hours, and then inoculated into a new 50mL of YPD medium at an initial OD of 0.01 for culture. 4g/L of pyruvic acid as an auxiliary carbon source was added to the culture, and the concentration was a ratio of the mass of pyruvic acid to the volume of the cultured system before the addition of pyruvic acid. Meanwhile, dodecane with different concentrations is added into the culture medium as an extracting agent to carry out two-phase fermentation.
After 3 days of two-phase fermentation, the dodecane phase was taken and the limonene content was measured by GC-MS, and the results are shown in FIG. 5 and Table 3. The results in Table 3 show that the highest limonene production reached 1.36mg/g DCW when 4mL dodecane was added to 50mL of the medium. Wherein the percentage is a volume percentage of the dodecane to the fermentation broth before the dodecane is added.
TABLE 3 limonene production after fermentation conditions optimization-2
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (13)
1. A genetically engineered yarrowia lipolytica strain producing limonene, characterized in that it is a yarrowia lipolytica auxotrophic for uracil and leucine transformed with a recombinant vector containing optimized gene LS and optimized gene NDPS 1(Yarrowia lipolytica) Polf, and the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2; the recombinant vector is a plasmid pINA1312LN, and the nucleotide sequence of the plasmid pINA1312LN is shown in a sequence table SEQ ID No. 5.
2. As claimed inThe yarrowia lipolytica genetically engineered bacterium for producing limonene of claim 1 is characterized in that the yarrowia lipolytica genetically engineered bacterium is constructed by transforming a transformant A with a plasmid pINA1269-HMG1, wherein the nucleotide sequence of the plasmid pINA1269-HMG1 is shown in a sequence table SEQ ID No. 3; the transformant A is a uracil and leucine auxotrophic yarrowia lipolytica transformed from the plasmid pINA1312LN (Yarrowia lipolytica) Polf was constructed.
3. The genetically engineered yarrowia lipolytica bacterium of claim 1, wherein it is constructed by transforming transformant A with plasmid pINA1269-HMG1-ERG12, the nucleotide sequence of said plasmid pINA1269-HMG1-ERG12 is shown in sequence table SEQ ID No. 4; the transformant A is a uracil and leucine auxotrophic yarrowia lipolytica transformed from the plasmid pINA1312LN (Yarrowia lipolytica) Polf was constructed.
4. A method for producing limonene, characterized in that it comprises the following steps: culturing the yarrowia lipolytica genetically engineered bacterium producing limonene according to any one of claims 1 to 3, adding dodecane to perform two-phase fermentation to obtain a fermentation broth, and extracting the dodecane phase of the fermentation broth.
5. The method of claim 4, wherein said culturing is further supplemented with pyruvate.
6. The method according to claim 5, wherein the pyruvic acid is at a concentration of 2 to 8g/L as a ratio of the mass of pyruvic acid to the volume of the cultured system before the addition of pyruvic acid.
7. The method according to claim 6, wherein the pyruvic acid is at a concentration of 4g/L as a ratio of the mass of pyruvic acid to the volume of the cultured system before the addition of pyruvic acid.
8. The method according to any one of claims 4 to 7, wherein the dodecane is present in a concentration of 2 to 10% by volume of the dodecane with respect to the volume of the fermentation broth before the addition of the dodecane.
9. The method of claim 8, wherein the dodecane is present in a concentration of 6 to 8 percent, by volume, of the dodecane with the fermentation broth prior to addition of the dodecane.
10. Use of the genetically engineered yarrowia lipolytica for producing limonene of any one of claims 1-3 in the preparation of limonene.
11. A method for preparing a yarrowia lipolytica gene engineering bacterium capable of producing limonene, which is characterized by comprising the following steps:
(1) constructing a recombinant vector containing an optimized gene LS and an optimized gene NDPS1, wherein the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2; the recombinant vector is a plasmid pINA1312LN, and the nucleotide sequence of the plasmid pINA1312LN is shown as a sequence table SEQ ID No. 5;
(2) transforming the recombinant vector prepared in step (1) into uracil and leucine auxotrophic yarrowia lipolytica (Yarrowia lipolytica) Polf, obtaining a transformant.
12. The method of claim 11, further comprising the steps of:
(3) constructing a plasmid pINA1269-HMG1 and a plasmid pINA1269-HMG1-ERG12, and transforming the plasmid pINA1269-HMG1 or the plasmid pINA1269-HMG1-ERG12 into the transformant obtained in the step (2), wherein the nucleotide sequence of the plasmid pINA1269-HMG1 is shown in a sequence table SEQ ID No. 3; the nucleotide sequence of the plasmid pINA1269-HMG1-ERG12 is shown in a sequence table SEQ ID No. 4.
13. A recombinant vector for preparing the yarrowia lipolytica genetically engineered bacterium capable of producing limonene according to any one of claims 1-3, characterized by comprising an optimized gene LS and an optimized gene NDPS1, wherein the nucleotide sequence of the optimized gene LS is shown in a sequence table SEQ ID No: 1; the nucleotide sequence of the optimized gene NDPS1 is shown in a sequence table SEQ ID No. 2; the recombinant vector is a plasmid pINA1312LN obtained by introducing the optimized gene LS and the optimized gene NDPS1 into a plasmid pINA1312, and the nucleotide sequence of the plasmid pINA1312LN is shown in a sequence table SEQ ID No. 5.
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| 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 |
| 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 |
| CN110499259B (en) * | 2019-07-22 | 2021-07-27 | 浙江工业大学 | Yarrowia lipolytica YW100-1 and application thereof |
| 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 |
| 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 |
| CN112626103B (en) * | 2020-10-21 | 2022-09-27 | 天津科技大学 | Yarrowia lipolytica engineering bacterium for producing limonene and application |
| CN115704038A (en) * | 2021-08-10 | 2023-02-17 | 西北农林科技大学 | Gene, recombinant vector, engineering bacterium and application thereof |
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