CN109913380B - Recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and construction method and application thereof - Google Patents
Recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and construction method and application thereof Download PDFInfo
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Abstract
The invention provides recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and a construction method and application thereof, relating to the field of bioengineering. The recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol is obtained by inserting (-) -alpha-bisabolol synthetase, farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase expression cassette into yarrowia lipolytica genome. The recombinant yarrowia lipolytica can efficiently synthesize (-) -alpha-bisabolol, and the construction method is efficient and simple to operate.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol and a construction method and application thereof.
Background
(-) - α -bisabolol is an unsaturated sesquiterpene alcohol mainly found in essential oils of eucalyptus brasiliensis (Eremanthus erythropapus) and chamomile germany (Matricaria recutita). Due to the characteristics of good anti-inflammation, antibiosis, antisepsis, moisture preservation and the like, the (-) -alpha-bisabolol is widely applied to oral hygiene products and skin care products, and is added into large-brand skin care products such as ashira, qianbui and the like at present. The U.S. Food and Drug Administration (FDA) has approved (-) - α -bisabolol as a safe ingredient for safe addition to various daily chemical products such as mouthwashes, skin care products, body washes, etc. (Waltimo T et al, Arch Oral biol.2013,58: 10-6).
At present, (-) -alpha-bisabolol is mainly obtained by a chemical synthesis method and a plant extraction method, and is mainly obtained by distilling and extracting Brazilian eucalyptus due to the complex chemical synthesis steps and the difficult subsequent chiral resolution of the (-) -alpha-bisabolol caused by the abnormal and complicated structure. However, the number of Brazilian eucalyptus is rapidly decreasing due to the limited resources, so the method of extracting (-) - α -bisabolol by distillation using Brazilian eucalyptus not only fails to satisfy the needs of people, but also is not sustainable. Therefore, in order to meet the demand of people for (-) -alpha-bisabolol, the development of an efficient sustainable manufacturing method is urgently needed to fill the market gap and benefit the human health.
Disclosure of Invention
The invention aims to provide a recombinant yarrowia lipolytica capable of efficiently synthesizing (-) -alpha-bisabolol.
The invention further aims to provide a construction method of the recombinant yarrowia lipolytica, which is efficient and simple to operate.
It is a further object of the present invention to provide the use of said recombinant yarrowia lipolytica for the production of (-) - α -bisabolol.
The purpose of the invention is realized by adopting the following technical scheme:
the recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol is obtained by inserting (-) -alpha-bisabolol synthetase, farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase expression cassette into yarrowia lipolytica genome.
The recombinant yarrowia lipolytica is characterized in that a 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and a fusion protein expression cassette are inserted into the genome of yarrowia lipolytica; the fusion protein is composed of (-) -alpha-bisabolol synthetase, connecting peptide and farnesyl pyrophosphate synthetase.
In a preferred technical scheme, the connecting peptide is GGGGS or GSG or EAAAK.
In a preferred technical scheme, the promoter of the expression cassette is P of yarrowia lipolyticaTEFinPromoter or PTEF1A promoter; the terminator is T of yarrowia lipolyticaxpr2And a terminator.
In a preferred embodiment, the recombinant yarrowia lipolytica further expresses 1 or more marker genes; the marker gene is selected from a3 (beta) -isopropylmalate dehydrogenase encoding gene expression cassette or an orotidine-5' -phosphate decarboxylase encoding gene expression cassette.
In a preferred technical scheme, the coding gene of the (-) -alpha-bisabolol synthetase is shown as SEQ ID NO. 1; the coding gene sequence of the 3-hydroxy-3-methylglutaryl CoA reductase is shown in SEQ ID NO. 2.
The invention also provides a construction method of the recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol, which comprises the steps of introducing the 3-hydroxy-3-methylglutaryl CoA reductase expression cassette, (-) -alpha-bisabolol synthetase expression cassette and farnesyl pyrophosphate synthetase expression cassette into the yarrowia lipolytica in a plasmid form, and then integrating the yarrowia lipolytica on the genome.
The invention also provides a construction method of the recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol, which comprises the steps of introducing the 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and the fusion protein expression cassette into the yarrowia lipolytica in a plasmid form, and then integrating the yarrowia lipolytica on the genome; the fusion protein is composed of (-) -alpha-bisabolol synthetase, connecting peptide and farnesyl pyrophosphate synthetase.
The invention also provides an application of the recombinant bacterium in the production of (-) -alpha-bisabolol, which comprises the following steps: (1) culturing the recombinant strain of any one of claims 1-9 in a fermentation medium to obtain a fermentation product; (2) and extracting the fermentation product by using an organic solution, and collecting an organic phase.
The recombinant yarrowia lipolytica of the invention can express (-) -alpha-bisabolol synthetase, overexpression endogenous farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase, experiments prove that the recombinant yarrowia lipolytica can efficiently ferment and produce (-) -alpha-bisabolol, and realizes the synthesis of natural products (-) -alpha-bisabolol in yarrowia lipolytica, and the recombinant yarrowia lipolytica of the invention has the following advantages:
(1) the recombinant yarrowia lipolytica constructed by the invention is based on yarrowia lipolytica strains which are responsible for coding non-homologous recombination genes ku70 and knocked out, so that the homologous recombination capability of the yarrowia lipolytica strains is enhanced, the integration of genes is realized through the homologous recombination function of the yarrowia lipolytica, and the genetic stability of the introduced genes can be greatly improved. The recombinant yarrowia lipolytica construction method is efficient and simple to operate.
