CN111154665A

CN111154665A - Recombinant yarrowia lipolytica and construction method and application thereof

Info

Publication number: CN111154665A
Application number: CN202010072154.7A
Authority: CN
Inventors: 纪晓俊; 王凯峰; 马一榕; 李文娟; 买洁
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-05-15
Anticipated expiration: 2040-01-21
Also published as: CN111154665B

Abstract

The invention provides a recombinant yarrowia lipolytica strain and a construction method and application thereof, relating to the field of bioengineering. The recombinant yarrowia lipolytica is yarrowia lipolytica (Yarrowia lipolytica) The strain XJ-8 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 19064. the construction method of the recombinant yarrowia lipolytica has simple and efficient operation and can efficiently synthesize β -elemene and/or germacrene A.

Description

Recombinant yarrowia lipolytica and construction method and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to recombinant yarrowia lipolytica for producing β -elemene and/or germacrene A, and a construction method and application thereof.

Background

β -elemene (β -elemene), with the scientific name of 1-methyl-1-vinyl-2, 4-diisopropylcyclohexane, is a volatile sesquiterpene, mainly exists in essential oil of plants such as Curcuma zedoary (Curcuma zedoaria), citronella (Cymbopogon citratus) and the like, β -elemene can pass through blood brain barrier, not only can directly resist tumor, but also has immune protection effect, has the characteristics of relieving cancer pain, increasing white blood cells and inhibiting platelet aggregation and the like (Edris, Current clinical Medicine, 2009,4(1): 43-46; Chen M et al, Evidence-Based comparative animal and D ingredient, 2012,2012.) compared with most medicines, the Medicine chemotherapy has small toxic and side effects, no obvious liver and kidney damage, no bone marrow inhibition, no drug resistance after long-term use (Zhai B, international patent publication, 20136.) and has taken as a new Medicine for clinical application, and has significant effect of oral administration, namely, and the Medicine has taken as a new drug injection for national Medicine.

β the elemene is obtained by steam distillation and supercritical CO distillation₂The extraction method is used for separating and extracting the β -elemene from the zedoary volatile oil, but the extraction method has the disadvantages of more material input, long time consumption, low purity and yield, complicated steps and incapability of completely meeting the market requirements due to low content of β -elemene in the zedoary, and the elemene extracted from plants has various isomers, so that the purification and the obtaining of the β -elemene with a single configuration are difficult.

Germacrene A Synthetase (GAS) catalyzes farnesyl pyrophosphate (FPP) to synthesize germacrene A, which undergoes Cope rearrangement at 250 ℃ to β -elemene (Goorest phone G et al, NaturalProduct Research,2017,31(15):1836-1839.) currently, this point has been exploited in the production of β -elemene in microorganisms, but industrial application has not been achieved due to low yield.

Disclosure of Invention

The invention aims to provide a recombinant yarrowia lipolytica which can efficiently synthesize β -elemene and/or germacrene A.

Still another object of the present invention is to provide a method for constructing the recombinant yarrowia lipolytica, which is efficient and easy to operate.

It is a further object of the present invention to provide the use of said recombinant yarrowia lipolytica for the production of β -elemene and/or germacrene A.

The purpose of the invention is realized by adopting the following technical scheme:

a recombinant Yarrowia lipolytica is Yarrowia lipolytica XJ-8 strain which is preserved in China general microbiological culture Collection center with the preservation number of CGMCCNo.19064.

In the invention, the recombinant Yarrowia lipolytica (Yarrowia lipolytica) XJ-8 strain is obtained by inserting germacrene A synthetase, farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase expression cassette into the Yarrowia lipolytica genome.

In the invention, the germacrene A synthetase is derived from chamomile (Chamaemelum nobile), the coding gene of the germacrene A synthetase is suitable for being expressed in yarrowia lipolytica after codon optimization, and the coding genes of farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase are derived from yarrowia lipolytica.

In the invention, the recombinant yarrowia lipolytica is obtained by inserting a 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and a fusion protein expression cassette into the yarrowia lipolytica genome; the fusion protein consists of germacrene A synthetase, connecting peptide and farnesyl pyrophosphate synthetase (genbank accession number XP _ 503599.1); the connecting peptide is GGGGS or GSG or EAAAK.

In the present invention, the promoter of the expression cassette is the TEFin promoter or TEF1 promoter of yarrowia lipolytica; the terminator is the xpr2t or cyc1 terminator of yarrowia lipolytica.

In the invention, the recombinant yarrowia lipolytica also expresses 1 or more marker genes selected from the group consisting of a 3(β) -isopropylmalate dehydrogenase encoding gene cassette and an orotidine-5' -phosphate decarboxylase encoding gene cassette.

In the invention, the coding gene of the germacrene A synthetase is shown as SEQ ID No: 1 is shown in the specification; the coding gene sequence of the 3-hydroxy-3-methylglutaryl CoA reductase is shown as SEQ ID No: 2, respectively.

In the present invention, the step of introducing said 3-hydroxy-3-methylglutaryl CoA reductase expression cassette, germacrene A synthase expression cassette and farnesyl pyrophosphate synthase expression cassette into said yarrowia lipolytica in the form of a plasmid and then integrating them on the genome of yarrowia lipolytica is included.

The invention also provides a construction method of the recombinant yarrowia lipolytica, 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 and the yarrowia lipolytica genome; the fusion protein is composed of germacrene A synthetase, connecting peptide and farnesyl pyrophosphate synthetase.

