CN111235047A - Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method - Google Patents
Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method Download PDFInfo
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Abstract
The invention discloses a recombinant yarrowia lipolytica for heterologously synthesizing α -citronellol and ursolic acid and a construction method thereof, wherein the construction method comprises the following steps of introducing an optimized mixed citronellol synthase encoding gene, an optimized ursolic acid synthase encoding gene and an optimized cytochrome-NADPH-reductase 1 encoding gene into yarrowia lipolytica to obtain a recombinant bacterium 2, and experiments prove that the yield of α -citronellol and ursolic acid is improved by obtaining the recombinant yarrowia lipolytica for producing α -citronellol and ursolic acid through a homologous recombination method, and the method provides a basis for artificially synthesizing α -citronellol and ursolic acid.
Description
Technical Field
The invention relates to the technical field of biology, in particular to recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumaryl alcohol and ursolic acid and a construction method thereof.
Background
Ursolic Acid (UA), a pentacyclic triterpene with a molecular formula of C30H48O3The molecular weight is 456.7, the ultraviolet absorption material is acid, white needle-shaped crystal, the melting point is 283-285 ℃, and the maximum ultraviolet absorption wavelength is about 450 nm. Ursolic acid is a triterpene compound composed of C-30 isoprenoid compounds. Insoluble in water, soluble in hot ethyl acetate and ethanolic sodium hydroxide solution. Biosynthesis of ursolic acid is mainly through folding and cyclization of squalene to form dammarane cation, which further undergoes ring expansion reaction and additional cyclization to form the fifth ring. The ursolic acid contains three oxygen atoms and can be used as a double ligand or a triple ligand, so that a lone pair of electrons can be easily transferred to a metal atom.
Ursolic acid can exist in both free acid and saponin forms. Ursolic acid is detected in many medicinal plants, such as loquat, rosemary, oldenlandia, glossy privet, Haemophilus, bearberry, cranberry, photinia glabra, etc., and in addition, ursolic acid is also detected in wax coatings of fruits such as apple, pear, plum, etc. Due to its various biological effects of sedation, blood sugar reduction, anti-inflammatory, antibacterial, antiulcer, antioxidant, antidiabetic etc., ursolic acid is widely used in medicine and cosmetics. Over the last decades, extensive research has been conducted to elucidate the medicinal value of ursolic acid, which has been shown to regulate signaling pathways associated with cancer, cardiovascular and neurological damage. Moreover, researchers have been able to synthesize derivatives of ursolic acid by chemical modification methods to further improve its therapeutic effect.
Yarrowia lipolytica is an oleaginous yeast, intracellular metabolic processes contain a large amount of acetyl-CoA production, and acetyl-CoA is a precursor substance shared in both lipid synthesis pathway and terpenoid pathway, which makes yarrowia lipolytica a potential strain for heterologous synthesis of terpenoids, like saccharomyces cerevisiae, yarrowia lipolytica contains an endogenous Mevalonate (MVA) pathway, which provides precursors for terpenoid synthesis, IPP and DMAPP, as well as squalene and 2, 3-oxidosqualene, which provides precursors for triterpene synthesis, and in recent years research on terpenoid synthesis by yarrowia lipolytica has increased 2016. shihiki task group by introducing both heterologous NPP synthase and limonene synthase NDPS1 (catalyzing DMAPP and IPP to form NPP), metabolic pathways for producing limonene in yarrowia lipolytica have been constructed, and further the yield of yarrowia lipolytica is increased by overexpressing 3-hydroxy-3-methylglutaryl CoA (HMG-glutaryl CoA) reductase genes, by optimizing the yield of yarrowia lipolytica in shake flask 56, by fermentation using the highest yield of phytolacca lipoxygenase synthesis of phytol 56 g, and fermentation of phytol.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method for heterogeneously synthesizing α -citronellol recombinant yarrowia lipolytica.
The second purpose of the invention is to provide a construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -citronellol and ursolic acid.
The third purpose of the invention is to provide a construction method of a second recombinant yarrowia lipolytica for heterologous synthesis of α -citronellol and ursolic acid.
The fourth purpose of the invention is to provide a construction method of a third recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The fifth purpose of the invention is to provide a construction method of a fourth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The sixth purpose of the invention is to provide a construction method of the fifth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The seventh purpose of the invention is to provide a construction method of the sixth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The eighth purpose of the invention is to provide a construction method of the seventh recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The ninth purpose of the present invention is to provide a construction method of the eighth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The tenth object of the present invention is to provide a method for constructing the ninth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The eleventh object of the present invention is to provide a method for constructing the tenth recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The twelfth object of the present invention is to provide a method for constructing the eleventh recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid.
The thirteenth object of the present invention is to provide a recombinant yarrowia lipolytica α -balsamic alcohol constructed by the above method for constructing recombinant yarrowia lipolytica for the heterologous synthesis of α -balsamic alcohol.
The fourteenth object of the present invention is to provide the recombinant yarrowia lipolytica for heterologous synthesis of α -balsam pear acid constructed by the construction method of the recombinant yarrowia lipolytica for heterologous synthesis of α -balsam pear alcohol and ursolic acid.
The fifteenth objective of the present invention is to provide the use of the recombinant yarrowia lipolytica fermentation of the above-mentioned heterologous syntheses of α -citronellol and ursolic acid for the production of α -citronellol and ursolic acid.
The technical scheme of the invention is summarized as follows:
a construction method of α -balsamic alcohol recombinant Yarrowia lipolytica by heterologous synthesis comprises the following steps of introducing an optimized mixed balsamic alcohol synthase encoding gene into Yarrowia lipolytica ATCC201249 to obtain a recombinant bacterium 1, wherein the nucleotide sequence of the optimized mixed balsamic alcohol synthase encoding gene is shown in SEQ ID NO. 1.
A first construction method of recombinant Yarrowia lipolytica for heterologous synthesis of α -citronellol and ursolic acid comprises the following steps of introducing an optimized mixed citronellol synthase encoding gene, an optimized ursolic acid synthase encoding gene and an optimized cytochrome-NADPH-reductase 1 encoding gene into Yarrowia lipolytica ATCC201249 to obtain a recombinant bacterium 2, wherein the nucleotide sequence of the optimized mixed citronellol synthase encoding gene is shown in SEQ ID NO.1, the nucleotide sequence of the optimized ursolic acid synthase encoding gene is shown in SEQ ID NO.2, and the nucleotide sequence of the optimized cytochrome-NADPH-reductase 1 encoding gene is shown in SEQ ID NO. 3.
The second construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -citronellol and ursolic acid comprises the following steps of introducing a gene HMG1 encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase into the recombinant bacterium 2 to obtain the recombinant bacterium 3, wherein the nucleotide sequence of the gene HMG1 is shown as SEQ ID NO. 4.
The third method for constructing the recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid comprises the following steps:
(1) removing 1500 nucleotides from the 5' -end of gene HMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase to obtain truncated gene tHMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase; the nucleotide sequence of the gene HMG1 is shown as SEQ ID NO. 4; the nucleotide sequence of the gene tHMG1 is shown as SEQ ID NO. 5;
(2) the gene tHMG1 was introduced into recombinant bacterium 2 to obtain recombinant bacterium 4.
A fourth construction method of the recombinant yarrowia lipolytica for the heterologous synthesis of α -coumarol and ursolic acid comprises the following steps of introducing an encoding gene ERG1 of oxidosqualene cyclase into the recombinant bacterium 2 to obtain a recombinant bacterium 5, wherein the nucleotide sequence of the gene ERG1 is shown as SEQ ID NO. 6.
The fifth construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -citronellol and ursolic acid comprises the following steps of introducing an encoding gene ERG8 of phosphomevalonate kinase into the recombinant bacterium 2 to obtain a recombinant bacterium 6, wherein the nucleotide sequence of the gene ERG8 is shown as SEQ ID No. 7.
A sixth construction method of recombinant yarrowia lipolytica for heterologous synthesis of α -citronellol and ursolic acid comprises the following steps of introducing a squalene synthase encoding gene ERG9 into a recombinant bacterium 2 to obtain a recombinant bacterium 7, wherein the nucleotide sequence of the gene ERG9 is shown in SEQ ID NO. 8.
The seventh construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -citronellol and ursolic acid comprises the following steps of introducing an encoding gene ERG10 of acetoacetyl-CoA thiolase into the recombinant bacterium 2 to obtain a recombinant bacterium 8, wherein the nucleotide sequence of the gene ERG10 is shown as SEQ ID No. 9.
The eighth construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid comprises the following steps of introducing a gene ERG12 encoding mevalonate kinase into the recombinant bacterium 2 to obtain a recombinant bacterium 9, wherein the nucleotide sequence of the gene ERG12 is shown in SEQ ID No. 10.
The ninth construction method of the recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid comprises the following steps of introducing a gene ERG19 encoding mevalonate diphosphate decarboxylase into the recombinant bacterium 2 to obtain a recombinant bacterium 10, wherein the nucleotide sequence of the gene ERG19 is shown as SEQ ID No. 11.
The tenth construction method of the recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid comprises the following steps of introducing an encoding gene ERG20 of farnesyl pyrophosphate synthase into the recombinant bacterium 2 to obtain a recombinant bacterium 11, wherein the nucleotide sequence of the gene ERG20 is shown as SEQ ID No. 12.
An eleventh construction method of the recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid comprises the step of introducing an expression cassette of an encoding gene IDI of isopentenyl pyrophosphate isomerase into the recombinant bacterium 2 to obtain the recombinant bacterium 12, wherein the nucleotide sequence of the gene IDI is shown in SEQ ID No. 13.
The recombinant yarrowia lipolytica of α -balsamic alcohol constructed by the construction method of the recombinant yarrowia lipolytica of the heterologous synthesis α -balsamic alcohol.
The recombinant yarrowia lipolytica for heterologous synthesis of α -balsam alcohol and ursolic acid is constructed by the construction method of the recombinant yarrowia lipolytica for heterologous synthesis of α -balsam alcohol and ursolic acid.
The use of recombinant yarrowia lipolytica fermentation of the above heterologous synthesis of α -balsam alcohol and ursolic acid for the production of α -balsam alcohol and ursolic acid.
Experiments prove that the recombinant yarrowia lipolytica for producing α -balsamic alcohol and ursolic acid obtained by the homologous recombination method improves the yield of α -balsamic alcohol and ursolic acid, and the method provides a basis for artificially synthesizing α -balsamic alcohol and ursolic acid.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The experimental procedures used in the following examples are all conventional ones unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The original strain is Yarrowia lipolytica ATCC201249, hereinafter abbreviated as Yarrowia lipolytica.