(2) The yarrowia lipolytica is oleaginous yeast which has strong lipid synthesis capacity, and the target product (-) -alpha-bisabolol is easy to dissolve in a fat-soluble environment, so that a good intracellular environment is provided for the production of the (-) -alpha-bisabolol in the yarrowia lipolytica.
(3) The recombinant yarrowia lipolytica realizes the high-efficiency synthesis of (-) -alpha-bisabolol by overexpressing a 3-hydroxy-3-methylglutaryl CoA reductase coding gene tHMG, a farnesyl pyrophosphate synthetase coding gene ERG20 and a (-) -alpha-bisabolol synthetase coding gene OptBBS.
Drawings
FIG. 1 is a structural diagram of a recombinant plasmid pUC-leu-A08-OptBBS, wherein A08-up represents the upstream sequence of A08 site, A08-dm represents the downstream sequence of A08 site, xpr2t represents a terminator, and TEFin represents a promoter.
FIG. 2 shows the structure of the recombinant plasmid pUC-HUH-IntC-tHMG wherein IntC-up denotes the sequence upstream of the IntC site, IntC-dm denotes the sequence downstream of the IntC site, xpr2t denotes the terminator and TEFin denotes the promoter.
FIG. 3 shows the structure of recombinant plasmid pUC-HUH-SCP2-ERG20, in which SCP2-up represents the upstream sequence of SCP2 site, SCP2-dm represents the downstream sequence of SCP2 site, xpr2t represents terminator, and TEFin represents promoter.
FIG. 4 is a GC analysis of (-) - α -bisabolol produced by recombinant bacterium 3.
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.
Yarrowia lipolytica Po1f delta ku70(MatA, delta ku70:: hisG, leu2-270, ura3-302, xpr2-322, axp1-2) is Yarrowia lipolytica, the coding gene ku70 responsible for non-homologous recombination is knocked out and purchased from the American type culture Collection.
The A08 site integration plasmid was obtained by inserting the upstream 2521bp and downstream 2031bp of the A08 site on chromosome A in the Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-leu vector (the construction method is shown in example 1), and the leu expression cassette was located between the upstream and downstream sequences of A08 site.
The IntC site integration plasmid is obtained by inserting sequences of 1402bp upstream and 1396bp downstream of the IntC site on chromosome C in Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-hisG-ura-hisG (construction method is shown in example 1) vector, and two hisG tag coding genes are arranged between sequences of the IntC site and the downstream.
The SCP2 site integration plasmid is obtained by inserting sequences of 1523bp upstream and 1524bp downstream of SCP2 site on chromosome E in Yarrowia lipolytica Po1f delta ku70 genome into pUC57-hisG-ura-hisG vector.
Example 1 amplification of Gene elements and preparation of target plasmids
(first) preparation of target Gene
According to the nucleotide sequence of the (-) -alpha-bisabolol synthetase encoding gene MrBBS provided by NCBI, after codon optimization, the optimized (-) -alpha-bisabolol synthetase encoding gene OptBBS is synthesized by Suzhou Jinzhi Biotechnology GmbH and inserted into a plasmid pUC57 to obtain a plasmid pUC 57-OptBBS. The nucleotide sequence of OptBBS is shown as SEQ ID NO. 1.
Based on the nucleotide sequence of leu, which is a3 (. beta. -isopropylmalate dehydrogenase-encoding gene in Yarrowia lipolytica provided at NCBI (M37309.1), Leu was synthesized by Cinzhi Biotechnology, Inc., Suzhou, and the expression cassette for the 3 (. beta. -isopropylmalate dehydrogenase-encoding gene was inserted into plasmid pUC57 to obtain plasmid pUC 57-leu.
Based on the nucleotide sequence of orotidine-5 '-phosphate decarboxylase encoding gene ura (genebank accession AJ306421.1) and hisG tag (genebank accession AF324729.1) in Yarrowia lipolytica provided at NCBI, Suzhou Jinzhi Biotechnology Limited was entrusted with the synthesis of two hisG tag encoding gene sequences inserted into plasmid pUC57 and orotidine-5' -phosphate decarboxylase encoding gene expression cassettes inserted into the two hisG tag encoding gene sequences to achieve ura marker recovery, resulting in plasmid pUC 57-hisG-ura-hisG.
3-hydroxy-3-methylglutaryl CoA reductase encoding gene tHMG is amplified by using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template and tHMG-F (SEQ ID NO.5) and tHMG-R (SEQ ID NO.6) as primers. the nucleotide sequence of tHMG is shown as SEQ ID NO. 2.
A farnesyl pyrophosphate synthetase encoding gene ERG20(genebank accession number YALI0E05753g) is amplified by using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template and ERG20-F (SEQ ID NO.7) and ERG20-R (SEQ ID NO.8) as primers.
Promoter PTEFinThe nucleotide sequence of (A) is shown as SEQ ID NO.3, and a terminator Txpr2The nucleotide sequence is shown as SEQ ID NO. 4.
(II) construction of recombinant plasmid
1. Construction of recombinant plasmid pUC-leu-A08-OptBBS
The recombinant plasmid pUC-leu-A08-OptBBS takes pUC57-leu as a framework, an upstream and a downstream homology arms at the A08 site in Yarrowia lipolytica Po1f delta ku70 are inserted, an OptBBS gene expression cassette is inserted between the upstream and downstream homology arms, and A3 (beta) -isopropylmalate dehydrogenase encoding gene expression cassette is inserted between the upstream and downstream homology arms, and the structure is shown in figure 1.