The invention also provides application of the recombinant bacteria in production of β -elemene and/or germacrene A, which is characterized by comprising the following steps of (1) culturing the recombinant bacteria in any one of claims 1-7 by adopting a fermentation culture medium to obtain a fermentation product, (2) extracting the fermentation product by using an organic solution, collecting an organic phase to obtain the germacrene A, and (3) heating the organic phase to 250-300 ℃ to obtain β -elemene.

The recombinant yarrowia lipolytica of the invention can express germacrene A synthetase, over-expression endogenous farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase, experiments prove that the recombinant yarrowia lipolytica can efficiently ferment and produce β -elemene and/or germacrene A, and realize the synthesis of natural products β -elemene and/or germacrene A in yarrowia lipolytica, the recombinant yarrowia lipolytica of the invention has the following advantages:

(1) the recombinant yarrowia lipolytica constructed by the invention is based on the yarrowia lipolytica which is used for coding the non-homologous recombination gene ku70 and is knocked out, so that the homologous recombination capability of the yarrowia lipolytica 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 construction method of the recombinant yarrowia lipolytica is efficient and simple to operate.

(2) The recombinant yarrowia lipolytica realizes the high-efficiency synthesis of β -elemene and/or germacrene A by over-expressing 3-hydroxy-3-methylglutaryl CoA reductase coding gene tHMG, farnesyl pyrophosphate synthetase coding gene ERG20 and germacrene A synthetase coding gene gas.

Drawings

FIG. 1 is a structural diagram of a recombinant plasmid pUC-leu-A08-Optgas, wherein A08-up represents the upstream homology arm at A08 site, A08-dm represents the downstream homology arm at A08 site, xpr2T represents the terminator T_xpr2tTEFin denotes the promoter P_TEFinLeu denotes the expression cassette for the gene encoding 3(β) -isopropylmalate dehydrogenase (containing the endogenous promoter and terminator of Yarrowia lipolytica).

FIG. 2 shows the structure of the recombinant plasmid pUC-HUH-IntC-tHMG wherein IntC-up denotes the homology arm upstream of the IntC site, IntC-dm denotes the homology arm downstream of the IntC site, xpr2T denotes the terminator T_xpr2tTEFin denotes the promoter P_TEFinURA denotes the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the promoter and terminator endogenous to Yarrowia lipolytica).

FIG. 3 shows the structure of recombinant plasmid pUC-HUH-SCP2-ERG20, in which SCP2-up represents the upstream homology arm at SCP2 site, SCP2-dm represents the downstream homology arm at SCP2 site, and xpr2T represents terminator T_xpr2tTEFin denotes the promoter P_TEFinURA denotes the orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing the promoter and terminator endogenous to Yarrowia lipolytica).

FIG. 4 is a structural diagram of recombinant plasmid pUC-leu-A08-Optgas-GSG-ERG20, wherein A08-up represents the upstream homology arm at A08 site, A08-dm represents the downstream homology arm at A08 site, and xpr2T represents terminator T_xpr2tTEFin denotes the promoter P_TEFinLeu denotes the expression cassette for the gene encoding 3(β) -isopropylmalate dehydrogenase (containing the endogenous promoter and terminator of Yarrowia lipolytica).

FIG. 5 is a structural diagram of recombinant plasmid pUC-leu-A08-ERG20-GSG-Optgas, wherein A08-up represents the upstream homology arm at A08 site, A08-dm represents the downstream homology arm at A08 site, and xpr2T represents terminator T_xpr2tTEFin denotes the promoter P_TEFinLeu denotes the expression cassette for the gene encoding 3(β) -isopropylmalate dehydrogenase (containing the endogenous promoter and terminator of Yarrowia lipolytica).

FIG. 6 is a structural diagram of recombinant plasmid pUC-leu-A08-ERG20-EAAAK-Optgas, wherein A08-up represents the upstream homology arm at A08 site, A08-dm represents the downstream homology arm at A08 site, and xpr2T represents terminator T_xpr2tTEFin denotes the promoter P_TEFinLeu denotes the expression cassette for the gene encoding 3(β) -isopropylmalate dehydrogenase (containing the endogenous promoter and terminator of Yarrowia lipolytica).

FIG. 7 shows a GC assay for β -elemene produced by Yarrowia lipolytica XJ-8 strain (recombinant 8).

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 (Yarrowia lipolytica) Po1f, available from American type culture Collection, accession number ATCC MYA-2613.

Yarrowia lipolytica (Yarrowia lipolytica) Po1f Δ ku70(MatA, &lTtT translation = Δ "&gTt Δ &lTt/T &gTt ku70:: hisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated Yarrowia lipolytica Po1f Δ ku 70. Yarrowia lipolytica Po1f Δ ku70 were constructed from Yarrowia lipolytica (Yarrowia lipolytica) Po1f by knocking out the coding gene ku70 responsible for non-homologous recombination (disclosed in Kretzschmar A et al, currentetics, 2013,59(1-2): 63-72).

The A08 site integration plasmid was obtained by inserting a sequence of 2521bp upstream of the start codon and 2031bp downstream of the stop codon of the A08 site on chromosome A of Yarrowia lipolytica Po1 f. delta. ku70 genome into pUC57-leu vector (the construction method is described in example 1), and the leu expression cassette (containing the Yarrowia lipolytica endogenous promoter and terminator) was located between the sequences upstream and downstream of the A08 site.