Time of purchase of yarrowia lipolytica, 2017.6
Website address: https:// www.atcc.org
Example 1 construction of heterologous Synthesis α -Carcinol recombinant yarrowia lipolytica YL-AAS (recombinant bacterium 1)
First, module construction
mixededAS gene (GenBank: AFJ19235.1) from Catharanthus roseus (Catharanthus roseus) is responsible for codon optimization and total chemical synthesis of yarrowia lipolytica by Wuhan Kingrui to obtain optimized mixedvanillyl alcohol synthase encoding gene, and the nucleotide sequence of the optimized mixedvanillyl alcohol synthase encoding gene is shown as SEQ ID NO. 1;
the promoter TEF1(SEQ ID NO.14), the terminator XPR2(SEQ ID NO.17), rDNA-up (SEQ ID NO.22) and rDNA-down (SEQ ID NO.23) are all from yarrowia lipolytica ATCC201249 genome, and the sequence of the screening marker gene URA3(SEQ ID NO.20) is from yarrowia lipolytica ATCC201249 and is synthesized by a chemical synthesis method by K.K.;
taking yarrowia lipolytica ATCC201249 genome as a template,
amplifying rDNA-up by using rDNA-up-F (SEQ ID NO.24) and rDNA-up-R-tef (SEQ ID NO.25) as primers; TEF1p-F-rdnaup (SEQ ID NO.26) and TEF1 p-R-opylaAAS (SEQ ID NO.27) are used as primers to amplify a promoter TEF 1; using opYLAAS-F-tef (SEQ ID NO.28) and opYLAAS-R-xpr (SEQ ID NO.29) as primers to amplify AAS (AAS is short for optimized mixed resinol synthase coding gene); XPR2 t-F-opylaAAS (SEQ ID NO.30) and XPR2t-R-ura (SEQ ID NO.31) are used as primer amplification terminators XPR 2; URA3 is amplified by using URA-F-xpr (SEQ ID NO.32) and URA-R-rdnadown (SEQ ID NO.33) as primers; rDNA-down was amplified using rDNA-F-ura (SEQ ID NO.34) and rDNA-down-R (SEQ ID NO.35) as primers.
The PCR enzyme used in the present invention is that of Nanjing NuoZan Biotech Co., Ltd
Max Super-Fidelity polymerase. A50. mu.L PCR amplification system was as follows: DNA template, 1. mu.L; 2 μ L of each of the front lead (10 μ M) and the rear lead (10 μ M); dNTP (10mM), 1. mu.L; 2 × Phanta Max Buffer, 25 μ L;
max Super-Fidelity polymerase, 1 μ L; finally, 50 mu L of double distilled water is used for supplementing. An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, and extension at 72 ℃ for 1min (34 cycles); extension at 72 ℃ for 5min (1 cycle).
The fusion PCR system used in the invention is as follows: the total amount of DNA fragments is 800ng, and the molar ratio is 1: 1; dNTP (10mM), 1. mu.L; 2 × Phanta Max Buffer, 25 μ L;
max Super-Fidelity polymerase, 1 μ L; finally, 50 mu L of double distilled water is used for supplementing. An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 1min (11 cycles), and extension at 72 ℃ for 5min (1 cycle).
Finally obtaining DNA fragments used for transforming yarrowia lipolytica, and respectively connecting with gene AAS expression cassette P of upstream homology arm rDNaupTEF1-AAS-TXPR2And a downstream homology arm URA3-rDNAdown containing the selection marker gene URA 3.
Yarrowia lipolytica transformation
The initial strain yarrowia lipolytica ATCC201249 was cultured in YPD medium for 12 hours, and 300. mu.L of the initial strain was added to 3mL of fresh YPD medium and cultured for 5 hours. Centrifuging at 3000rpm for 5min, collecting thallus, discarding supernatant, and sterilizing with ddH2The cells were washed with O, centrifuged at 3000rpm for 5min, the cells were collected, and the supernatant was discarded. Then, 1mL of 100mM lithium acetate was added to the cells, the cells were suspended uniformly by gentle pipetting, the cells were left at room temperature for 5min and then centrifuged at 3000rpm at room temperature for 5min, and the cells were collected to prepare yeast competent cells. The transformation mixed system comprises 240 mu L of PEG (50% W/V), 36 mu L of 1.0M lithium acetate and 10 mu L of ss-DNA, and transformation fragments are respectively connected with a gene AAS expression cassette P of an upstream homology arm rDNaupTEF1-AAS-TXPR2And two fragments containing the downstream homology arm URA3-rDNAdown of the selection marker gene URA3, each 400 ng. Finally using sterilized ddH2The content of O is filled to 360 mu L. Adding the above components into the just-prepared yarrowia lipolytica competent cell in sequence, blowing and beating uniformly with a pipette, placing in 42 deg.C water bath for 30min, centrifuging at 4000rpm for 2min, discarding supernatant, adding 1mLYPD liquid culture medium, and culturing at 30 deg.C and 220rpm for 2 h. Then centrifuging at 4000rpm for 5min at normal temperature, discarding the supernatant, washing with sterile water for 2 times, finally resuspending the cells with 100 μ L of sterile water, and screening by coating URA-deficient plates. The screening culture condition is 30 ℃, and the culture lasts more than 48 h. And (3) selecting a transformant for overnight culture, extracting a genome, verifying by pcr to obtain a correct clone of a correct target fragment, and naming the correct clone as a strain YL-AAS (recombinant strain 1).
Example 2 construction of recombinant yarrowia lipolytica YL-UA (recombinant bacterium 2) for heterologous Synthesis of α -Turesinol and Ursolic acid
mixededAS gene (GenBank: AFJ19235.1) from Catharanthus roseus (Catharanthus roseus) is responsible for codon optimization and total chemical synthesis of yarrowia lipolytica by Wuhan Kingrui to obtain optimized mixedvanillyl alcohol synthase encoding gene, and the nucleotide sequence of the optimized mixedvanillyl alcohol synthase encoding gene is shown as SEQ ID NO. 1;
CYP716A12 (GenBank: CBN88268.1) derived from Medicago truncatula (Medicago truncatula) is responsible for codon optimization and full chemical synthesis of yarrowia lipolytica by Wuhan Kingkurui company to obtain an optimized ursolic acid synthase coding gene, and the nucleotide sequence of the optimized ursolic acid synthase coding gene is shown as SEQ ID NO. 2.
The coding gene AtCPR1 of cytochrome-NADPH-reductase 1 is derived from Arabidopsis thaliana, the gene is synthesized by Wuhan King Kerui bioengineering GmbH by a chemical synthesis method, and codon optimization is carried out on yarrowia lipolytica to obtain the optimized coding gene of cytochrome-NADPH-reductase 1, and the nucleotide sequence of the optimized coding gene of cytochrome-NADPH-reductase 1 is shown as SEQ ID NO. 3.
The promoters TEF1(SEQ ID NO.14), EXP1(SEQ ID NO.15), GPD1(SEQ ID NO. 16);
terminator XPR2(SEQ ID NO.17), MIG1(SEQ ID NO.18), LIP2(SEQ ID NO.19), rDNA-up (SEQ ID NO.22) and rDNA-down (SEQ ID NO.23) are all from yarrowia lipolytica ATCC201249 genome, and screening marker gene URA3(SEQ ID NO.20) sequence is from yarrowia lipolytica ATCC201249, and the genes are synthesized by Wuhan gold Kerre bioengineering GmbH by a chemical synthesis method;
using yarrowia lipolytica ATCC201249 genome as template,
amplifying rDNA-up by using rDNA-up-F (SEQ ID NO.24) and rDNA-up-R-tef (SEQ ID NO.25) as primers; TEF1p-F-rdnaup (SEQ ID NO.26) and TEF1 p-R-opylaAAS (SEQ ID NO.27) are used as primers to amplify a promoter TEF 1; using opYLAAS-F-tef (SEQ ID NO.28) and opYLAAS-R-xpr (SEQ ID NO.29) as primers to amplify AAS; XPR2 t-F-opylaAAS (SEQ ID NO.30) and XPR2t-R-exp (SEQ ID NO.36) are used as primer amplification terminators XPR 2; EXP1p-F-xpr (SEQ ID NO.37) and EXP1 p-R-yCYP 716A12(SEQ ID NO.38) are used as primers to amplify a promoter EXP 1; the optimized ursolic acid synthase coding gene CYP716A12 is amplified by taking YLYP 716A12-F-exp (SEQ ID NO.39) and YLYP 716A12-R-mig (SEQ ID NO.40) as primers; MIG1 t-F-yCYP 716A12(SEQ ID NO.41) and MIG1t-R-gpd (SEQ ID NO.42) are used as primers to amplify a terminator MIG 1; the GPDp-F-mig (SEQ ID NO.43) and the GPDp-R-ylatr1(SEQ ID NO.44) are used as primers to amplify a promoter GPD 1; amplifying an optimized cytochrome-NADPH-reductase 1 encoding gene AtCPR1 by using YLATR1-F-gpd (SEQ ID NO.45) and YLATR1-R-lip (SEQ ID NO.46) as primers; amplifying a terminator LIP2 by using LIP2t-F-ylatr1(SEQ ID NO.47) and LIP2t-R-ura (SEQ ID NO.48) as primers; URA3 was amplified using URA3-F-lip (SEQ ID NO.49) and URA-R-rDNAdown (SEQ ID NO.33) as primers, and rDNA-down was amplified using rDNdowno-F-URA (SEQ ID NO.34) and rDNdownR (SEQ ID NO.35) as primers.
The PCR enzyme used in the present invention is that of Nanjing NuoZan Biotech Co., Ltd
Max Super-Fidelity polymerase. A50. mu.L PCR amplification system was as follows: DNA template, 1. mu.L; 2 μ L of each of the front lead (10 μ M) and the rear lead (10 μ M); dNTP (10mM), 1. mu.L; 2 × Phanta Max Buffer, 25 μ L;
max Super-Fidelity polymerase, 1 μ L; finally, 50 mu L of double distilled water is used for supplementing. An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, and extension at 72 ℃ for 1min (34 cycles); extension at 72 ℃ for 5min (1 cycle).
The fusion PCR system used in the invention is as follows: the total amount of DNA fragments is 800ng, and the molar ratio is 1: 1; dNTP (10mM), 1. mu.L; 2 × Phanta Max Buffer, 25 μ L;
max Super-Fidelity polymerase, 1 μ L; finally supplementing with double distilled waterThe volume is 50 mu L. An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 1min (11 cycles), and extension at 72 ℃ for 5min (1 cycle).
Finally obtaining DNA fragments used for transforming yarrowia lipolytica, and respectively connecting with gene AAS expression cassette P of upstream homology arm rDNaupTEF1-AAS-TXPR2Cytochrome CYP716A12 encoding Gene expression cassette PEXP1p-CYP716A12-TMIG1tCytochrome NADPH reductase 1 encoding Gene expression cassette PGPD1p-AtCPR1-TLIP2tAnd rDNA site downstream homology arm URA3-rDNA-down containing selection marker gene URA 3;
using the same yeast transformation method as in example 1, AAS expression cassettes P of genes respectively linked to upstream homology arms rDNaup were transformedTEF1-AAS-TXPR2Cytochrome CYP716A12 encoding Gene expression cassette PEXP1p-CYP716A12-TMIG1tCytochrome NADPH reductase 1 encoding Gene expression cassette PGPD1p-AtCPR1-TLIP2tAnd four fragments containing homologous arm URA3-rDNA-down at the downstream of rDNA locus of selection marker gene URA3, each 400 ng. Culturing on URA-deleted yeast synthetic culture medium, selecting culture condition of culturing at 30 deg.C for more than 48h, selecting transformant for overnight culture, extracting genome, verifying with pcr to obtain correct clone of all correct target fragments, and naming the correct clone as strain YL-UA (recombinant bacterium 2).
Example 3 cloning of Gene elements and construction of plasmids containing the corresponding Gene elements
PCR amplification gene element
HMG1, tHMG1, ERG1, ERG8, ERG9, ERG10, ERG12, ERG19, ERG20 and IDI were amplified respectively using yarrowia lipolytica ATCC201249 genome as a template and using the primers shown in Table 1. And purifying and recovering the gene fragment obtained after amplification for later use.