A08-TEFinp-F (SEQ ID NO.9) and TEFinp-OptBBS-R (SEQ ID NO.10) are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and an OptBBS expression cassette promoter P is amplifiedTEFin. OptBBS expression cassette terminator T is amplified by taking OptBBS-xpr2T-F (SEQ ID NO.11) and A08-xpr2T-R (SEQ ID NO.12) as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a templatexpr2。
Plasmid pUC57-OptBBS is taken as a template, TEFinp-OptBBS-F (SEQ ID NO.13) and OptBBS-xpr2t-R (SEQ ID NO.14) are taken as primers, and promoters P are respectively arranged at two amplified endsTEFinAnd a terminator Txpr2The OptBBS gene of the homology arm.
The PCR enzyme used in the above-mentioned PCR reaction was PrimeSTAR Max DNA polymerase from Baozi medical technology (Beijing) Ltd. The PCR amplification system is as follows:
| components | Volume of |
| PrimerSTAR Max Premix | 25ul |
| Form panel | 1ul |
| Primer1 | 2ul |
| Primer2 | 2ul |
| Distilled water | 20ul |
The procedure for the above PCR was as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition.
Each Fragment was purified and recovered by TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Baisai Biotechnology Ltd.).
The A08 site integration plasmid was digested with the NEB restriction enzyme SnaB I, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.
The linearized A08 site integration plasmid and each element in the OptBBS gene expression cassette constructed in title 1 of this example were cloned in One Step using the Clon express Multi S One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd, the reaction system is shown in the following table. After incubating the reaction system at 37 ℃ for 30min, a circular recombinant vector was obtained, and an OptBBS gene expression cassette and a3 (. beta. -isopropylmalate dehydrogenase-encoding gene expression cassette (selection marker) were inserted between the upper and lower homology arms at A08 site.
The system for one-step cloning is as follows:
| components | Volume of |
| 5×CE MultiS Buffer | 4ul |
| Exnase MultiS | 2ul |
| Linearized vector | x ng |
| Insert fragment | y ng |
| Distilled water | Make up the volume to 10ul |
The amount of the linearized vector (x) and the insert (y) used can be calculated from the following formula: the optimum amount of the fragment or linearized vector used was [0.02 × base number of fragment or linearized vector ] ng.
The circular recombinant vector is transformed into escherichia coli DH5 alpha competent cells, and positive recombinant plasmid pUC-leu-A08-OptBBS is obtained through ampicillin resistance plate screening and colony PCR and sequencing verification.
2. Construction of recombinant plasmid pUC-HUH-IntC-tHMG
The recombinant plasmid pUC-HUH-IntC-tHMG takes pUC57-hisG-ura-hisG as a framework, an upstream and a downstream homology arms at the IntC site in Yarrowia lipolytica Po1f delta ku70 are inserted, a tHMG expression cassette is inserted between the upstream and downstream homology arms, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette is also inserted between the upstream and downstream homology arms, and the specific structure is shown in figure 2.
The intC-TEFinp-F (SEQ ID NO.15) and the TEFinp-tHMG-R (SEQ ID NO.16) are used as primers, the Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and a tHMG expression cassette promoter P is amplifiedTEFin. The tHMG expression cassette terminator T is amplified by using tHMG-xpr2T-F (SEQ ID NO.17) and IntC-xpr2T-R (SEQ ID NO.18) as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a templatexpr2。
Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as template, uses TEFinp-tHMG-F (SEQ ID NO.19) and tHMG-xpr2t-R (SEQ ID NO.20) as primer to amplify two ends with promoter PTEFinAnd a terminator Txpr2tHMG gene of homologous arm。
After the IntC site integration plasmid was digested with the restriction enzyme Pac1 of NEB, the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis.
The linearized IntC site integration plasmid and each element in the tHMG gene expression cassette constructed under the heading 2 of this example were cloned in One Step using the Clon express Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Co., Ltd to obtain the recombinant plasmid pUC-HUH-IntC-tHMG.
3. Construction of recombinant plasmid pUC-HUH-SCP2-ERG20
The recombinant plasmid pUC-HUH-SCP2-ERG20 is characterized in that pUC57-hisG-ura-hisG is used as a framework, upstream and downstream homology arms at SCP2 site in Yarrowia lipolytica Po1f delta ku70 are inserted, an ERG20 expression cassette is inserted between the upstream and downstream homology arms, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette is also inserted between the upstream and downstream homology arms.
Amplifying the promoter P of the ERG20 expression cassette by taking SCP2-TEFinp-F (SEQ ID NO.21) and TEFinp-ERG20-R (SEQ ID NO.22) as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a templateTEFin. ERG20-xpr2T-F (SEQ ID NO.23) and SCP2-xpr2T-R (SEQ ID NO.24) are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and an ERG20 expression cassette terminator T is amplifiedxpr2。
Using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template, TEFinp-ERG20-F (SEQ ID NO.25) and ERG20-xpr2t-R (SEQ ID NO.26) as primers, and respectively carrying promoters P at two ends of amplificationTEFinAnd a terminator Txpr2ERG20 gene of homologous arm.
The SCP2 site integration plasmid was digested with the NEB restriction enzyme Hind III, and the linearized SCP2 site integration plasmid was recovered by agarose gel electrophoresis.