The IntC site integration plasmid is obtained by inserting sequences of 1402bp upstream of the initiation codon and 1396bp downstream of the termination codon of the IntC site on chromosome C in Yarrowia lipolytica Po1f delta ku70 genome into a pUC57-hisG-ura-hisG (the construction method is shown in example 1) vector, and two hisG tag encoding genes are arranged between the sequences of the upstream and downstream of the IntC site.

The SCP2 site integration plasmid is obtained by inserting a sequence of 1523bp upstream of an initiation codon and 1524bp downstream of a termination codon of an SCP2 site on chromosome E in a Yarrowia lipolytica Po1f delta ku70 genome into a pUC57-hisG-ura-hisG vector, and two hisG tag coding genes are arranged between the upstream and downstream sequences of the SCP2 site.

The primer sequences in the present invention are shown in tables 2 and 4-1, tables 4-2, tables 4-3 and tables 4-4.

Example 1 amplification of Gene elements and preparation of target plasmids

(first) preparation of target Gene

According to the nucleotide sequence of the germacrene A synthetase encoding gene gas (genbank accession number KU589283.1) from Roman chamomile provided at NCBI, after codon optimization, Suzhou Jinzhi Biotechnology limited was entrusted to synthesize the optimized germacrene A synthetase encoding gene Optgas, and inserted into plasmid pUC57, to obtain plasmid pUC 57-Optgas. The nucleotide sequence of Optgas is shown in SEQ ID No: 1 is shown.

Based on the nucleotide sequence of 3(β) -isopropylmalate dehydrogenase-encoding gene leu in Yarrowia lipolytica (genbank accession number M37309.1) provided at NCBI, Suzhou Limited public Biotechnology LimitedSynthesizing the gene leu, an expression cassette for the gene encoding 3(β) -isopropylmalate dehydrogenase (containing the endogenous promoter P of Yarrowia lipolytica)_TEFinA terminator T_xpr2t) Inserted into plasmid pUC57 to obtain plasmid pUC 57-leu. Based on the nucleotide sequence of orotidine-5 '-phosphate decarboxylase encoding gene ura (genbank accession number AJ306421.1) and hisG tag (genbank accession number AF324729.1) in Yarrowia lipolytica provided at NCBI, the synthesis of Suzhou Jinzhi Biotechnology Limited was entrusted, two hisG tag encoding gene sequences were inserted into plasmid pUC57, and an orotidine-5' -phosphate decarboxylase encoding gene expression cassette (containing Yarrowia lipolytica endogenous promoter P) was inserted into the two hisG tag encoding gene sequences_TEFinA terminator T_xpr2t) To achieve ura-tag recovery, resulting in plasmid pUC 57-hisG-ura-hisG.

3-hydroxy-3-methylglutaryl CoA reductase coding gene tHMG is amplified by using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template and using tHMG-F and tHMG-R as primers. the nucleotide sequence of tHMG is shown as SEQ ID No: 2, respectively.

The farnesyl pyrophosphate synthetase encoding gene ERG20(genbank accession number XP _503599.1) was amplified using Yarrowia lipolytica Po1F delta ku70 genomic DNA as a template and ERG20-F and ERG20-R as primers.

Endogenous promoter P of Yarrowia lipolytica_TEFinThe nucleotide sequence of (A) is shown in SEQ ID No: 3, endogenous terminator T of Yarrowia lipolytica_xpr2tThe nucleotide sequence is shown as SEQ ID No: 4, respectively.

(II) construction of recombinant plasmid

The structure of the recombinant plasmid is shown in Table 1 and FIGS. 1-5.

1. Construction of recombinant plasmid pUC-leu-A08-Optgas

The recombinant plasmid pUC-leu-A08-Optgas takes pUC57-leu as a framework, an upstream homology arm A08-up at the initiation codon of A08 site in yarrowia lipolytica Po1f delta ku70 and a downstream homology arm A08-dm at the termination codon are inserted, and an Optgas gene expression cassette (P08-dm) is inserted between the upstream and downstream homology arms_TEFin-Optgas-T_xpr2t) 3(β) -isopropylmalate dehydrogenase-encoding Gene expression cassetteBetween the downstream homology arms, the structure is as in FIG. 1.

Amplifying an Optgas expression cassette promoter P by taking A08-TEFinp-F and TEFinp-Optgas-R as primers and Yarrowia lipolytica Po1F delta ku70 genome DNA as a template_TEFin. Optgas expression cassette terminator T is amplified by taking Optgas-xpr2T-F and A08-xpr2T-R as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a template_xpr2t。

Plasmid pUC57-Optgas is used as a template, TEFinp-Optgas-F and Optgas-xpr2t-R are used as primers, and promoters P are respectively arranged at two amplified ends_TEFinAnd a terminator T_xpr2tThe Optgas gene of the homology arm.

The PCR enzyme used in the above PCR reaction was PrimeSTAR MaxDNA polymerase from Baozi medical technology (Beijing) Ltd. The PCR amplification system is as follows:

components	Volume of
		PrimerSTAR Max Premix	25ul
Form panel	1ul
		Primer 1	2ul
Primer 2	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 (promoter P) in the Optgas gene expression cassette constructed in title 1 of this example_TEFinOptgas fragment and terminator T_xpr2t) One-Step Cloning is realized by using the Clonexpress Multi S One Step Cloning Kit of Nanjing Novowed Biotechnology Co., Ltd, and a reaction system is shown in the following table. After incubating the reaction system at 37 ℃ for 30min, the circular recombinant vector is obtained.