TABLE 1PCR amplification Gene primers Table
Hair brushThe PCR enzyme used is that of Nanjing Novozam Biotech Co., Ltd
Max Super-Fidelity polymerase. A50. mu.L PCR amplification system was as follows: DNA template, 1. mu.L; 2 μ L of each of the front lead (10 μ M) and the rear lead (10 μ M); dNTP (10mM), 1. mu.L; 2 × Phanta Max Buffer, 25 μ L;
max Super-Fidelity polymerase, 1 μ L; finally, 50 mu L of double distilled water is used for supplementing. An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, and extension at 7 ℃ for 1min (34 cycles); extension at 72 ℃ for 5min (1 cycle).
Second, pHMG1 plasmid construction
The PmlI enzyme cuts the plasmid pINA1269 (general Tatin biotechnology (Beijing) Co., Ltd.), and the fragment is purified and recovered to be about 7259 bp.
The plasmid was constructed using a one-step cloning kit of Nanjing Novozam Biotechnology Co., Ltd, and a reaction system shown in the following table was prepared in an ice-water bath.
TABLE 2pHMG1 plasmid construction reaction System
After the system is prepared, a micro liquid transfer gun is used for gentle blowing and even mixing. The reaction mixture was left at 37 ℃ for 30 min. Immediately thereafter, the reaction was cooled in an ice-water bath for 5 min.
Chemical transformation method (Heat shock method) of competent cells of Escherichia coli
1. Taking out the Trans5 α chemical competent cell finished product from a refrigerator at the temperature of-80 ℃, placing the finished product in an ice box for thawing for 10min, and sucking 50 mu L of bacterial liquid into a sterilized 1.5mL PE centrifuge tube in an aseptic environment;
2. adding 5 mu L of recombinant system solution into a centrifuge tube, gently mixing uniformly, and placing in an ice box for 30 min;
3. taking out the centrifugal tube from the ice box, thermally shocking in a constant-temperature water bath at 42 ℃ for 30sec, taking out and immediately placing in the ice box for 2 min; 4. adding 500 mu L of sterilized LB liquid culture medium into a centrifuge tube, and putting the centrifuge tube into a shaking table with the set conditions of 37 ℃ and 200rpm for resuscitation for 1 h;
5. centrifuging for 3min under 4000rmp, sucking out 350 μ L of supernatant, collecting and resuspending thallus;
6. the bacterial liquid was spread on LB plate medium containing antibiotic Amp under aseptic conditions, and cultured overnight in an incubator at 37 ℃ by inversion.
The colony PCR of the transformant is selected for verification, the obtained correct target fragment is the correct transformant, and the plasmid which is verified to be correct by single enzyme digestion of the plasmid PmlI is extracted and named as the correct plasmid pHMG 1.
Plasmid construction of ptHMG1, pERG1, pERG8, pERG9, pERG10, pERG12, pERG19, pERG20 and pIDI
By using the same method of the construction of the plasmid of the second pHMG1, the amplified gene fragments of tHMG1, ERG1, ERG8, ERG9, ERG10, ERG12, ERG19, ERG20 and IDI are recombined with pINA1269 plasmid after PmlI linearization by using a one-step cloning kit respectively, and are transformed by escherichia coli, the transformant is verified, and plasmid enzyme digestion verification is extracted to obtain correct recombinant plasmids which are named as ptHMG1, pERG1, pERG8, pERG9, pERG10, pERG12, pERG19, pERG20 and pIDI respectively.
Example 4 construction of recombinant yarrowia lipolytica YL-UA-01 (recombinant bacterium 3) for heterologous Synthesis of α -Turesinol and Ursolic acid
The BsrGI single-restriction enzyme plasmid pHMG1 was used to purify and recover a linearized plasmid fragment (containing the expression cassette P) of about 10259bphp4d-HMG1-TXPR2)。
The preparation and transformation of YL-UA competent cells were carried out in the same manner as in example 1, and then transferred to linearized pHMG1, followed by selection and culture in LEU and URA-deficient medium to obtain transformants. The screening culture condition is 30 ℃, the culture is carried out for more than 48h, a transformant is selected for overnight culture, a genome is extracted, check primers are used for verification, and the obtained correct target fragment is a correct clone and is named as a strain YL-UA-01 (recombinant strain 3). The nucleotide sequence of LEU2 is shown in SEQ ID NO. 21.
Example 5 construction of recombinant yarrowia lipolytica YL-UA-02 (recombinant bacterium 4) to YL-UA-10 (recombinant bacterium 12) for heterologous Synthesis of α -Tupistinol and Ursolic acid
Plasmid ptHMG1, pERG1, pERG8, pERG9, pERG10, pERG12, pERG19, pERG20 and pIDI were digested with BsrGI, and a linearized plasmid fragment of about 10259bp was purified and recovered.
The preparation and transformation of YL-UA competent cells were carried out in the same manner as in example 1, and transformants were obtained by screening and culturing linearized ptHMG1, pERG1, pERG8, pERG9, pERG10, pERG12, pERG19, pERG20 and pIDI in a LEU and URA-deleted medium. Screening culture conditions are 30 ℃, culturing for more than 48h, selecting transformants for overnight culture, extracting genome, verifying by check primer, and obtaining correct target fragments which are correct clones, and are named as strains YL-UA-02 (recombinant bacteria 4), YL-UA-03 (recombinant bacteria 5), YL-UA-04 (recombinant bacteria 6), YL-UA-05 (recombinant bacteria 7), YL-UA-06 (recombinant bacteria 8), YL-UA-07 (recombinant bacteria 9), YL-UA-08 (recombinant bacteria 10), YL-UA-09 (recombinant bacteria 11) and YL-UA-10 (recombinant bacteria 12).
Example 6 recombinant production of α -Turkey alcohol and Ursolic acid
1. Recombinant bacterium culture and product extraction
The recombinant yarrowia lipolytica strains YL-AAS, YL-UA, YL-UA-01, YL-UA-02, YL-UA-03, YL-UA-04, YL-UA-05, YL-UA-06, YL-UA-07, YL-UA-08, YL-UA-09, YL-UA-10 obtained in examples 1, 2, 4, 5, together with ATCC201249, were activated on solid screening media; fermentation seed solutions were prepared in YPD liquid medium (30 ℃ C., 220rmp, 16 hours), and the cells were collected by centrifugation, transferred to a 250mL Erlenmeyer flask containing 30mL YPD fermentation medium, and cultured while adjusting OD to 0.5 (30 ℃ C., 220 rmp). Centrifuging after 6d, collecting cells, adding acetone, performing ultrasonic disruption in ice water bath for 10min, centrifuging at 12000rpm at normal temperature for 2min, and collecting supernatant and passing through 0.22mm organic membrane for use.
2. LC-MS identification α -citronellol and ursolic acid
α qualitative determination of balsam alcohol and ursolic acid by LC-MS liquid chromatography with sample volume of 5 μ L, ZoRBAX SB-Aq, and mobile phase of 90% acetonitrileFlow rate: 0.2 mL/min. Mass spectrum conditions: the atomizing gas and the drying gas are both N2(ii) a Collision voltage: -70V; spraying voltage: 3.8 kV; an ion source: APCI; ion source temperature: 120 ℃; desolventizing temperature: 300 ℃; post column effluent introduction ion source rate: 5 mu L/min; mass spectrometry scan mass number range: 200 and 1000 Da.
α quantification of citronellol and ursolic acid was performed by HPLC using liquid chromatography conditions of 20. mu.L sample size, ZORBAX SB-Aq Agilent, acetonitrile as a mobile phase 4:6, flow rate 1mL/min, α Standard of citronellol from Yunnan Xili Biotechnology Co., Ltd (www.biobiopha.com) and ursolic acid from Beijing Solebao science Co., Ltd (www.solarbio.cn).
3. Results
Yarrowia lipolytica ATCC201249 did not synthesize α -coumarol or ursolic acid.
Detection of an extraction product of YL-AAS (recombinant bacteria 1) and YL-UA (recombinant bacteria 2) by LC-MS:
the retention time of the extracted product of the recombinant bacterium 1 and the α -coumarol standard product is the same, and the mass spectrogram is the same, which shows that α -coumarol exists in the extracted product of the recombinant bacterium 1;
the retention time of the extracted product of the recombinant bacterium 2 is the same as that of the ursolic acid standard product, and the mass spectrogram is the same, which indicates that the extracted product of the recombinant bacterium 2 contains the ursolic acid.
The result shows that the fermentation yield of the recombinant bacterium 1 α -balsamic alcohol reaches 20.5mg/L, and the fermentation yield of YL-UA (recombinant bacterium 2) ursolic acid reaches 23.8 mg/L.
Based on YL-UA, recombinant bacteria for improving the yield of the ursolic acid of the target product are obtained by up-regulating the expression of HMG1, tHMG1, ERG1, ERG8, ERG9, ERG10, ERG12, ERG19, ERG20 and IDI genes in yarrowia lipolytica, and the yield of each recombinant bacterium after fermentation for 6 days is shown in Table 3. The data presented in the table are averaged over three parallel experiments.