The linearized SCP2 site integration plasmid and each element in the ERG20 gene expression cassette constructed under the title 3 of this example were cloned in One Step using the Clon express Multi S One Step Cloning Kit from Biotech, Inc., Nanjing Novowed to obtain the recombinant plasmid pUC-HUH-SCP2-ERG 20.
4. Construction of pUC-leu-A08-OptBBS-GSG-ERG20
The recombinant plasmid pUC-leu-A08-OptBBS-GSG-ERG20 takes pUC57-leu as a framework, an upstream homology arm and a downstream homology arm of an A08 site in Yarrowia lipolytica Po1f delta ku70 are inserted, an expression cassette of a fusion protein OptBBS-GSG-ERG20 is inserted between the upstream homology arm and the downstream homology arm, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase encoding gene is also inserted between the upstream homology arm and the downstream homology arm. The N end of the fusion protein OptBBS-GSG-ERG20 is (-) -alpha-bisabolol synthetase, the C end is farnesyl pyrophosphate synthetase, and the (-) -alpha-bisabolol synthetase and the farnesyl pyrophosphate synthetase are connected by a connecting peptide with the amino acid sequence of GSG.
A08-TEFinp-F (SEQ ID NO.9) and TEFinp-OptBBS-R (SEQ ID NO.10) are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and an OptBBS-GSG-ERG20 expression cassette promoter P is amplifiedTEFin. ERG20-xpr2T-F (SEQ ID NO.23) and A08-xpr2T-R (SEQ ID NO.12) are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and the terminator T of the OptBBS expression cassette is amplifiedxpr2。
The OptBBS gene was amplified using plasmid pUC57-OptBBS as a template and TEFinp-OptBBS-F (SEQ ID NO.13) and OptBBS-ERG20-R (SEQ ID NO.28) as primers.
The ERG20 gene is amplified by using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template and using OptBBS-ERG20-F (SEQ ID NO.27) and ERG20-xpr2t-R (SEQ ID NO.26) as primers.
The A08 site integration plasmid was digested with the NEB restriction enzyme SnaB I, and the linearized A08 site integration plasmid was recovered by agarose gel electrophoresis.
Each gene element and linearized A08 site integration plasmid in the OptBBS-GSG-ERG20 expression cassette obtained by amplifying the title 4 of the example were cloned in One Step by using the Clon express MultiS One Step Cloning Kit of Nanjing Novokation Biotechnology Co., Ltd to obtain the recombinant plasmid pUC-leu-A08-OptBBS-GSG-ERG 20.
5. Construction of recombinant plasmid pUC-leu-A08-ERG20-GSG-OptBBS
The recombinant plasmid pUC-leu-A08-ERG20-GSG-OptBBS takes pUC57-leu as a framework, an upstream and a downstream homology arms of an A08 site (Yarrowia lipolytica Po1f delta ku70) are inserted, an expression cassette of a fusion protein ERG20-GSG-OptBBS is inserted between the upstream and downstream homology arms, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase encoding gene is also inserted between the upstream and downstream homology arms. The N end of the fusion protein ERG20-GSG-OptBBS is farnesyl pyrophosphate synthetase, the C end is (-) -alpha-bisabolol synthetase, and the farnesyl pyrophosphate synthetase and the (-) -alpha-bisabolol synthetase are connected by a connecting peptide with the amino acid sequence of GSG.
A08-TEFinp-F (SEQ ID NO.9) and TEFinp-ERG20-R (SEQ ID NO.22) are used as primers, Yarrowia lipolytica delta ku70 genome is used as a template, and a promoter P of an ERG20-GSG-OptBBS expression cassette is amplifiedTEFin. The terminator T of the ERG 20-GSG-OptibBS expression cassette is amplified by taking OptBBS-xpr2T-F (SEQ ID NO.11) and A08-xpr2T-R (SEQ ID NO.12) as primers and Yarrowia lipolytica delta ku70 genome as a templatexpr2。
The ERG20 gene is amplified by taking Yarrowia lipolytica Po1F delta ku70 genome as a template and TEFinp-ERG20-F (SEQ ID NO.25) and ERG20-OptBBS-R (SEQ ID NO.29) as primers.
The OptBBS gene was amplified using a plasmid pUC57-OptBBS with the synthesized OptBBS gene as a template and ERG20-OptBBS-F (SEQ ID NO.30) and OptBBS-xpr2t-R (SEQ ID NO.14) as primers.
The A08 site integration plasmid was digested with the NEB restriction enzyme SnaB I, and the linearized A08 site integration plasmid was recovered from the gel after agarose gel electrophoresis.
Each gene element and linearized A08 site integration plasmid in the ERG20-GSG-OptBBS expression cassette obtained by amplifying the title 5 of this example were cloned in One Step using the Clon express Multi S One Step Cloning Kit from Nanjing Novokation Biotechnology Ltd to obtain the recombinant plasmid pUC-leu-A08-ERG 20-GSG-OptBBS.