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

Wherein, the usage amount of the linearized vector (x) and the insert (y) can be calculated by 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 α competent cells, and positive recombinant plasmid pUC-leu-A08-Optgas 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 homology arm of an IntC site initiation codon and a downstream homology arm of a termination codon in yarrowia lipolytica Po1f delta ku70 are inserted, and a tHMG expression cassette (promoter P) is inserted between the upstream and downstream homology arms_TEFinA terminator T_xpr2t) Orotidine-5' -phosphate decarboxylase encoding gene expression cassettes (containing the Yarrowia lipolytica endogenous promoter P)_TEFinA terminator T_xpr2t) Also between the upstream and downstream homology arms, the specific structure is shown in FIG. 2.

The IntC-TEFinp-F and TEFinp-tHMG-R are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and a tHMG expression cassette promoter P is amplified_TEFin. Amplifying a terminator T of the tHMG expression cassette by using tHMG-xpr2T-F and IntC-xpr2T-R as primers and Yarrowia lipolytica Po1F delta ku70 genome DNA as a template_xpr2t。

Uses Yarrowia lipolytica Po1F delta ku70 genome DNA as template, uses TEFinp-tHMG-F and tHMG-xpr2t-R as primer to amplify two ends respectively with promoter P_TEFinAnd a terminator T_xpr2ttHMG 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 (fragment P) of the tHMG gene expression cassette constructed in title 2 of this example_TEFintHMG and T_xpr2t) One-step Cloning was carried out using the Clonexpress MultiS OneStep Cloning Kit of Nanjing Novowed Biotechnology Ltd to obtain 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, an upstream homology arm of an initiation codon and a downstream homology arm of a termination codon of an SCP2 site in Yarrowia lipolytica Po1f delta ku70 are inserted, and an ERG20 expression cassette (a promoter P20) is inserted between the upstream and downstream homology arms_TEFinA terminator T_xpr2t) The expression cassette of the gene coding orotidine-5' -phosphate decarboxylase is also between the upstream and downstream homology arms, and the specific structure is shown in figure 3.

Amplifying an ERG20 expression cassette promoter P by taking SCP2-TEFinp-F and TEFinp-ERG20-R as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a template_TEFin. ERG20-xpr2T-F and SCP2-xpr2T-R are used as primers, Yarrowia lipolytica Po1F delta ku70 genome DNA is used as a template, and an ERG20 expression cassette terminator T is amplified_xpr2t。

The genomic DNA of Yarrowia lipolytica Po1F delta ku70 is taken as a template, TEFinp-ERG20-F and ERG20-xpr2t-R are taken as primers, and promoters P are respectively arranged at two ends of amplification_TEFinAnd a terminator T_xpr2tERG20 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 (fragment P) in the ERG20 gene expression cassette constructed in title 3 of this example_TEFinERG20 and T_xpr2t) one-Step Cloning was carried out using the Clon express MultiSOne Step Cloning Kit of Nanjing Novowed Biotechnology Ltd to obtain recombinant plasmid pUC-HUH-SCP2-ERG 20.

4. Construction of pUC-leu-A08-Optgas-GSG-ERG20

The recombinant plasmid pUC-leu-A08-Optgas-GSG-ERG20 is constructed by pUC57-leu, and is inserted into upstream homology arm of A08 site initiation codon and downstream homology arm of termination codon in Yarrowia lipolytica Po1f delta ku70, and an expression cassette (P-GSG-ERG 20) of fusion protein Optgas-GSG is inserted between the upstream and downstream homology arms_TEFin-Optgas-GSG-ERG20-T_xpr2) The N end of the fusion protein Optgas-GSG-ERG20 is germacrene A synthetase, the C end is farnesyl pyrophosphate synthetase, and the germacrene A synthetase and the farnesyl pyrophosphate synthetase are connected by a connecting peptide with the amino acid sequence of GSG, and the specific structure is shown in figure 4.

Amplifying Optgas-GSG-ERG20 expression cassette promoter P by taking A08-TEFinp-F and TEFinp-Optgas-R as primers and Yarrowia lipolytica Po1F delta ku70 genome DNA as a template_TEFin. The Optgas-GSG-ERG20 expression cassette terminator T is amplified by taking ERG20-xpr2T-F and A08-xpr2T-R as primers and Yarrowia lipolytica Po1F delta ku70 genomic DNA as a template_xpr2t。

The Optgas gene was amplified using plasmid pUC57-Optgas as template and TEFinp-Optgas-F and Optgas-ERG20-R as primers.

The ERG20 gene is amplified by using Yarrowia lipolytica Po1F delta ku70 genome DNA as a template and Optgas-ERG20-F and ERG20-xpr2t-R as primers.

Each gene element (fragment P) in the Optgas-GSG-ERG20 expression cassette obtained by amplifying the title 4 of this example_TEFinOptgas gene, ERG20 gene and T_xpr2t) And linearized A08 site integration plasmid was cloned in One Step using the Clon express MultiS One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd to obtain the recombinant plasmid pUC-leu-A08-Optgas-GSG-ERG 20.