TABLE 3 recombinant Ursolic acid yields
| Engineered bacterial strains | Up-regulated gene | Ursolic acid yield (mg/L) |
| YL-UA-01 (recombinant bacterium 3) | HMG1 | 49.7 |
| YL-UA-02 (recombinant bacterium 4) | tHMG1 | 54.2 |
| YL-UA-03 (recombinant bacterium 5) | ERG1 | 25.8 |
| YL-UA-04 (recombinant bacterium 6) | ERG8 | 24.3 |
| YL-UA-05 (recombinant bacterium 7) | ERG9 | 64.2 |
| YL-UA-06 (recombinant bacterium 8) | ERG10 | 40.1 |
| YL-UA-07 (recombinant bacterium 9) | ERG12 | 38.9 |
| YL-UA-08 (recombinant bacterium 10) | ERG19 | 46.8 |
| YL-UA-09 (recombinant bacterium 11) | ERG20 | 26.9 |
| YL-UA-10 (recombinant bacterium 12) | IDI | 27.4 |
Sequence listing
<110> Tianjin university
<120> recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method
<160>69
<170>SIPOSequenceListing 1.0
<210>1
<211>2310
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atgtggaagc tgaaggtcgc ccagggtaac gacccttacc tgtactccac caacaacttc 60
gtcggccgac agtactggga gttccagccc gacgccggca cccccgagga gcgagaggag 120
gtcgagaagg cccgaaagga ttacgtcaac aacaagaagc tgcacggcat ccacccctgc 180
tctgatatgc tgatgcgacg acagctgatc aaggagtccg gcatcgacct gctgtccatc 240
ccccctctgc gactggacga gaacgagcag gttaactacg acgccgtcac caccgctgtc 300
aagaaggctc tgcgactgaa ccgagccatc caggctcatg acggtcactg gcctgctgag 360
aacgctggtt ctctgctgta caccccccct ctgatcatcg ctctgtacat ctccggcacc 420
atcgacacca tcctgaccaa gcagcacaag aaggagctga tccgattcgt ctacaaccac 480
cagaacgagg acggcggttg gggttcctac atcgagggtc attccaccat gatcggctcc 540
gtcctgtcct acgtcatgct gcgactgctg ggtgagggtc tggctgagtc cgacgacggt 600
aacggtgctg ttgagcgagg tcgaaagtgg atcttggacc acggcggtgc tgctggtatc 660
ccttcctggg gtaagaccta cctggccgtt ctgggtgtct acgagtggga gggttgcaac 720
cctctgcctc ctgagttctg gctgttccct tcctccttcc ccttccaccc tgctaagatg 780
tggatctact gccgatgcac ctacatgccc atgtcctacc tgtacggcaa gcgataccac 840
ggccctatca ccgatctggt cctgtctctg cgacaggaga tctacaacat cccctacgag 900
cagatcaagt ggaaccagca gcgacacaac tgctgcaagg aggacctgta ctacccccac 960
accctggttc aggacctggt ttgggacggt ctgcactact tctccgagcc tttcctgaag 1020
cgatggccct tcaacaagct gcgaaagcga ggcctgaagc gagtcgttga gctgatgcga 1080
tacggcgcta ccgagactcg attcatcacc accggcaacg gcgagaaggc tctgcagatt 1140
atgtcctggt gggccgagga ccctaacggt gacgagttta agcaccacct ggcccgaatc 1200
cccgactttc tgtggatcgc tgaggacggt atgaccgtcc agtccttcgg ttcccagctg 1260
tgggattgca tcctggctac ccaggctatc atcgccacca acatggtcga ggagtacggc 1320
gattccctga agaaggccca cttcttcatc aaggagtccc agatcaagga gaacccccga 1380
ggtgacttcc tgaagatgtg ccgacagttc accaagggcg cctggacctt ttccgaccag 1440
gatcatggct gcgtcgtctc tgactgcacc gctgaggctc tgaagtgcct gctgctgctg 1500
tctcagatgc cccaggatat cgtcggcgag aagcctgagg tcgagcgact gtacgaggct 1560
gttaacgtcc tgctgtacct gcagtcccga gtctctggtg gtttcgctgt ctgggagcct 1620
cctgttccta agccttacct ggagatgctg aacccctccg agatcttcgc cgacatcgtc 1680
gttgagcgag agcacatcga gtgcaccgct tctgtcatca agggcctgat ggccttcaag 1740
tgcctgcacc ctggtcaccg acagaaggag atcgaggact ccgtcgctaa ggccatccga 1800
tacctggagc gaaaccagat gcccgacggt tcttggtacg gcttctgggg tatctgcttc 1860
ctgtacggca ccttcttcac cctgtccggt ttcgcttccg ccggtcgaac ctacgataac 1920
tccgaggctg tccgaaaggg cgtcaagttc ttcctgtcca cccagaacga ggagggcggt 1980
tggggtgagt ccctggagtc ctgcccttcc gagaagttca cccctctgaa gggcaaccga 2040
accaacctgg tccagacctc ttgggctatg ctgggtctga tgttcggcgg tcaggctgag 2100
cgagatccta ctcctctgca ccgagctgct aagctgctga tcaacgccca gatggacaac 2160
ggcgacttcc ctcagcagga gatcaccggt gtctactgca agaactccat gctgcactac 2220
gccgagtacc gaaacatctt ccccctgtgg gccctgggtg agtaccgaaa gcgagtttgg 2280
ctgcccaagc accagcagct gaagatctaa 2310
<210>2
<211>1449
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atggtcctgt tcttctccct gtccctgctg ctgctgcctc tgctgctgct gtttgcttac 60
ttctcctaca ccaagcgaat cccccagaag gagaacgact ccaaggcccc cctgcctcct 120
ggtcagaccg gttggcctct gattggtgag accctgaact acctgtcctg cgtcaagtcc 180
ggcgtctctg agaacttcgt caagtaccga aaggagaagt actcccccaa ggtcttccga 240
acctccctgc tgggtgagcc tatggctatc ctgtgcggtc ctgagggtaa caagttcctg 300
tactccaccg agaagaagct ggtccaggtc tggttcccct cctctgttga gaagatgttc 360
ccccgatccc acggcgagtc taacgctgac aacttctcca aggtccgagg caagatgatg 420
ttcctgctga aggtcgatgg catgaagaag tacgtcggcc tgatggaccg agtcatgaag 480
cagttcctgg agaccgactg gaaccgacag cagcagatca acgtccacaa caccgtcaag 540
aagtacaccg tcaccatgtc ctgccgagtc ttcatgtcca tcgacgacga ggagcaggtc 600
acccgactgg gttcctccat tcagaacatc gaggccggcc tgctggctgt ccctatcaac 660
attcccggca ccgctatgaa ccgagccatc aagaccgtca agctgctgac ccgagaggtc 720
gaggctgtta tcaagcagcg aaaggtcgat ctgctggaga acaagcaggc ctcccagcct 780
caggatctgc tgtcccacct gctgctgact gctaaccagg acggtcagtt cctgtccgag 840
tccgatatcg cctcccacct gatcggtctg atgcagggtg gttacaccac cctgaacggc 900
actatcacct tcgtcctgaa ctacctggcc gagttccccg atgtctacaa ccaggtcctg 960
aaggagcagg tcgagatcgc taactccaag caccccaagg agctgctgaa ctgggaggat 1020
ctgcgaaaga tgaagtactc ctggaacgtc gcccaggagg tcctgcgaat tatccctcct 1080
ggcgtcggta ctttccgaga ggctatcacc gacttcacct acgccggtta cctgatcccc 1140
aagggttgga agatgcacct gatcccccac gacacccaca agaaccctac ctacttcccc 1200
tcccccgaga agttcgaccc tactcgattc gagggcaacg gccctgctcc ttacactttc 1260
acccccttcg gcggtggtcc tcgaatgtgc cctggtattg agtacgcccg actggtcatc 1320
ctgatcttca tgcacaacgt cgtcaccaac ttccgatggg agaagctgat ccccaacgag 1380
aagatcctga ccgaccccat cccccgattc gctcacggtc tgcctatcca tctgcaccct 1440
cacaactaa 1449
<210>3
<211>2079
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
atgacctccg ctctgtacgc ttccgacctg ttcaagcagc tgaagtccat catgggcacc 60
gactccctgt ccgatgacgt tgttctggtc atcgccacca cctccctggc cctggtcgcc 120
ggcttcgtcg tcctgctgtg gaagaagacc accgctgacc gatccggtga gctgaagccc 180
ctgatgatcc ctaagtccct gatggccaag gacgaggacg acgatctgga cctgggttct 240
ggtaagaccc gagtctccat cttcttcggc acccagaccg gtactgccga gggcttcgcc 300
aaggccctgt ccgaggagat caaggcccga tacgagaagg ccgctgtcaa ggttatcgac 360
ctggacgact acgccgccga tgatgatcag tacgaggaga agctgaagaa ggagaccctg 420
gccttcttct gcgtcgctac ttacggcgac ggcgagccca ccgacaacgc cgcccgattc 480
tacaagtggt tcaccgagga gaacgagcga gacatcaagc tgcagcagct ggcttacggc 540
gtcttcgccc tgggcaaccg acagtacgag catttcaaca agatcggcat cgtcctggac 600
gaggagctgt gtaagaaggg cgctaagcga ctgatcgagg tcggtctggg tgacgatgac 660
cagtctatcg aggacgactt caacgcctgg aaggagtccc tgtggtccga gctggataag 720
ctgctgaagg acgaggacga caagtccgtc gctaccccct acactgctgt catccctgag 780
taccgagtcg tcacccacga cccccgattt actacccaga agtccatgga gtccaacgtc 840
gccaacggca acaccactat cgacatccac cacccctgcc gagtcgatgt tgctgttcag 900
aaggagctgc acacccacga gtccgatcga agctgtatcc acctggagtt cgacatctcc 960
cgaaccggta tcacctacga gaccggtgac cacgtcggtg tttacgctga gaaccacgtc 1020
gagatcgtcg aggaggctgg taagctgctg ggtcattccc tggacctggt cttctccatc 1080
cacgctgaca aggaggacgg ctctcctctg gagtctgctg ttcctcctcc cttccctggt 1140
ccttgtaccc tgggcaccgg cctggcccga tacgccgacc tgctgaaccc ccctcgaaag 1200
tccgctctgg ttgctctggc tgcttacgct accgagcctt ctgaggctga gaagctgaag 1260
cacctgacct cccctgacgg taaggacgag tactcccagt ggatcgtcgc ctctcagcga 1320
tctctgctgg aggttatggc cgctttcccc tctgctaagc cccctctggg tgttttcttc 1380
gccgctatcg cccctcgact gcagcctcga tactactcta tctcctcctc cccccgactg 1440
gctccttccc gagtccacgt cacctccgct ctggtctacg gtcctactcc taccggtcga 1500
atccacaagg gcgtctgttc cacctggatg aagaacgccg tccccgctga gaagtcccat 1560
gagtgctctg gtgcccctat cttcatccga gcctccaact tcaagctgcc ctccaaccct 1620
tccaccccta tcgttatggt cggccctggt actggcctgg ctcctttccg aggtttcctg 1680
caggagcgaa tggccctgaa ggaggatggt gaggagctgg gttcctccct gctgtttttc 1740
ggctgccgaa accgacagat ggacttcatc tacgaggacg agctgaacaa cttcgtcgat 1800
cagggcgtca tctccgagct gatcatggct ttctcccgag agggcgctca gaaggagtac 1860
gttcagcaca agatgatgga gaaggccgcc caggtctggg atctgattaa ggaggagggc 1920
tacctgtacg tctgcggtga tgctaagggc atggcccgag atgtccaccg aactctgcac 1980
actatcgtcc aggagcagga gggtgtctcc tcttctgagg ctgaggctat cgtcaagaag 2040
ctgcagaccg agggccgata cctgcgagac gtctggtaa 2079
<210>4
<211>3000
<212>DNA
<213> Yarrowia lipolytica
<400>4
atgctacaag cagctattgg aaagattgtg ggatttgcgg tcaaccgacc catccacaca 60
gttgtcctga cgtccatcgt ggcgtcaacc gcatacctcg ccatcctcga cattgccatc 120
ccgggtttcg agggcacaca acccatctca tactaccacc ctgcagcaaa atcttacgac 180
aaccctgctg attggaccca cattgcagag gccgacatcc cttcagacgc ctaccgactt 240
gcatttgccc agatccgtgt cagtgatgtt cagggcggag aggcccccac catccctggc 300
gccgtggccg tgtctgatct cgaccacaga atcgtcatgg actacaaaca gtgggccccc 360
tggaccgcca gcaacgagca gatcgcctcg gagaaccaca tctggaagca ctccttcaag 420
gaccacgtgg ccttcagctg gatcaagtgg ttccgatggg cctacctgcg tttgtccact 480
ctcatccagg gggcagacaa cttcgacatt gccgtggtcg cccttggcta tcttgccatg 540
cactacacct tcttcagtct cttccgatcc atgcgaaagg ttggctcgca cttttggctt 600
gcctccatgg ctctggtctc ttccaccttc gctttcctgc ttgcggtggt ggcttcctct 660
agcctgggtt accgacctag catgatcacc atgtccgagg gcctgccctt cctcgtggtc 720