TABLE 1 insertion sequence in each recombinant plasmid
TABLE 2 primer sequences
Example 2 construction of recombinant bacteria
(first) construction of recombinant bacterium 1
(1) And (3) introducing a plasmid pUC-leu-A08-OptBBS containing an OptBBS gene expression cassette into Yarrowia lipolytica Po1f delta ku70, and integrating the OptBBS expression cassette into a genome A08 site to obtain the recombinant bacterium 1. The specific method comprises the following steps: (1) competent cells were prepared after overnight culture of Yarrowia lipolytica Po1 f. delta. ku70 in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose). (2) pUC-leu-A08-OptBBS was transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination. (3) Screening by adopting a screening culture medium SD-Leu, and identifying a positive clone with correct PCR identification, wherein the positive clone is named as a recombinant bacterium 1. Wherein the screening medium SD-Leu contains: 20g/L glucose, 6.7g/L Yeast Nitrogen Base (YNB, Yeast ammonium sulfate), 0.67g/L CSM-Leu, and 23g/L agar powder.
(II) construction of recombinant bacterium 2
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-HUH-SCP2-ERG20 are introduced into the recombinant bacterium 1, a tHMG expression cassette is integrated into a genome IntC locus, and an ERG20 expression cassette is integrated into a genome SCP2 locus to obtain a recombinant bacterium 2. The specific method comprises the following steps: recombinant bacterium 1 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose) to prepare competence, recombinant plasmids pUC-HUH-IntC-tHMG and pUC-HUH-SCP2-ERG20 were transformed into recombinant bacterium 1 using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, and screened using screening medium SD-Leu-Ura, and positive clones identified correctly by PCR were designated as recombinant bacterium 2.
Wherein the components of the screening culture medium SD-Leu-Ura are as follows: 20g/L of glucose, 6.7g/L of YNB, 0.67g/L of CSM-Leu-Ura and 23g/L of agar powder.
(III) construction of recombinant bacterium 3
The pUC-HUH-IntC-tHMG and pUC-leu-A08-OptBBS-GSG-ERG20 plasmids were introduced into Yarrowia lipolytica Po1 f. delta. ku70, the tHMG expression cassette was integrated into the IntC site in the genome, and the OptBBS-GSG-ERG20 expression cassette was integrated into the A08 site to obtain recombinant bacterium 3. The specific method comprises the following steps: after overnight culture of Yarrowia lipolytica Po1 f. delta. ku70 in YPD liquid medium (containing 2% peptone, 1% Yeast extract, 2% glucose), pUC-HUH-intC-tHMG and pUC-Leu-A08-OptBBS-GSG-ERG20 was transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROZEN EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination, and correct positive clones were identified by PCR using selection medium SD-Leu-Ura selection, and named recombinant bacterium 3.
The screened culture medium SD-Leu-Ura comprises the following components: 20g/L of glucose, 6.7g/L of YNB, 0.67g/L of CSM-Leu-Ura and 23g/L of agar powder.
(IV) construction of recombinant bacterium 4
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-leu-A08-ERG20-GSG-OptBBS are introduced into Yarrowia lipolytica Po1f delta ku70, a tHMG expression cassette is integrated into an IntC site in a genome, and an ERG20-GSG-OptBBS expression cassette is integrated into an A08 site to obtain a recombinant bacterium 4.
The specific method comprises the following steps: after overnight culture of Yarrowia lipolytica Po1 f. delta. ku70 in YPD liquid medium (2% peptone, 1% Yeast extract, 2% glucose), recombinant plasmids pUC-HUH-intC-tHMG and pUC-Leu-A08-ERG20-GSG-OptBBS were transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROZEN EZ Yeast Transformation Kit II from Zymo Research Corporation, and correct positive clones were identified by PCR using selection medium SD-Leu-Ura to obtain recombinant bacterium 4.
Wherein the components of the screening culture medium SD-Leu-Ura are as follows: 20g/L of glucose, 6.7g/L of YNB, 0.67g/L of CSM-Leu-Ura and 23g/L of agar powder.
(V) construction of recombinant bacterium 5-8
Respectively carrying out PCR amplification by using genome DNA of plasmids pUC57-OptBBS and Yarrowia lipolytica Po1f delta ku70 as templates and using primers in a table 4 (tables 4-1, 4-2, 4-3 and 4-4) to obtain OptBBS and ERG20 fragments containing different homology arms corresponding to different primers; then respectively cloning the amplification products corresponding to different primers to form fusion protein expression box plasmids, and respectively replacing connecting peptides GSG between two enzymes contained in the fusion protein in the vector with GGGGS and EAAAK. The plasmid and pUC-HUH-IntC-tHMG plasmid are transferred into Yarrowia lipolytica Po1f delta ku70 for homologous recombination to respectively obtain recombinant bacteria 5-8.
The OptBBS gene was PCR-amplified using plasmid pUC57-OptBBS with the synthesized OptBBS gene as a template and each pair of primers OptBBS-F and OptBBS-R with the linker peptide GGGGS/EAAAK in Table 4 (tables 4-1, 4-2, 4-3, and 4-4) to give OptBBS amplified fragments corresponding to the primer pair SEQ ID NO.31 and SEQ ID NO.32, the primer pair SEQ ID NO.31 and SEQ ID NO.35, the primer pair SEQ ID NO.39 and SEQ ID NO.40, and the primer pair SEQ ID NO.42 and SEQ ID NO. 40.
The genome of Yarrowia lipolytica Po1F delta ku70 is used as a template, and each corresponding primer ERG20-F and ERG20-R with the connecting peptide of GGGGS/EAAAK in Table 4 (tables 4-1, 4-2, 4-3 and 4-4) is used for carrying out PCR amplification on the ERG20 gene to obtain ERG20 fragments amplified by corresponding primer pairs SEQ ID NO.33 and SEQ ID NO.34, primer pairs SEQ ID NO.36 and SEQ ID NO.34, primer pair SEQ ID NO.37 and SEQ ID NO.38 and primer pair SEQ ID NO.37 and SEQ ID NO. 41.