5. Construction of recombinant plasmid pUC-leu-A08-ERG20-GSG-Optgas

The recombinant plasmid pUC-leu-A08-ERG20-GSG-Optgas takes pUC57-leu as a framework,the upstream homology arm of the initiation codon of A08 site and the downstream homology arm of the termination codon of Yarrowia lipolytica Po1f delta ku70 were inserted, and an expression cassette (P) of the fusion protein ERG20-GSG-Optgas was inserted between the upstream and downstream homology arms_TEFin-ERG20-GSG-Optgas-T_xpr2t) The N end of the fusion protein ERG20-GSG-Optgas is farnesyl pyrophosphate synthetase, the C end is germacrene A synthetase, and the farnesyl pyrophosphate synthetase and the germacrene A synthetase are connected by a connecting peptide with the amino acid sequence being GSG, and the specific structure is shown in figure 5.

A08-TEFinp-F and TEFinp-ERG20-R are used as primers, Yarrowia lipolytica delta ku70 genome is used as a template, and a promoter P of an ERG20-GSG-Optgas expression cassette is amplified_TEFin. Amplifying terminator T of ERG20-GSG-Optgas expression cassette by using Optgas-xpr2T-F and A08-xpr2T-R as primers and Yarrowia lipolytica delta ku70 genome as template_xpr2t。

The ERG20 gene is amplified by taking Yarrowia lipolytica Po1F delta ku70 genome as a template and TEFinp-ERG20-F and ERG20-Optgas-R as primers.

The Optgas gene was amplified using plasmid pUC57-Optgas with the synthesized Optgas gene as a template and ERG20-Optgas-F and Optgas-xpr2t-R 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 of the gene elements (fragment P) in the ERG20-GSG-Optgas expression cassette obtained by amplifying the title 5 of this example_TEFinERG20 gene, Optgas gene and T_xpr2t) And linearizing an A08 site integration plasmid, and utilizing a Clon express MultiS One Step Cloning Kit of Nanjing Novowed Biotechnology Limited to realize One-Step Cloning to obtain a recombinant plasmid pUC-leu-A08-ERG 20-GSG-Optgas.

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) Plasmid pUC-leu-A08-Optgas containing the Optgas gene expression cassette is introduced into yarrowia lipolytica Po1f delta ku70, and the Optgas expression cassette is integrated at the site A08 of the genome to obtain 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-Optgas was transformed into Yarrowia lipolytica Po1 f. delta. ku70 competent cells using Zymogen FROzen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination.

(2) 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: glucose 20g/L, Yeast Nitrogen Base (YNB, ammonium sulfate, available from BBI Life Sciences)6.7g/L, CSM-Leu (complete supplement mixture to remove leucine, available from MPBiomedia) 0.67g/L, agar powder 23 g/L.

(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: glucose 20g/L, YNB 6.7g/L, CSM-Leu-Ura (complete supplement mixture to remove leucine and uracil, from MP Biomedicals)0.67g/L, agar powder 23 g/L.

(III) construction of recombinant bacterium 3

The pUC-HUH-IntC-tHMG and pUC-leu-A08-Optgas-GSG-ERG20 plasmids were introduced into yarrowia lipolytica Po1 f. delta. ku70, the tHMG cassette was integrated into the IntC site in the genome, and the Optgas-GSG-ERG20 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-Optgas-GSG-ERG20 were transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROZEN Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination, and correct positive clones were identified by PCR using selection medium SD-Leu-Ura selection, which was designated as recombinant bacterium 3.

The screened culture medium SD-Leu-Ura comprises the following components: glucose 20g/L, YNB (Yeast Nitrogen alkali ammonium sulfate, from BBILIfe Sciences)6.7g/L, CSM-Leu-Ura (complete supplement mixture to remove leucine and uracil, from MPBiomedia) 0.67g/L, agar powder 23 g/L.

(IV) construction of recombinant bacterium 4

Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-leu-A08-ERG20-GSG-Optgas are introduced into yarrowia lipolytica Po1 f. delta. ku70, the tHMG expression cassette is integrated into the IntC site in the genome, and the ERG20-GSG-Optgas expression cassette is integrated into the A08 site, so as to obtain recombinant bacterium 4.

The specific method comprises the following steps: after the Yarrowia lipolytica Po1 f. delta. ku70 was cultured overnight in YPD liquid medium (2% peptone, 1% Yeast extract, 2% glucose), recombinant plasmids pUC-HUH-intC-tHMG and pUC-Leu-A08-ERG 20-GSG-Optigas were transformed into Yarrowia lipolytica Po1 f. delta. ku70 using Zymogen FROZEN EZ Yeast Transformation Kit II of 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-Optgas and Yarrowia lipolytica Po1f delta ku70 as templates and using primers in a table 4 (comprising tables 4-1, 4-2, 4-3 and 4-4) to obtain Optgas and ERG20 fragments which contain different homology arms and correspond 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 Optgas gene was PCR-amplified using plasmid pUC57-Optgas with the synthesized Optgas gene as a template, and using pairs of primers Optgas-F and Optgas-R in Table 4 (tables 4-1, 4-2, 4-3, 4-4) with the linker peptide GGGGS/EAAAK to obtain fragments corresponding to the primer pairs Optgas-F (Optgas-GGGGS-ERG20) and Optgas-R (Optgas-GGGGS-ERG20), the primer pairs Optgas-F (Optgas-EAAAK-ERG20) and Optgas-R (Optgas-EAAAK-ERG20), the primer pairs Optgas-F (ERG 20-GGS-Optgas) and Optgas-R (Optgas 63-GGS-Optgas), the primer pairs Optgas-OPgaS-OPGAS (Optgas-EAK-ERG 8678), and Optgas-R (Optgas-EAK-ERG-8678).