gccattggct ttgaccgaaa ggtcaacctg gctagcgagg tgctcacatc caagagcagc 780
cagctcgctc ccatggtgca ggtgatcaca aagatcgcct ccaaggcgct gtttgagtac 840
agccttgagg tggccgccct gtttgctggc gcctataccg gagttcctcg actgtcccag 900
ttttgcttct tatctgcttg gatcctcatc ttcgactaca tgtttttgct gaccttctac 960
tctgctgtcc ttgctatcaa gtttgagatc aatcacatta agcgaaaccg aatgatccag 1020
gatgctctca aggaggatgg tgtatctgct gctgttgccg agaaggtagc cgactcttct 1080
cccgacgcca agctcgaccg aaagtccgac gtttctcttt ttggagcctc tggcgccatt 1140
gcggtgttca agatcttcat ggtccttggg ttccttggtc tcaacctcat caacctgact 1200
gccatccctc accttggcaa ggcggccgcc gctgcccagt ctgtgactcc catcaccctc 1260
tcccccgagc ttctccatgc catccccgcc tctgtgcccg ttgttgtcac ctttgtgccc 1320
agcgttgtgt acgagcactc ccagctcatt ctgcagctgg aggacgccct cactaccttc 1380
ctggctgcct gctccaaaac tattggtgac cccgtcatct ccaagtacat cttcctgtgc 1440
ctgatggtct ccaccgccct gaacgtctac ctgtttggag ccacccgaga agttgtgcga 1500
acccagtctg tgaaggtggt tgagaagcac gttcctatcg tcattgagaa gcccagcgag 1560
aaggaggagg acacctcttc tgaagactcc attgagctga ctgtcggaaa gcagcccaag 1620
cccgtgaccg agacccgttc tctggacgac ctagaggcta tcatgaaggc aggtaagacc 1680
aagcttctgg aggaccacga ggttgtcaag ctctctctcg agggcaagct tcctttgtat 1740
gctcttgaga agcagcttgg tgacaacacc cgagctgttg gcatccgacg atctatcatc 1800
tcccagcagt ctaataccaa gactttagag acctcaaagc ttccttacct gcactacgac 1860
tacgaccgtg tttttggagc ctgttgcgag aacgttattg gttacatgcc tctccccgtt 1920
ggtgttgctg gccccatgaa cattgatggc aagaactacc acattcctat ggccaccact 1980
gagggttgtc ttgttgcctc aaccatgcga ggttgcaagg ccatcaacgc cggtggcggt 2040
gttaccactg tgcttactca ggacggtatg acacgaggtc cttgtgtttc cttcccctct 2100
ctcaagcggg ctggagccgc taagatctgg cttgattccg aggagggtct caagtccatg 2160
cgaaaggcct tcaactccac ctctcgattt gctcgtctcc agtctcttca ctctaccctt 2220
gctggtaacc tgctgtttat tcgattccga accaccactg gtgatgccat gggcatgaac 2280
atgatctcca agggcgtcga acactctctg gccgtcatgg tcaaggagta cggcttccct 2340
gatatggaca ttgtgtctgt ctcgggtaac tactgcactg acaagaagcc cgcagcgatc 2400
aactggatcg aaggccgagg caagagtgtt gttgccgaag ccaccatccc tgctcacatt 2460
gtcaagtctg ttctcaaaag tgaggttgac gctcttgttg agctcaacat cagcaagaat 2520
ctgatcggta gtgccatggc tggctctgtg ggaggtttca atgcacacgc cgcaaacctg 2580
gtgaccgcca tctaccttgc cactggccag gatcctgctc agaatgtcga gtcttccaac 2640
tgcatcacgc tgatgagcaa cgtcgacggt aacctgctca tctccgtttc catgccttct 2700
atcgaggtcg gtaccattgg tggaggtact attttggagc cccagggggc tatgctggag 2760
atgcttggcg tgcgaggtcc tcacatcgag acccccggtg ccaacgccca acagcttgct 2820
cgcatcattg cttctggagt tcttgcagcg gagctttcgc tgtgttctgc tcttgctgcc 2880
ggccatcttg tgcaaagtca tatgacccac aaccggtccc aggctcctac tccggccaag 2940
cagtctcagg ccgatctgca gcgtctacaa aacggttcga atatttgcat acggtcatag 3000
<210>5
<211>1503
<212>DNA
<213> Yarrowia lipolytica
<400>5
atgacccagt ctgtgaaggt ggttgagaag cacgttccta tcgtcattga gaagcccagc 60
gagaaggagg aggacacctc ttctgaagac tccattgagc tgactgtcgg aaagcagccc 120
aagcccgtga ccgagacccg ttctctggac gacctagagg ctatcatgaa ggcaggtaag 180
accaagcttc tggaggacca cgaggttgtc aagctctctc tcgagggcaa gcttcctttg 240
tatgctcttg agaagcagct tggtgacaac acccgagctg ttggcatccg acgatctatc 300
atctcccagc agtctaataccaagacttta gagacctcaa agcttcctta cctgcactac 360
gactacgacc gtgtttttgg agcctgttgc gagaacgtta ttggttacat gcctctcccc 420
gttggtgttg ctggccccat gaacattgat ggcaagaact accacattcc tatggccacc 480
actgagggtt gtcttgttgc ctcaaccatg cgaggttgca aggccatcaa cgccggtggc 540
ggtgttacca ctgtgcttac tcaggacggt atgacacgag gtccttgtgt ttccttcccc 600
tctctcaagc gggctggagc cgctaagatc tggcttgatt ccgaggaggg tctcaagtcc 660
atgcgaaagg ccttcaactc cacctctcga tttgctcgtc tccagtctct tcactctacc 720
cttgctggta acctgctgtt tattcgattc cgaaccacca ctggtgatgc catgggcatg 780
aacatgatct ccaagggcgt cgaacactct ctggccgtca tggtcaagga gtacggcttc 840
cctgatatgg acattgtgtc tgtctcgggt aactactgca ctgacaagaa gcccgcagcg 900
atcaactgga tcgaaggccg aggcaagagt gttgttgccg aagccaccat ccctgctcac 960
attgtcaagt ctgttctcaa aagtgaggtt gacgctcttg ttgagctcaa catcagcaag 1020
aatctgatcg gtagtgccat ggctggctct gtgggaggtt tcaatgcaca cgccgcaaac 1080
ctggtgaccg ccatctacct tgccactggc caggatcctg ctcagaatgt cgagtcttcc 1140
aactgcatca cgctgatgag caacgtcgac ggtaacctgc tcatctccgt ttccatgcct 1200
tctatcgagg tcggtaccat tggtggaggt actattttgg agccccaggg ggctatgctg 1260
gagatgcttg gcgtgcgagg tcctcacatc gagacccccg gtgccaacgc ccaacagctt 1320
gctcgcatca ttgcttctgg agttcttgca gcggagcttt cgctgtgttc tgctcttgct 1380
gccggccatc ttgtgcaaag tcatatgacc cacaaccggt cccaggctcc tactccggcc 1440
aagcagtctc aggccgatct gcagcgtcta caaaacggtt cgaatatttg catacggtca 1500
tag 1503
<210>6
<211>1470
<212>DNA
<213> Yarrowia lipolytica
<400>6
atggtcaccc aacagtctgc agcagagacc agcgccaccc agaccaacga gtacgacgtg 60
gtcattgtcg gagctggtat tgccgggccc gctctggccg tggctcttgg aaatcagggc 120
agaaaggttc ttgttgtgga acgagatctc tccgaaccgg accgaatcgt gggagagctg 180
cttcagcccg gaggagtcgc tgctctcaag actctgggtc tcggctcttg tatcgaggat 240
atcgacgcga tcccctgcca gggatacaac gtgatctact ctggagaaga gtgcgttctc 300
aaatacccca aggtcccccg agacatccag caggactaca acgagctgta cagaagcgga 360
aagtctgccg acatctccaa cgaggctccc cgaggagtat ccttccacca cggccgattt 420
gtcatgaact tgcgaagggc cgcacgagac acacccaatg tgactctgct ggaggccaca 480
gtcaccgagg tggtcaagaa cccttacacc ggccacatta ttggagtcaa gaccttctct 540
aaaactggag gcgccaaaat ctacaagcac ttctttgctc ctctcaccgt cgtctgtgat 600
ggaacttttt ccaagttccg aaaggacttt agcaccaaca agacgtctgt gcgttcgcat 660
ttcgccggtc tgattctcaa ggacgctgtt ctgccctccc cccagcatgg ccacgtgatt 720
ctgtcgccca actcgtgtcc cgttcttgtc taccaggttg gagctcgaga gacccgaatt 780
ctgtgtgaca ttcagggacc cgtcccctct aatgcaaccg gagccctcaa ggaacacatg 840
gagaagaacg tcatgcccca cctgcctaag tccatccagc cgtctttcca agccgctctc 900
aaggagcaga ccattcgagt catgcccaac tctttcctgt cggcctccaa gaacgatcac 960
cacggtttga ttctgctggg tgacgcactc aacatgcgac atccacttac cggaggagga 1020
atgaccgttg ctctcaatga tgcccttcta ctcagcagac ttctcaccgg cgttaacctg 1080
gaagacacct atgccgtgtc ctccgtcatg agctcgcagt tccactggca gcgaaaacac 1140
ctcgactcca tcgtcaacat tctctccatg gccctctact cgctcttcgc cgccgactcg 1200
gactacctgc gaatcctgca gctcggatgc ttcaactact tcaagctggg aggcatctgt 1260
gtggaccacc ccgtcatgct gttggctgga gttctccccc gacccatgta cctgtttacg 1320
catttcttcg tagtggccat ctacggcgga atctgcaaca tgcaggccaa cggcattgcc 1380
aagctgcccg cgtcgctact gcaatttgtc gcctctctgg tcaccgcttg catcgtcatc 1440
ttcccttaca tttggagcga gctgacttag 1470
<210>7
<211>1257
<212>DNA
<213> Yarrowia lipolytica
<400>7
atgaccacct attcggctcc gggaaaggcc ctcctttgcg gcggttattt ggttattgat 60
ccggcgtatt cagcatacgt cgtgggcctc tcggcgcgta tttacgcgac agtttcggct 120
tccgaggcct ccaccacctc tgtccatgtc gtctctccgc agtttgacaa gggtgaatgg 180
acctacaact acacgaacgg ccagctgacg gccatcggac acaacccatt tgctcacgcg 240
gccgtcaaca ccgttctgca ttacgttcct cctcgaaacc tccacatcaa catcagcatc 300
aaaagtgaca acgcgtacca ctcgcaaatt gacagcacgc agagaggcca gtttgcatac 360
cacaaaaagg cgatccacga ggtgcctaaa acgggcctcg gtagctccgc tgctcttacc 420
accgttcttg tggcagcttt gctcaagtca tacggcattg atcccttgca taacacccac 480
ctcgttcaca acctgtccca ggttgcacac tgctcggcac agaagaagat tgggtctgga 540
tttgacgtgg cttcggccgt ttgtggctct ctagtctata gacgtttccc ggcggagtcc 600
gtgaacatgg tcattgcagc tgaagggacc tccgaatacg gggctctgtt gagaactacc 660
gttaatcaaa agtggaaggt gactctggaa ccatccttct tgccgccggg aatcagcctg 720
cttatgggag acgtccaggg aggatctgag actccaggta tggtggccaa ggtgatggca 780
tggcgaaaag caaagccccg agaagccgag atggtgtgga gagatctcaa cgctgccaac 840
atgctcatgg tcaagttgtt caacgacctg cgcaagctct ctctcactaa caacgaggcc 900
tacgaacaac ttttggccga ggctgctcct ctcaacgctc taaagatgat aatgttgcag 960
aaccctctcg gagaactagc acgatgcatt atcactattc gaaagcatct caagaagatg 1020
acacgggaga ctggtgctgc tattgagccg gatgagcagt ctgcattgct caacaagtgc 1080
aacacttata gtggagtcat tggaggtgtt gtgcctggag caggaggcta cgatgctatt 1140
tctcttctgg tgatcagctc tacggtgaac aatgtcaagc gagagagcca gggagtccaa 1200
tggatggagc tcaaggagga gaacgagggt ctgcggctcg agaaggggtt caagtag 1257
<210>8
<211>1338
<212>DNA
<213> Yarrowia lipolytica
<400>8
atgggaaaac tcatcgaact gctcttgcac cctagcgaac tgtctgctgc tatccactac 60
aagctgtggc gtcagcctct gcatccccgc gatctttcca aggagtccac tgagctgcga 120
cgatgctatg agcttctaga cgtgtgctca cgatcatttg cagccgttat tcgagaactg 180
catcctgagg tgcgagacgc tgtaatgctg ttctatctga ttcttcgtgc tctcgacacg 240
attgaagacg atatgactct gtcgcgtgac atcaagatcc caattcttcg agacttcacg 300
aagtgcatga agacacctgg ctggaagttc accgactctg atcccaacga gcgagatcgt 360
gtggtgctac aggagtttcc tgtggttatg actgagttca acaagctcaa gcccaagtac 420
caggaagtaa tctacgacat taccgacaga atgggaaacg gaatggccga ttacgtcatt 480
gatgacgact tcaacaacaa cggcgtggac accattgccg cttatgatct gtactgtcat 540
catgttgccg gcatcgtggg tgagggcctt acccgaatta cgattctcgc tggttttgga 600
accgacgtgt tgcacgaaaa cccccgactt caggagtcta tgggcttgtt cttgcaaaag 660
gtcaacatca tccgagacta cagagaagac attgacgtga acagagcttt ctggcctcga 720