Using one-step cloning to respectively use OptBBS amplified fragments and ERG20 amplified fragments corresponding to different primer pairs as fusion protein expression cassette elements to construct recombinant plasmids pUC-leu-A08-OptBBS-GGGGS-ERG20, pUC-leu-A08-OptBBS-EAAAK-ERG20, pUC-leu-A08-ERG20-GGGGS-OptBBS, pUC-leu-A08-ERG 20-EAAAK-OptBBS.
The recombinant plasmid pUC-leu-A08-OptBBS-GGGGS-ERG20 takes pUC57-leu as a framework, an upstream homology arm and a downstream homology arm of Yarrowia lipolytica Po1f delta ku70A08 site are inserted, an expression cassette of a fusion protein OptBBS-GGGGS-ERG20 is inserted between the upstream homology arm and the downstream homology arm, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase encoding gene responsible for synthesizing leucine is also arranged between the upstream homology arm and the downstream homology arm. The N end of the fusion protein OptBBS-GGGGS-ERG20 is (-) -alpha-bisabolol synthetase, the C end is farnesyl pyrophosphate synthetase, and the (-) -alpha-bisabolol synthetase and the farnesyl pyrophosphate synthetase are connected by a connecting peptide with the amino acid sequence of GGGGS.
The recombinant plasmid pUC-leu-A08-OptBBS-EAAAK-ERG20 takes pUC57-leu as a framework, an upstream homology arm and a downstream homology arm of Yarrowia lipolytica Po1f delta ku70A08 site are inserted, an expression cassette of a fusion protein OptBBS-EAAAK-ERG20 is inserted between the upstream homology arm and the downstream homology arm, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase encoding gene responsible for synthesizing leucine is arranged between the upstream homology arm and the downstream homology arm. The N end of the fusion protein OptBBS-EAAAK-ERG20 is (-) -alpha-bisabolol synthetase, the C end is farnesyl pyrophosphate synthetase, and the (-) -alpha-bisabolol synthetase and the farnesyl pyrophosphate synthetase are connected by a connecting peptide with an amino acid sequence of EAAAK.
The recombinant plasmid pUC-leu-A08-ERG 20-GGGGGGS-OptBBS takes pUC57-leu as a framework, an upstream homology arm and a downstream homology arm of Yarrowia lipolytica Po1f delta ku70A08 site are inserted, an expression cassette of fusion protein ERG20-GGGGS-OptBBS is inserted between the upstream homology arm and the downstream homology arm, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase coding gene responsible for synthesizing leucine is arranged between the upstream homology arm and the downstream homology arm. The N end of the fusion protein ERG20-GGGGS-OptBBS is farnesyl pyrophosphate synthetase, the C end is (-) -alpha-bisabolol synthetase, and the farnesyl pyrophosphate synthetase and the (-) -alpha-bisabolol synthetase are connected by a connecting peptide with the amino acid sequence of GGGGS.
The recombinant plasmid pUC-leu-A08-ERG20-EAAAK-OptBBS takes pUC57-leu as a framework, an upstream homology arm and a downstream homology arm of Yarrowia lipolytica Po1f delta ku70A08 site are inserted, an expression cassette of fusion protein ERG20-EAAAK-OptBBS is inserted between the upstream homology arm and the downstream homology arm, and an expression cassette of A3 (beta) -isopropylmalate dehydrogenase coding gene responsible for synthesizing leucine is arranged between the upstream homology arm and the downstream homology arm. The N end of the fusion protein ERG20-EAAAK-OptBBS is farnesyl pyrophosphate synthetase, the C end is (-) -alpha-bisabolol synthetase, and the farnesyl pyrophosphate synthetase and the (-) -alpha-bisabolol synthetase are connected by a connecting peptide with an amino acid sequence of EAAAK.
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-OptBBS-GGGGS-ERG20 are introduced into Yarrowia lipolytica Po1f delta ku70 for homologous recombination, a tHMG expression cassette is integrated at an IntC site of a genome, an OptBBS-GGGGS-ERG20 expression cassette is integrated at a genome A08 site, screening is carried out by adopting a screening medium SD-Leu-Ura, and a positive clone which is correctly identified by PCR is named as a recombinant bacterium 5.
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-OptBBS-EAAAK-ERG20 are introduced into Yarrowia lipolytica Po1f delta ku70 for homologous recombination, a tHMG expression cassette is integrated to a genome IntC site, an OptBBS-EAAAK-ERG20 expression cassette is integrated to a genome A08 site, screening is carried out by adopting a screening medium SD-Leu-Ura, and a positive clone which is correctly identified by PCR is named as recombinant bacteria 6.
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-ERG20-GGGGS-OptBBS are introduced into Yarrowia lipolytica Po1f delta ku70 for homologous recombination, a tHMG expression cassette is integrated at an IntC site of a genome, an ERG20-GGGGS-OptBBS expression cassette is integrated at a genome A08 site, screening is carried out by adopting a screening medium SD-Leu-Ura, and a positive clone which is correctly identified by PCR is named as recombinant bacterium 7.
Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-ERG20-EAAAK-OptBBS are introduced into Yarrowia lipolytica Po1f delta ku70 for homologous recombination, a tHMG expression cassette is integrated into an IntC site in a genome, an ERG20-EAAAK-OptBBS expression cassette is integrated into an A08 site, screening is carried out by adopting a screening culture medium SD-Leu-Ura, and a positive clone which is correctly identified by PCR is named as a recombinant bacterium 8.
Table 3 shows the nucleotide and amino acid sequences of the linker peptides
| Amino acid sequence | Nucleotide sequence (5'→ 3') |
| GGGGS | GGCGGTGGTGGCTCC |
| GSG | GGTTCTGGT |
| EAAAK | GAAGCTGCCGCCAAA |
Table 4-1 shows the primers required for constructing recombinant plasmid pUC-leu-A08-OptBBS-GGGGS-ERG20
Table 4-2 shows the primers required for constructing recombinant plasmid pUC-leu-A08-OptBBS-EAAAK-ERG20
Table 4-3 shows the primers required for constructing recombinant plasmid pUC-leu-A08-ERG20-GGGGS-OptBBS
Table 4-4 shows the primers required for constructing recombinant plasmid pUC-leu-A08-ERG20-EAAAK-OptBBS
Example 3 application of recombinant bacteria 1-8 in production of (-) -alpha-bisabolol
1. Engineering bacteria culture and product extraction
The recombinant bacteria 1-8 in example 2 were used to produce (-) -alpha-bisabolol, respectively. The specific method comprises the following steps: activating the recombinant bacteria, and culturing in YPD liquid culture medium at 30 deg.C and 220rpm for 16h to obtain seed liquid. Inoculating the seed liquid into 50ml of fermentation medium with the inoculation amount of 1%, performing shake culture at 30 ℃ and 220rpm for 1 day, adding n-dodecane with the volume of 10% of the fermentation liquid, and continuing to perform shake culture for 3 days. After the fermentation is finished, transferring the fermentation liquor to a 50ml centrifuge tube, centrifuging for 15min at 5000rpm, and collecting an organic phase for later use.
Wherein the fermentation medium contains 55g/L glucose, 10g/L yeast extract and 20g/L tryptone. 2. Qualitative and quantitative analysis of (-) -alpha-bisabolol
And (3) passing the organic phase in the fermentation liquor of each recombinant bacterium through an organic nylon filter membrane (0.22um), and detecting by GC. Detection conditions are as follows: the temperature of a sample inlet is 250 ℃, the sample injection volume is 1ul, and the flow is not split; a chromatographic column: HP-5ms (30m 0.25 mM); chromatographic conditions are as follows: the initial temperature was 60 deg.C, ramped up to 160 deg.C at a rate of 10 deg.C/min, then ramped up to 200 deg.C at 5 deg.C/min, and finally ramped up to 230 deg.C at 10 deg.C/min. Qualitative and quantitative determinations were performed with a standard of (-) - α bisabolol.
FIG. 4 is a GC analysis of (-) - α -bisabolol produced by recombinant bacterium 3. After fermentation for 5 days, the yields of the (-) -alpha-bisabolol of the recombinant bacteria 3 and the recombinant bacteria 4 are the highest and reach 120mg/L, namely 120mg of the (-) -alpha-bisabolol is produced in each liter of fermentation liquor.
The yields of the recombinant bacteria 1, the recombinant bacteria 2, the recombinant bacteria 5, the recombinant bacteria 6, the recombinant bacteria 7 and the recombinant bacteria 8 are 76mg/L, 98mg/L, 83mg/L, 67mg/L, 103mg/L and 70mg/L respectively.
SEQUENCE LISTING
<110> Nanjing university of industry
<120> recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol, and construction method and application thereof
<130> 20190325
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1719
<212> DNA
<213> artificial
<220>
<223> OptBBS
<400> 1
atgtctaccc tctccgtgtc cactccttcc ttctcctcct cccccctctc ttccgtgaac 60
aaaaactcca ccaagcagca cgtcacccga aactccgtga tcttccatga ctctatctgg 120
ggcgaccagt tcctcgaata taaagagaag tttaatgtcg ctactgaaaa gcagctgatc 180
gaggaactca aggaggaggt ccgaaacgaa ctcatgatcc gagcttgcaa cgaggcctcc 240
cgatacatta aactcatcca gctgatcgac gtcgtggaac gactgggcct cgcttatcac 300
ttcgagaagg agatcgaaga gtccctccaa cacatctacg tcacctacgg ccacaagtgg 360
accaactaca acaacatcga gtctctctct ctctggtttc gactcctccg acagaacggt 420
ttcaacgtgt cttccgatat ctttgaaaac catatcgacg aaaagggcaa ctttcaagaa 480
tctttatgca acgaccccca aggtatgctg gctttatacg aggccgccta catgagagtc 540
gagggtgaga tcattttaga taaggccctc gagtttacca agctgcacct cggcatcatt 600
tctaacgacc cctcttgcga ttcctctctg cgaactgaga tcaagcaagc tttaaaacag 660
cctctccgac gaagactgcc tcgactggag gccgtccgat atatcgctat ttaccagcag 720
aaggcctccc actctgaggt tttactcaag ctcgctaaac tggactttaa cgtgctccaa 780
gaaatgcaca aggatgagct gtcccagatc tgcaaatggt ggaaggacct cgacatccga 840