ERG20 gene was PCR amplified using Yarrowia lipolytica Po 1F. delta. ku70 genome as template and using respective primers ERG20-F and ERG20-R with the linker peptide GGGGS/EAAAK in Table 4 (tables 4-1, 4-2, 4-3, 4-4) to obtain corresponding primer pairs ERG20-F (Optgas-GGGGS-ERG20) and ERG20-R (Optgas-GGGGS-ERG20), primer pair ERG20-F (Optgas-AAAAK-ERG 20) and ERG20-R (Optgas-EAAAK-ERG20), primer pair ERG20-F (ERG 20-GGS-Optgas) and ERG20-R (ERG 20-GGS-ERG 20), primer pair ERG-20-ERG-IRG 5848-ERG-IRG-20-IRG-20, and the amplified fragments thereof.

Respectively carrying out one-step cloning on Optgas amplified fragments and ERG20 amplified fragments corresponding to different primer pairsRecombinant plasmids pUC-leu-A08-Optgas-GGGGS-ERG20, pUC-leu-A08-Optgas-EAAAK-ERG20, pUC-leu-A08-ERG20-GGGGS-Optgas, pUC-leu-A08-ERG20-EAAAK-Optgas were constructed for the fusion protein expression cassette elements. Wherein the promoter of each fusion protein expression cassette element is Yarrowia lipolytica endogenous promoter P_TEFinThe terminators are all Yarrowia lipolytica endogenous terminator T_xpr2t. Wherein the expression cassette promoter P of the fusion protein ERG20-EAAAK-Optgas_TEFinThe amplification primers are A08-TEFinp-F and TEFinp-Optgas-R, and the terminator T_xpr2tThe amplification primers are ERG20-xpr2t-F and A08-xpr2 t-R. ERG20, Optgas fragments and P obtained by amplification with the primers in tables 4-4_TEFin、T_xpr2tIs the original of fusion protein ERG 20-EAAAK-Optgas.

The recombinant plasmid pUC-leu-A08-Optgas-GGGGS-ERG20 is constructed by pUC57-leu, and has inserted upstream homology arm of Yarrowia lipolytica Po1f delta ku 70A 08 site start codon and downstream homology arm of stop codon, and inserted expression cassette (P-leu-GGGGS-ERG 20) of fusion protein between the upstream and downstream homology arms_TEFin-Optgas-GGGGS-ERG20-T_xpr2t) The N-terminal of the fusion protein Optgas-GGGGS-ERG20 is germacrene A synthetase, the C-terminal is farnesyl pyrophosphate synthetase, and the germacrene A synthetase and the farnesyl pyrophosphate synthetase are connected by a connecting peptide with an amino acid sequence of GGGGS.

The recombinant plasmid pUC-leu-A08-Optgas-EAAAK-ERG20 takes pUC57-leu as a framework, an upstream homology arm of an initiation codon and a downstream homology arm of a termination codon of Yarrowia lipolytica Po1f delta ku 70A 08 site are inserted, and an expression cassette (P-Optgas-EAAAK-ERG 20) of the fusion protein is inserted between the upstream and downstream homology arms_TEFin-Optgas-EAAAK-ERG20-T_xpr2t) The N-terminal of the fusion protein Optgas-EAAAK-ERG20 is germacrene A synthetase, the C-terminal is farnesyl pyrophosphate synthetase, and a connecting peptide with an amino acid sequence of EAAAK is arranged between the germacrene A synthetase and the farnesyl pyrophosphate synthetaseAre connected.

The recombinant plasmid pUC-leu-A08-ERG 20-GGGGGGS-Optgas takes pUC57-leu as a framework, is inserted with upstream homology arm of Yarrowia lipolytica Po1f delta ku 70A 08 site start codon and downstream homology arm of stop codon, and is inserted with expression cassette (P20-GGGGS-Optgas) of fusion protein ERG20 between the upstream and downstream homology arms_TEFin-ERG20-GGGGS-Optgas-T_xpr2t) The N-terminal of the fusion protein ERG20-GGGGS-Optgas is farnesyl pyrophosphate synthetase, the C-terminal is germacrene A synthetase, and the farnesyl pyrophosphate synthetase and the germacrene A synthetase are connected by a connecting peptide with an amino acid sequence of GGGGS.

The recombinant plasmid pUC-leu-A08-ERG20-EAAAK-Optgas takes pUC57-leu as a framework, is inserted with an upstream homology arm of an initiation codon of Yarrowia lipolytica Po1f delta ku 70A 08 site and a downstream homology arm of a termination codon, and an expression cassette (P20-EAAAK-Optgas) of fusion protein ERG20 is inserted between the upstream and downstream homology arms_TEFin-ERG20-EAAAK-Optgas-T_xpr2tThe N-terminal of the fusion protein ERG20-EAAAK-Optgas is farnesyl pyrophosphate synthetase, the C-terminal is germacrene A synthetase, and the farnesyl pyrophosphate synthetase and the germacrene A synthetase are connected by a connecting peptide with an amino acid sequence of EAAAK, and the specific structure is shown in figure 6.

Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-Optgas-GGGGS-ERG20 are introduced into Yarrowia lipolytica Po1f delta ku70 for homologous recombination, a tHMG expression cassette is integrated at the site of intC of a genome, an Optgas-GGGGS-ERG20 expression cassette is integrated at the site of A08 of the genome, 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 5.

Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-Optgas-EAAAK-ERG20 are introduced into Yarrowia lipolytica Po1 f. delta. ku70 for homologous recombination, a tHMG expression cassette is integrated at the site of intC of the genome, an Optgas-EAAAK-ERG20 expression cassette is integrated at the site of A08 of the genome, and a screening medium SD-Leu-Ura is adopted for screening, so that a positive clone which is correct for PCR identification is named as recombinant bacterium 6.

Recombinant plasmids pUC-HUH-IntC-tHMG and pUC-Leu-A08-ERG20-GGGGS-Optgas are introduced into Yarrowia lipolytica Po1 f. delta. ku70 for homologous recombination, a tHMG expression cassette is integrated at the site of intC of the genome, an ERG20-GGGGS-Optgas expression cassette is integrated at the site of A08 of the genome, 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-Optgas are introduced into Yarrowia lipolytica Po1 f. delta. ku70 for homologous recombination, a tHMG expression cassette is integrated into an IntC site in a genome, an ERG20-EAAAK-Optgas expression cassette is integrated into an 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 8.

Wherein, the recombinant bacterium 8 has been preserved in China general microbiological culture Collection center (CGMCC) in 2019 at 12 months and 02 days, and the address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, classified and named: yarrowia lipolytica, according to the biological materials (strains): XJ-8, accession number: CGMCC No. 19064.

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-Optgas-GGGGS-ERG20

TABLE 4-2 primers required for construction of recombinant plasmid pUC-leu-A08-Optgas-EAAAK-ERG20

Table 4-3 shows the primers required for constructing recombinant plasmid pUC-leu-A08-ERG20-GGGGS-Optgas

Tables 4-4 show the primers required for construction of recombinant plasmid pUC-leu-A08-ERG20-EAAAK-Optgas

Example 3 application of recombinant bacteria 1-8 in β -elemene production

1. Engineering bacteria culture and product extraction

The method comprises the steps of activating the recombinant bacteria, culturing the activated recombinant bacteria in a YPD liquid culture medium for 16 hours at 30 ℃ and 220rpm to obtain seed liquid, inoculating the seed liquid into 50ml of a fermentation culture medium according to the inoculation amount of 1%, performing shake culture at 30 ℃ and 220rpm for 1 day, adding n-dodecane accounting for 10% of the volume of the fermentation liquid, continuing to perform shake culture for 3 days, transferring the fermentation liquid to a 50ml centrifuge tube after the fermentation is finished, centrifuging the fermentation liquid at 5000rpm for 15min, 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.β qualitative and quantitative analysis of elemene

1) β transformation of elemene

And (3) heating the organic phase sample in a ventilation kitchen for 1 hour by using an oil bath at 220 ℃ to remove n-dodecane, thereby obtaining a substance converted by each recombinant bacterium.

2) β -elemene detection

Diluting the substance converted by each recombinant bacterium by using n-hexane for 10 times, filtering the diluted substance through an organic nylon filter membrane (0.22um), detecting by using GC under the detection conditions that the injection port temperature is 250 ℃, the injection volume is 1ul, and no shunt is generated, a chromatographic column is HP-5ms (30m x 0.25mM), the initial temperature is 60 ℃, the initial temperature is increased to 160 ℃ according to the speed of 10 ℃/min, then the initial temperature is increased to 200 ℃ at 5 ℃/min, finally the initial temperature is increased to 230 ℃, and qualitative and quantitative determination is carried out by using β -elemene standard products.

FIG. 7 is a GC test chart of β -elemene produced by recombinant bacterium 8 after 2 days of fermentation, after 5 days of fermentation, β -elemene yield of recombinant bacterium 8 is the highest, which reaches 1052mg/L, namely, 1052mg of β -elemene is produced per liter of fermentation liquid.

The yields of the recombinant bacteria 1, 2, 3, 4, 5, 6 and 7 are respectively 61mg/L, 201mg/L, 254mg/L, 187mg/L, 79mg/L, 246mg/L and 413 mg/L.

SEQUENCE LISTING

<110> Nanjing university of industry

<120> recombinant yarrowia lipolytica, construction method and application thereof