gaaatctggc acaagtacgc cgaagaaatg cgagatttca aggacccgaa gtattccaag 780
aaggccttgc attgcacctc cgatctggtt gcaaatgccc tcggacatgc cacagactgc 840
ctcgattacc tcgacaacgt caccgatcct tcaaccttca ctttctgcgc cattccccag 900
gtcatggcca ttgctaccct ggacttggtc taccgaaacc ccgacgtttt ccagaagaac 960
gtcaagttgc gcaagggaac tactgtcagc ctgattcttg aggccagcaa cgtttctgga 1020
gtatgtgaca ttttcactcg atacgcccgg aaggtgtaca agaagtccga ccccaatgac 1080
cccaactact tccgagtgtc tgtgctctgc ggtaagatcg agcagcatgc ggctctgatc 1140
aagagacagc gaggaccccc cgctaaaacc attgcacaac tggaaggtga acgaaaagag 1200
atggccctgt cgctaattgt ctgtttagca gttatcttct cgatgtctgg actgatggct 1260
tatatcgcct acgtgtctgg attcagatgg tcaccccgag agattttcga ctctaagatg 1320
tttcctctga gagattag 1338
<210>9
<211>1179
<212>DNA
<213> Yarrowia lipolytica
<400>9
atggagcccg tctacattgt ttctactgct cgaaccccca ttggttcttt tctgagctcg 60
ctgagcggcc agacctacgt ggatctggga gcccatgccg tgaaggccgc actcgccaag 120
accgacatca agcccgacca ggtcgacgag atcatcttcg gtaacgttct ctccgccggc 180
gtcggacagg cccctgctcg acaggttgcc ctcaaggctg gtctccctga caccattgtc 240
gctaccaccg tcaacaaggt ctgtgcctcc ggtatgaagg ccatcatcca gggtgcccag 300
gccatcatga ccggatctgc cgacattgtc attgctggcg gtgccgagtc catgtccaac 360
gtgccccact acgtgcaggc ccgtgtcgct aacaagtacg gcaacggctc tctggtcgac 420
ggtatccagc gagacggtct gtttgacgcc tacgatggcc aggccatggg tgtggccgct 480
gaggtctgtg ctgacaccca ctccatctcc cgagaggagc aggacgagtt tgccattggc 540
tcttacaaga agacccaggc tgcctatgcc gccggcaagtttaaggacga gattgccccc 600
attgagctgc ccggcttccg aggcaagcct ggtgtcgttg tctccgagga cgaggagtac 660
aagaacctca acgaggacaa gctcaagtct gcccgaactg tttttaagaa ggacggtacc 720
gtgactgccc ccaacgcctc ccctatcaac gacggtggag ctgccgtcat tctggcctct 780
gctgccaagg tcaaggagct tggtcttaag cccctgctca agattgtctc ttggggcgag 840
gctgccaacg agcccgtcaa gttcaccact gctcccgctc tggccgtccc cgttgccctc 900
aagcgagctg gtctcgaggc caaggacatt gacttttacg agttcaacga ggccttctct 960
gttgttggta tcgccaacac caagctgctt ggtctggact ccagcaaggt caacgtctac 1020
ggaggtgctg ttgccattgg tcaccctctg ggctgctctg gtgcccgagt cattgtcact 1080
ctcaactctg ttctgcacca ggaaggcggc aagtacggat gtgctgccat ctgcaacggt 1140
ggtggtggcg cttcctctat cattgttgag aagtgttag 1179
<210>10
<211>1350
<212>DNA
<213> Yarrowia lipolytica
<400>10
atggactaca tcatttcggc gccaggcaaa gtgattctat ttggtgaaca tgccgctgtg 60
tttggtaagc ctgcgattgc agcagccatc gacttgcgaa catacctgct tgtcgaaacc 120
acaacatccg acaccccgac agtcacgttg gagtttccag acatccactt gaacttcaag 180
gtccaggtgg acaagctggc atctctcaca gcccagacca aggccgacca tctcaattgg 240
tcgactccca aaactctgga taagcacatt ttcgacagct tgtctagctt ggcgcttctg 300
gaagaacctg ggctcactaa ggtccagcag gccgctgttg tgtcgttctt gtacctctac 360
atccacctat gtcccccttc tgtgtgcgaa gattcatcaa actgggtagt tcgatcaacg 420
ctgcctatcg gcgcgggcct gggctcttcc gcatccattt gtgtctgttt ggctgcaggt 480
cttctggttc tcaacggcca gctgagcatt gaccaggcaa gagatttcaa gtccctgacc 540
gagaagcagc tgtctctggt ggacgactgg tccttcgtcg gtgaaatgtg cattcacggc 600
aacccgtcgg gcatcgacaa tgctgtggct actcagggag gtgctctgtt gttccagcga 660
cctaacaacc gagtccctct tgttgacatt cccgagatga agctgctgct taccaatacg 720
aagcatcctc gatctaccgc agacctggtt ggtggagtcg gagttctcac taaagagttt 780
ggctccatca tggatcccat catgacttca gtaggcgaga tttccaacca ggccatggag 840
atcatttcta gaggcaagaa gatggtggac cagtctaacc ttgagattga gcagggtatc 900
ttgcctcaac ccacctctga ggatgcctgc aacgtgatgg aagatggagc tactcttcaa 960
aagttgagag atatcggttc ggaaatgcag catctagtga gaatcaatca cggcctgctt 1020
atcgctatgg gtgtttccca cccgaagctc gaaatcattc gaactgcctc cattgtccac 1080
aacctgggtg agaccaagct cactggtgct ggaggaggag gttgcgccat cactctagtc 1140
acttctaaag acaagactgc gacccagctg gaggaaaatg tcattgcttt cacagaggag 1200
atggctaccc atggcttcga ggtgcacgag actactattg gtgccagagg agttggtatg 1260
tgcattgacc atccctctct caagactgtt gaagccttca agaaggtgga gcgggcggat 1320
ctcaaaaaca tcggtccctg gacccattag 1350
<210>11
<211>1164
<212>DNA
<213> Yarrowia lipolytica
<400>11
atgatccacc aggcctccac caccgctccg gtgaacattg cgacactcaa gtactggggc 60
aagcgagacc ctgctctcaa tctgcccact aacaactcca tctccgtgac tttgtcgcag 120
gatgatctgc ggaccctcac cacagcctcg tgttcccctg atttcaccca ggacgagctg 180
tggctcaatg gcaagcagga ggacgtgagc ggcaaacgtc tggttgcgtg tttccgagag 240
ctgcgggctc tgcgacacaa aatggaggac tccgactctt ctctgcctaa gctggccgat 300
cagaagctca agatcgtgtc cgagaacaac ttccccaccg ccgctggtct cgcctcatcg 360
gctgctggct ttgccgccct gatccgagcc gttgcaaatc tctacgagct ccaggagacc 420
cccgagcagc tgtccattgt ggctcgacag ggctctggat ccgcctgtcg atctctctac 480
ggaggctacg tggcatggga aatgggcacc gagtctgacg gaagcgactc gcgagcggtc 540
cagatcgcca ccgccgacca ctggcccgag atgcgagccg ccatcctcgt tgtctctgcc 600
gacaagaagg acacgtcgtc cactaccggt atgcaggtga ctgtgcacac ttctcccctc 660
ttcaaggagc gagtcaccac tgtggttccc gagcggtttg cccagatgaa gaagtcgatt 720
ctggaccgag acttccccac ctttgccgag ctcaccatgc gagactcaaa ccagttccac 780
gccacctgtc tggactcgta tcctcccatt ttctacctca acgacgtgtc gcgagcctcc 840
attcgggtag ttgaggccat caacaaggct gccggagcca ccattgccgc ctacaccttt 900
gatgctggac ccaactgtgt catctactac gaggacaaga acgaggagct ggttctgggt 960
gctctcaagg ccattctggg ccgtgtggag ggatgggaga agcaccagtc tgtggacgcc 1020
aagaagattg atgttgacga gcggtgggag tccgagctgg ccaacggaat tcagcgggtg 1080
atccttacca aggttggagg agatcccgtg aagaccgctg agtcgcttat caacgaggat 1140
ggttctctga agaacagcaa gtag 1164
<210>12
<211>1035
<212>DNA
<213> Yarrowia lipolytica
<400>12
atgtccaagg cgaaattcga aagcgtgttc ccccgaatct ccgaggagct ggtgcagctg 60
ctgcgagacg agggtctgcc ccaggatgcc gtgcagtggt tttccgactc acttcagtac 120
aactgtgtgg gtggaaagct caaccgaggc ctgtctgtgg tcgacaccta ccagctactg 180
accggcaaga aggagctcga tgacgaggag tactaccgac tcgcgctgct cggctggctg 240
attgagctgc tgcaggcgtt tttcctcgtg tcggacgaca ttatggatga gtccaagacc 300
cgacgaggcc agccctgctg gtacctcaag cccaaggtcg gcatgattgc catcaacgat 360
gctttcatgc tagagagtgg catctacatt ctgcttaaga agcatttccg acaggagaag 420
tactacattg accttgtcga gctgttccac gacatttcgt tcaagaccga gctgggccag 480
ctggtggatc ttctgactgc ccccgaggat gaggttgatc tcaaccggtt ctctctggac 540
aagcactcct ttattgtgcg atacaagact gcttactact ccttctacct gcccgttgtt 600
ctagccatgt acgtggccgg cattaccaac cccaaggacc tgcagcaggc catggatgtg 660
ctgatccctc tcggagagta cttccaggtc caggacgact accttgacaa ctttggagac 720
cccgagttca ttggtaagat cggcaccgac atccaggaca acaagtgctc ctggctcgtt 780
aacaaagccc ttcagaaggc cacccccgag cagcgacaga tcctcgagga caactacggc 840
gtcaaggaca agtccaagga gctcgtcatc aagaaactgt atgatgacat gaagattgag 900
caggactacc ttgactacga ggaggaggtt gttggcgaca tcaagaagaa gatcgagcag 960
gttgacgaga gccgaggctt caagaaggag gtgctcaacg ctttcctcgc caagatttac 1020
aagcgacaga agtag 1035
<210>13
<211>813
<212>DNA
<213> Yarrowia lipolytica
<400>13
atgacgacgt cttacagcga caaaatcaag agtatcagcg tgagctctgt ggctcagcag 60
tttcctgagg tggcgccgat tgcggacgtg tccaaggcta gccggcccag cacggagtcg 120
tcggactcgt cggccaagct atttgatggc cacgacgagg agcagatcaa gctgatggac 180
gagatctgtg tggtgctgga ctgggacgac aagccgattg gcggcgcgtc caaaaagtgc 240
tgtcatctga tggacaacat caacgacgga ctggtgcatc gggccttttc cgtgttcatg 300
ttcaacgacc gcggtgagct gcttctgcag cagcgggcgg cggaaaaaat cacctttgcc 360
aacatgtgga ccaacacgtg ctgctcgcat cctctggcgg tgcccagcga gatgggcggg 420
ctggatctgg agtcccggat ccagggcgcc aaaaacgccg cggtccggaa gcttgagcac 480
gagctgggaa tcgaccccaa ggccgttccg gcagacaagt tccatttcct cacccggatc 540
cactacgccg cgccctcctc gggcccctgg ggcgagcacg agattgacta cattctgttt 600
gtccggggcg accccgagct caaggtggtg gccaacgagg tccgcgatac cgtgtgggtg 660
tcgcagcagg gactcaagga catgatggcc gatcccaagc tggttttcac cccttggttc 720
cggctcattt gtgagcaggc gctgtttccc tggtgggacc agttggacaa tctgcccgcg 780
ggcgatgacg agattcggcg gtggatcaag tag 813
<210>14
<211>406
<212>DNA
<213> Yarrowia lipolytica
<400>14
agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaa 406
<210>15
<211>999
<212>DNA
<213> Yarrowia lipolytica
<400>15
gagtttggcg cccgtttttt cgagccccac acgtttcggt gagtatgagc ggcggcagat 60
tcgagcgttt ccggtttccg cggctggacg agagcccatg atgggggctc ccaccaccag 120
caatcagggc cctgattaca cacccacctg taatgtcatg ctgttcatcg tggttaatgc 180
tgctgtgtgc tgtgtgtgtg tgttgtttgg cgctcattgt tgcgttatgc agcgtacacc 240
acaatattgg aagcttatta gcctttctat tttttcgttt gcaaggctta acaacattgc 300
tgtggagagg gatggggata tggaggccgc tggagggagt cggagaggcg ttttggagcg 360
gcttggcctg gcgcccagct cgcgaaacgc acctaggacc ctttggcacg ccgaaatgtg 420
ccacttttca gtctagtaac gccttaccta cgtcattcca tgcatgcatg tttgcgcctt 480
ttttcccttg cccttgatcg ccacacagta cagtgcactg tacagtggag gttttggggg 540
ggtcttagat gggagctaaa agcggcctag cggtacacta gtgggattgt atggagtggc 600
atggagccta ggtggagcct gacaggacgc acgaccggct agcccgtgac agacgatggg 660
tggctcctgt tgtccaccgc gtacaaatgt ttgggccaaa gtcttgtcag ccttgcttgc 720
gaacctaatt cccaattttg tcacttcgca cccccattga tcgagcccta acccctgccc 780
atcaggcaat ccaattaagc tcgcattgtc tgccttgttt agtttggctc ctgcccgttt 840
cggcgtccac ttgcacaaac acaaacaagc attatatata aggctcgtct ctccctccca 900
accacactca cttttttgcc cgtcttccct tgctaacaca aaagtcaaga acacaaacaa 960