aacaagctcc cctacgtcag agaccgactg attgagggct acttctggat tttaggcatc 900
tactttgagc cccagcactc ccgaacccga atgtttttaa tgaagacttg tatgtggctc 960
atcgttttag acgacacctt tgacaactac ggcacttacg aggagctgga gatcttcacc 1020
caagctgtcg agcgatggtc catcacttgt ctggacgagc tgcccgagta catgaagctc 1080
atctaccatg agcagttccg agtccatcaa gagatggagg agtccctcga gaaagagggc 1140
aaggcctacc agatccatta catcaaagag atggccaagg agggcactcg atccttatta 1200
ctggaggcca agtggctgaa ggagggttat atgcccaccc tcgacgagta cctctccaac 1260
tctttagtca cttgtggcta cgctctcatg actgccagat cttacgtcgc tcgagacgat 1320
ggtatcgtga ccgaggacgc tttcaaatgg gtcgctactc acccccctat cgtgaaggcc 1380
gcttgtaaga ttctccgact catggacgac atcgccaccc ataaggagga gcaagaacga 1440
ggccacatcg cctcttctat cgagtgctac agaaaggaga ccggcgcctc tgaggaggag 1500
gcttgcatgg actttctcaa gcaagttgaa gatggctgga aggtcattaa ccaagaatct 1560
ctcatgccta ccgacgtgcc cttcccctta ttaattcccg ctatcaacct cgcccgagtg 1620
tccgacactt tatataaaga caacgacggc tacaatcacg ccgataagga ggtgatcggc 1680
tacatcaagt ccctcttcgt ccaccctatg attgtgtaa 1719
<210> 2
<211> 1503
<212> DNA
<213> Yarrowia lipolytica Po1f Δku70
<400> 2
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 agtctaatac caagacttta 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> 3
<211> 531
<212> DNA
<213> Yarrowia lipolytica Po1f Δku70
<400> 3
agagaccggg ttggcggcgc 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 ttcaaaatgg tgagtttcag 420
aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480
acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531
<210> 4
<211> 516
<212> DNA
<213> Yarrowia lipolytica Po1f Δku70
<400> 4
gatccaacta cggaacttgt gttgatgtct ttgcccccgg ctccgatatc atctctgcct 60
cttaccagtc cgactctggt actttggtct actccggtac ctccatggcc tgtccccacg 120
ttgccggtct tgcctcctac tacctgtcca tcaatgacga ggttctcacc cctgcccagg 180
tcgaggctct tattactgag tccaacaccg gtgttcttcc caccaccaac ctcaagggct 240
ctcccaacgc tgttgcctac aacggtgttg gcatttaggc aattaacaga tagtttgccg 300
gtgataattc tcttaacctc ccacactcct ttgacataac gatttatgta acgaaactga 360
aatttgacca gatattgttg taaatagaaa atctggcttg taggtggcaa aatcccgtct 420
ttgttcatca attccctctg tgactactcg tcatcccttt atgttcgact gtcgtatttt 480
tattttccat acatacgcaa gtgagatgcc cgtgtc 516
Claims (6)
1. The recombinant yarrowia lipolytica for producing (-) -alpha-bisabolol is characterized in that the recombinant yarrowia lipolytica is inserted with a 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and a fusion protein expression cassette in the genome of yarrowia lipolytica; the fusion protein expression cassette consists of (-) -alpha-bisabolol synthetase, connecting peptide and farnesyl pyrophosphate synthetase, wherein the coding gene of the (-) -alpha-bisabolol synthetase is shown as SEQ ID NO. 1; the coding gene sequence of the 3-hydroxy-3-methylglutaryl CoA reductase is shown in SEQ ID NO. 2; the genebank accession number of the farnesyl pyrophosphate synthetase encoding gene ERG20 is YALI0E05753 g.
2. The recombinant yarrowia lipolytica for producing (-) - α -bisabolol of claim 1, wherein said linker peptide is GGGGS or GSG or EAAAK.
3. The recombinant yarrowia lipolytica producing (-) - α -bisabolol of claim 2, wherein the promoter of said fusion protein expression cassette is the PTEFin promoter or PTEF1 promoter of yarrowia lipolytica and the terminator is the Txpr2 terminator of yarrowia lipolytica; the sequence of the PTEFin promoter is shown in SEQ ID NO. 3; the sequence of the Txpr2 terminator is shown in SEQ ID NO. 4.
4. The recombinant yarrowia lipolytica for the production of (-) - α -bisabolol of claim 3, wherein said recombinant yarrowia lipolytica further expresses 1 or more marker genes; the marker gene is selected from a3 (beta) -isopropylmalate dehydrogenase encoding gene or an orotidine-5' -phosphate decarboxylase encoding gene.
5. The method of claim 1 for constructing recombinant yarrowia lipolytica producing (-) - α -bisabolol comprising the step of introducing the expression cassette for 3-hydroxy-3-methylglutaryl CoA reductase and the expression cassette for the fusion protein into yarrowia lipolytica in the form of plasmids, followed by integration into the genome of yarrowia lipolytica; the fusion protein expression box is composed of (-) -alpha-bisabolol synthetase, connecting peptide and farnesyl pyrophosphate synthetase.
6. Use of the recombinant yarrowia lipolytica of any one of claims 1-4 for the production of (-) - α -bisabolol, comprising the steps of: (1) culturing the recombinant yarrowia lipolytica of any one of claims 1-4 in a fermentation medium to obtain a fermentation product; (2) and extracting the fermentation product by using an organic solution, and collecting an organic phase.
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