ccaccccaac ccccttacac acaagacata tctacagca 999
<210>16
<211>931
<212>DNA
<213> Yarrowia lipolytica
<400>16
cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg tgcttggaga 60
tggaagccgg tagaaccggg ctgcttgggg ggatttgggg ccgctgggct ccaaagaggg 120
gtaggcattt cgttggggtt acgtaattgc ggcatttggg tcctgcgcgc atgtcccatt 180
ggtcagaatt agtccggata ggagacttat cagccaatca cagcgccgga tccacctgta 240
ggttgggttg ggtgggagca cccctccaca gagtagagtc aaacagcagc agcaacatga 300
tagttggggg tgtgcgtgtt aaaggaaaaa aaaagaagct tgggttatat tcccgctcta 360
tttagaggtt gcgggataga cgccgacgga gggcaatggc gccatggaac cttgcggata 420
tcgatacgcc gcggcggact gcgtccgaac cagctccagc agcgtttttt ccgggccatt 480
gagccgactg cgaccccgcc aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg 540
ggaggccact ttttaagtag cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag 600
aagcggctgc agtggtgcaa acggggcgga aacggcggga aaaagccacg ggggcacgaa 660
ttgaggcacg ccctcgaatt tgagacgagt cacggcccca ttcgcccgcg caatggctcg 720
ccaacgcccg gtcttttgca ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa 780
gcttaacata ttataccgaa cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac 840
atttatataa gggtctgcat cgccggctca attgaatctt ttttcttctt ctcttctcta 900
tattcattct tgaattaaac acacatcaac a 931
<210>17
<211>411
<212>DNA
<213> Yarrowia lipolytica
<400>17
cctgtcccca cgttgccggt cttgcctcct actacctgtc catcaatgac gaggttctca 60
cccctgccca ggtcgaggct cttattactg agtccaacac cggtgttctt cccaccacca 120
acctcaaggg ctctcccaac gctgttgcct acaacggtgt tggcatttag gcaattaaca 180
gatagtttgc cggtgataat tctcttaacc tcccacactc ctttgacata acgatttatg 240
taacgaaact gaaatttgac cagatattgt tgtaaataga aaatctggct tgtaggtggc 300
aaaatcccgt ctttgttcat caattccctc tgtgactact cgtcatccct ttatgttcga 360
ctgtcgtatt tttattttcc atacatacgc aagtgagatg cccgtgtccg a 411
<210>18
<211>502
<212>DNA
<213> Yarrowia lipolytica
<400>18
cactggccgg tcgataattt aacgtgctga gctcagcaca cgcattgccc attggctgta 60
tatagatgaa tgtaatgata ccgtaagaga atgagagcac ggtattgtat tacaggggat 120
taagtacaca ttacttggag ttctgtacca gaagacacta ctatacatgg tatcacttac 180
attagagtcg gtgaccgtat tcgtctcgta tagacataat attttcctac cccacattgt 240
tcctgggcct tcggagcaca tctacagtga gtgactgttt cagttgagct tgaggggtta 300
agtaagtggg ggaagggttt gcgattctga aaaagagcat gactaatctc tctgtggagg 360
agcaatgaag tcacgtgatg caatcatacc ggtgtatcgg atctgcctgg gtgtctgatt 420
actaatcatt tactcacctg ttttccccag ctatctcatc catctcagag cctcggccca 480
gccttcggcc cttttgggtt tc 502
<210>19
<211>200
<212>DNA
<213> Yarrowia lipolytica
<400>19
gctatttatc actctttaca acttctacct caactatcta ctttaataaa tgaatatcgt 60
ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc agcatgcgat 120
cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc aggcagcttg 180
agaagattga cacaggtgga 200
<210>20
<211>1907
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
ggtgtgttct gtggagcatt ctcacttttg gtaaacgaca ttgcttcaag tgcagcggaa 60
tcaaaaagta taaagtgggc agcgagtata cctgtacaga ctgtaggcga taactcaatc 120
caattacccc ccacaacatg actggccaaa ctgatctcaa gactttattg aaatcagcaa 180
caccgattct caatgaaggc acatacttct tctgcaacat tcacttgacg cctaaagttg 240
gtgagaaatg gaccgacaag acatattctg ctatccacgg actgttgcct gtgtcggtgg 300
ctacaatacg tgagtcagaa gggctgacgg tggtggttcc caaggaaaag gtcgacgagt 360
atctgtctga ctcgtcattg ccgcctttgg agtacgactc caactatgag tgtgcttgga 420
tcactttgac gatacattct tcgttggagg ctgtgggtct gacagctgcg ttttcggcgc 480
ggttggccga caacaatatc agctgcaacg tcattgctgg ctttcatcat gatcacattt 540
ttgtcggcaa aggcgacgcc cagagagcca ttgacgttct ttctaatttg gaccgatagc 600
cgtatagtcc agtctatcta taagttcaac taactcgtaa ctattaccat aacatatact 660
tcactgcccc agataaggtt ccgataaaaa gttctgcaga ctaaatttat ttcagtctcc 720
tcttcaccac caaaatgccc tcctacgaag ctcgagctaa cgtccacaag tccgcctttg 780
ccgctcgagt gctcaagctc gtggcagcca agaaaaccaa cctgtgtgct tctctggatg 840
ttaccaccac caaggagctc attgagcttg ccgataaggt cggaccttat gtgtgcatga 900
tcaagaccca tatcgacatc attgacgact tcacctacgc cggcactgtg ctccccctca 960
aggaacttgc tcttaagcac ggtttcttcc tgttcgagga cagaaagttc gcagatattg 1020
gcaacactgt caagcaccag tacaagaacg gtgtctaccg aatcgccgag tggtccgata 1080
tcaccaacgc ccacggtgta cccggaaccg gaatcattgc tggcctgcga gctggtgccg 1140
aggaaactgt ctctgaacag aagaaggagg acgtctctga ctacgagaac tcccagtaca 1200
aggagttcct ggtcccctct cccaacgaga agctggccag aggtctgctc atgctggccg 1260
agctgtcttg caagggctct ctggccactg gcgagtactc caagcagacc attgagcttg 1320
cccgatccga ccccgagttt gtggttggct tcattgccca gaaccgacct aagggcgact 1380
ctgaggactg gcttattctg acccccgggg tgggtcttga cgacaaggga gacgctctcg 1440
gacagcagta ccgaactgtt gaggatgtca tgtctaccgg aacggatatc ataattgtcg 1500
gccgaggtct gtacggccag aaccgagatc ctattgagga ggccaagcga taccagaagg 1560
ctggctggga ggcttaccag aagattaact gttagaggtt agactatgga tatgtaattt 1620
aactgtgtat atagagagcg tgcaagtatg gagcgcttgt tcagcttgta tgatggtcag 1680
acgacctgtc tgatcgagta tgtatgatac tgcacaacct gtgtatccgc atgatctgtc 1740
caatggggca tgttgttgtg tttctcgata cggagatgct gggtacaagt agctaatacg 1800
attgaactac ttatacttat atgaggcttg aagaaagctg acttgtgtat gacttattct 1860
caactacatc cccagtcaca ataccaccac tgcactacca ctacacc 1907
<210>21
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
cgcttttatg tatcatgatc tagtgatccc ccacacacca ta 42
<210>22
<211>700
<212>DNA
<213> Yarrowia lipolytica
<400>22
tcgatcctaa ggggtggcat aactgtcgcg tacggcccga taagggcctt ctccaaaagg 60
gaagccggtt gaaattccgg cacttggatg tggattctcc acggcaacgt aactgaatgt 120
ggggacggtg gcacaagtct tggaaggagt tatcttttct ttttaacgga gtcaacaccc 180
tggaattagt ttgtctagag atagggtatc gttccggaag aggggggcag ctttgtcccc 240
tccgatgcac ttgtgacgcc ccttgaaaac ccgcaggaag gaatagtttt cacgccaagt 300
cgtactgata accgcagcag gtctccaagg tgaacagcct ctagttgata gaataatgta 360
gataagggaa gtcggcaaaa tagatccgta acttcgggataaggattggc tctgggggtt 420
ggtggatgga agcgtgggag accccaaggg actggcggct gggcaactgg cagccggacc 480
cgcggcagac actgcgtcgc tccgtccaca tcatcaaccg ccccagaact ggtacggaca 540
aggggaatct gactgtctaa ttaaaacata gctttgcgat ggttgtaaaa caatgttgac 600
gcaaagtgat ttctgcccag tgctctgaat gtcaaagtga agaaattcaa ccaagcgcgg 660
gtaaacggcg ggagtaacta tgactctctt aaggtagcca 700
<210>23
<211>599
<212>DNA
<213> Yarrowia lipolytica
<400>23
aatgcctcgt catctaatta gtgacgcgca tgaatggatt aacgagattc ccactgtccc 60
tatctactat ctagcgaaac cacagccaag ggaacgggct tggcagaatc agcggggaaa 120
gaagaccctg ttgagcttga ctctagtttg acattgtgaa gagacatagg gggtgtagaa 180
taagtgggag cttcggcgcc ggtgaaatac cactaccctt atcgtttctt tacttattta 240
gtaagtggaa gtggtttaac aaccattttc tagcattcct ttccaggctg aagacattgt 300
caggtgggga gtttggctgg ggcggcacat ctgttaaaag ataacgcaga tgtcctaagg 360
gggactcaat gagaacagaa atctcatgta gaacaaaagg gtaaaagtcc ccttgatttt 420
gattttcagt gtgaatacaa accatgaaag tgtggcctat cgatccttta gttgttcgga 480
gtttgaacct agaggtgcca gaaaagttac cacagggata actggcttgt ggcagtcaag 540
cgttcatagc gacattgctt tttgatcctt cgatgtcggc tcttcctatc ataccgaag 599
<210>24
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
tcgatcctaa ggggtggcat aactgtcgc 29
<210>25
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
acgccgccaa cccggtctct tggctacctt aagagagtca t 41
<210>26
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
atgactctct taaggtagcc aagagaccgg gttggcggcg ta 42
<210>27
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gcgaccttca gcttccacat tttgaatgat tcttatactc a 41
<210>28
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gagtataaga atcattcaaa atgtggaagc tgaaggtcgc c 41
<210>29
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
accggcaacg tggggacagg ttagatcttc agctgctggt g 41
<210>30
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
accagcagct gaagatctaa cctgtcccca cgttgccggt c 41
<210>31
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
aatgtcgttt accaaaagtg agaatgctcc acagaacaca cctcggacac gggcatctca 60
ct 62
<210>32
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
ttttattttc catacatacg caagtgagat gcccgtgtcc gaggtgtgtt ctgtggagca 60
ttc 63
<210>33
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gtcactaatt agatgacgag gcattggtgt agtggtagtg cagtgg 46
<210>34
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
accactgcac taccactaca ccaatgcctc gtcatctaat tag 43
<210>35
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
cttcggtatg ataggaagag ccgacatcg 29
<210>36
<211>65
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
tcaccgaaac gtgtggggct cgaaaaaacg ggcgccaaac tctcggacac gggcatctca 60
cttgc 65
<210>37
<211>66
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
tatttttatt ttccatacat acgcaagtga gatgcccgtg tccgagagtt tggcgcccgt 60
tttttc 66
<210>38
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
agggagaaga acaggaccat tgctgtagat atgtcttgtg t 41
<210>39
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
cacaagacat atctacagca atggtcctgt tcttctccct g 41
<210>40
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
taaattatcg accggccagt gttagttgtg agggtgcaga tg 42
<210>41
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
atctgcaccc tcacaactaa cactggccgg tcgataattt a 41
<210>42
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
tccacacccc acaaaaagac ccgtgcagga catcctactg cgaaacccaa aagggccgaa 60
gg 62
<210>43
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
tccatctcag agcctcggcc cagccttcgg cccttttggg tttcgcagta ggatgtcctg 60
cac 63
<210>44
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
gcgtacagag cggaggtcat tgttgatgtg tgtttaattc a 41
<210>45
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gaattaaaca cacatcaaca atgacctccg ctctgtacgc t 41
<210>46
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
tgtaaagagt gataaatagc ttaccagacg tctcgcaggt a 41
<210>47
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
tacctgcgag acgtctggta agctatttat cactctttac aa 42
<210>48
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
aatgtcgttt accaaaagtg agaatgctcc acagaacaca cctccacctg tgtcaatctt 60
ctc 63
<210>49
<211>63
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
gtcctgatag tcaggcagct tgagaagatt gacacaggtg gaggtgtgtt ctgtggagca 60
ttc 63
<210>50
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
acaaccacac acatccacgt gatgctacaa gcagctattg gaaag 45
<210>51
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
ttagtttcgg gttcccacgt gctatgaccg tatgcaaata ttcgaa 46
<210>52
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
acaaccacac acatccacgt gatgacccag tctgtgaagg tgg 43
<210>53
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
ttagtttcgg gttcccacgt gctatgaccg tatgcaaata ttcgaa 46
<210>54
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
acaaccacac acatccacgt gatggtcacc caacagtctg ca 42
<210>55
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
ttagtttcgg gttcccacgt gctaagtcag ctcgctccaa atgt 44
<210>56
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
acaaccacac acatccacgt gatgaccacc tattcggctc cg 42
<210>57
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
ttagtttcgg gttcccacgt gctacttgaa ccccttctcg agc 43
<210>58
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
acaaccacac acatccacgt gatgggaaaa ctcatcgaac tgc 43
<210>59
<211>51
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
ttagtttcgg gttcccacgt gctaatctct cagaggaaac atcttagagt c 51
<210>60
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
acaaccacac acatccacgt gatgcgactc actctgcccc g 41
<210>61
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
ttagtttcgg gttcccacgt gctactcgac agaagagacc t 41
<210>62
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
acaaccacac acatccacgt gatggactac atcatttcgg cg 42
<210>63
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
ttagtttcgg gttcccacgt gctaatgggt ccagggaccg a 41
<210>64
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
acaaccacac acatccacgt gatgatccac caggcctcca 40
<210>65
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
ttagtttcgg gttcccacgt gctacttgct gttcttcaga gaacc 45
<210>66
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
acaaccacac acatccacgt gatgtccaag gcgaaattcg a 41
<210>67
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
ttagtttcgg gttcccacgt gctacttctg tcgcttgtaa atcttgg 47
<210>68
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
acaaccacac acatccacgt gatgacgacg tcttacagcg aca 43
<210>69
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
ttagtttcgg gttcccacgt gctacttgat ccaccgccga a 41
Claims (15)
1. A construction method of heterologous synthesis α -resinol recombinant yarrowia lipolytica is characterized by comprising the following steps of introducing an optimized mixed resinol synthase coding gene into yarrowia lipolytica to obtain a recombinant bacterium 1, wherein the nucleotide sequence of the optimized mixed resinol synthase coding gene is shown in SEQ ID NO. 1.
2. The construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -balsam alcohol and ursolic acid is characterized by comprising the following steps:
introducing an optimized mixed balsam resin alcohol synthase encoding gene, an optimized ursolic acid synthase encoding gene and an optimized cytochrome-NADPH-reductase 1 encoding gene into yarrowia lipolytica to obtain a recombinant bacterium 2; the nucleotide sequence of the optimized mixed resinol synthase coding gene is shown in SEQ ID NO. 1; the nucleotide sequence of the optimized ursolic acid synthase coding gene is shown by SEQ ID NO. 2; the nucleotide sequence of the optimized cytochrome-NADPH-reductase 1 coding gene is shown by SEQ ID NO. 3.
3. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid is characterized by comprising the following steps of introducing a coding gene HMG1 of 3-hydroxy-3-methylglutaryl coenzyme A reductase into a recombinant bacterium 2 to obtain a recombinant bacterium 3, wherein the nucleotide sequence of the gene HMG1 is shown as SEQ ID No. 4.
4. The construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -balsam alcohol and ursolic acid is characterized by comprising the following steps:
(1) removing 1500 nucleotides from the 5' -end of gene HMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase to obtain truncated gene tHMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase; the nucleotide sequence of the gene HMG1 is shown as SEQ ID NO. 4; the nucleotide sequence of the gene tHMG1 is shown as SEQ ID NO. 5;
(2) the gene tHMG1 was introduced into recombinant bacterium 2 to obtain recombinant bacterium 4.
5. The construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -balsam alcohol and ursolic acid is characterized by comprising the following steps:
introducing an encoding gene ERG1 of oxidosqualene cyclase into the recombinant bacterium 2 to obtain a recombinant bacterium 5; the nucleotide sequence of the gene ERG1 is shown in SEQ ID NO. 6.
6. The construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -balsam alcohol and ursolic acid is characterized by comprising the following steps:
introducing an encoding gene ERG8 of phosphomevalonate kinase into the recombinant bacterium 2 to obtain a recombinant bacterium 6; the nucleotide sequence of the gene ERG8 is shown in SEQ ID NO. 7.
7. The construction method of the recombinant yarrowia lipolytica for heterogeneously synthesizing α -balsam alcohol and ursolic acid is characterized by comprising the following steps:
introducing a squalene synthase encoding gene ERG9 into the recombinant strain 2 to obtain a recombinant strain 7; the nucleotide sequence of the gene ERG9 is shown in SEQ ID NO. 8.
8. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid is characterized by comprising the following steps of introducing an encoding gene ERG10 of acetoacetyl-CoA thiolase into a recombinant bacterium 2 to obtain a recombinant bacterium 8, wherein the nucleotide sequence of the gene ERG10 is shown as SEQ ID No. 9.
9. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid is characterized by comprising the following steps of introducing a mevalonate kinase coding gene ERG12 into a recombinant bacterium 2 to obtain a recombinant bacterium 9, wherein the nucleotide sequence of the gene ERG12 is shown as SEQ ID No. 10.
10. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid is characterized by comprising the following steps of introducing an encoding gene ERG19 of mevalonate diphosphate decarboxylase into a recombinant bacterium 2 to obtain a recombinant bacterium 10, wherein the nucleotide sequence of the gene ERG19 is shown as SEQ ID No. 11.
11. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid is characterized by comprising the following steps of introducing an encoding gene ERG20 of farnesyl pyrophosphate synthase into a recombinant bacterium 2 to obtain a recombinant bacterium 11, wherein the nucleotide sequence of the gene ERG20 is shown as SEQ ID No. 12.
12. A construction method of recombinant yarrowia lipolytica for heterogeneously synthesizing α -citronellol and ursolic acid is characterized by comprising the following steps of introducing an expression cassette of an encoding gene IDI of isopentenyl pyrophosphate isomerase into a recombinant bacterium 2 to obtain a recombinant bacterium 12, wherein the nucleotide sequence of the gene IDI is shown as SEQ ID No. 13.
13. The method of claim 1 of constructing a heterologous synthetic α -balsamic alcohol recombinant yarrowia lipolytica heterologous synthetic α -balsamic alcohol.
14. The method for constructing the recombinant yarrowia lipolytica for the heterologous synthesis of α -citronellol and ursolic acid of any one of claims 2 to 12.
15. Use of the recombinant yarrowia lipolytica fermentation of the heterologous synthesis of α -citronellol and ursolic acid of claim 14 to produce α -citronellol and ursolic acid.
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