<130>20200121

<160>4

<170>PatentIn version 3.3

<210>1

<211>1683

<212>DNA

<213> Roman Chamomile

<400>1

atggccgctg tccaagccaa cgtcaccacc ggcgtccaag ccaacaccaa gacctctgcc 60

aagcccgtca gacctctggc caacttcccc ccttccgtgt ggggtaccag attcctctcc 120

ttctctctgg acaagtccga gttcgagcga tatgccatgg ccatggaaaa gcccaaggag 180

gatctgcgaa atctgattgt ggaccccaag atggactcca acgaaaagct cggtctgatc 240

tactccgtgc atcgactcgg tctgacctac attttcctcg aggagatcga gtctcagctg 300

gacaagctct tcaacgagtt ctctctgcaa gactacgaag acgtggatct gtacaccatc 360

tctatcaact tccaagtctt tcgacatctg ggctacaagc tcgcttgcga cgtgttcaat 420

aaattcaagg acgtgtcctc cggcaccttc aaagagtcca ttacctccga cgtccgaggt 480

atgctgggtc tgtacgagtc tgcccagctg cggatccgag gcgaaaagat tctggacgag 540

gcttccgtgt tcatcgaggg taagctgaag tctgtcgtga acactctgga gtgcaatctc 600

gcccagcaag tcaagcagtc tctgaagcga cccttccacc aaggcatgcc cattgtggag 660

gcccgactct acttttccaa ctacgaggag gaatgttcct cccacgactc tctgttcaag 720

ctcgccaaac tgcacttcaa gtatctggag ctccagcaga aggaggagct ccgaatcgtg 780

accaagtggt acaaggacat gcgattccaa gaggacaccc cttacgtccg agatcgagtc 840

cccgagatct atctctggat cctcggcctc tactttgagc cccattactc tctcgctcga 900

atcatcgcca ccaagatcac tctgtttctc ctcatcctcg atgacaccta cgacgcttac 960

gccaccatcg acgagattcg actgctgact tatgccatta ataagtggga catttccgct 1020

atggagcaga tccccgaata catccgaccc ttttacaaag tcctcatcaa cgagtacgcc 1080

gagatcgaga agaagatggc taaggagggt cgagccaaca ccgtgatcgc ctccaaggag 1140

gccttccaag acattgctcg aggccatctg gaagaggccg agtggaccaa ctctggttat 1200

gtggcctcct tccccgacta catgaagaac ggtctgatta cctccggcta caacctcatc 1260

tccaagtctg ctctggtggg catgggtgac aacgtcggtg aggacgctct ggcttggtac 1320

gagtctcacc ccaagcctct gcaagcctcc gaactgattt ccagactgca agacgacgtg 1380

atgacctacc agttcgaacg agaacgaggt cagtctgcca cctccgtcga cgcctatatc 1440

aagacctacg gcgtgtccga aaagaaagcc attgacgagc tgaagtccat gatcgagaat 1500

gcttggaagg acatcaacga gggctgtctc aagccccgac aagtccctat ggatctgctg 1560

gccatcattc tgaacctcgc ccgaatgatc gacgtgatct accgatacga cgacggtttc 1620

accttccaag gctctaccat gaaggagtac attaacattc tgttcgtcga ctctctgccc 1680

gtg 1683

<210>2

<211>1503

<212>DNA

<213> yarrowia lipolytica

<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

<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

<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

1. A recombinant yarrowia lipolytica is yarrowia lipolytica (yarrowia lipolytica) (A)Yarrowia lipolytica) The strain XJ-8 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 19064.

2. The recombinant yarrowia lipolytica of claim 1, wherein said recombinant yarrowia lipolytica (Yarrowia lipolytica) The XJ-8 strain is obtained by inserting an expression cassette of germacrene A synthetase, farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase into the genome of yarrowia lipolytica.

3. The recombinant yarrowia lipolytica of claim 2, wherein said germacrene A synthase is derived from Chamomile (R) (Roman ™)Chamaemelum nobile) The coding gene is suitable for expressing in yarrowia lipolytica after codon optimization, and the coding genes of farnesyl pyrophosphate synthetase and 3-hydroxy-3-methylglutaryl CoA reductase are derived from yarrowia lipolytica.

4. The recombinant yarrowia lipolytica of claim 3, wherein said recombinant yarrowia lipolytica is obtained by inserting a 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and a fusion protein expression cassette into the genome of yarrowia lipolytica; the fusion protein consists of germacrene A synthetase, connecting peptide and farnesyl pyrophosphate synthetase; the connecting peptide is GGGGS or GSG or EAAAK.

5. The recombinant yarrowia lipolytica of claim 4, wherein said promoter of said expression cassette is the TEFin promoter or TEF1 promoter of yarrowia lipolytica; the terminator is the xpr2t or cyc1 terminator of yarrowia lipolytica.

6. The recombinant yarrowia lipolytica of claim 5, wherein said recombinant yarrowia lipolytica further expresses 1 or more marker genes selected from the group consisting of a 3(β) -isopropylmalate dehydrogenase encoding gene cassette and an orotidine-5' -phosphate decarboxylase encoding gene cassette.

7. The recombinant yarrowia lipolytica of claim 6, wherein said gene encoding said germacrene A synthase is as set forth in SEQ ID No: 1 is shown in the specification; the coding gene sequence of the 3-hydroxy-3-methylglutaryl CoA reductase is shown as SEQID No: 2, respectively.

8. The method of claim 7, wherein said method comprises the step of introducing said 3-hydroxy-3-methylglutaryl CoA reductase expression cassette, germacrene A synthase expression cassette and farnesyl pyrophosphate synthase expression cassette into said yarrowia lipolytica in the form of a plasmid and then integrating them into the genome of said yarrowia lipolytica.

9. The method of claim 8, wherein said recombinant yarrowia lipolytica is constructed by introducing said 3-hydroxy-3-methylglutaryl CoA reductase expression cassette and said fusion protein expression cassette into said yarrowia lipolytica in the form of a plasmid and then integrating into the genome of yarrowia lipolytica; the fusion protein is composed of germacrene A synthetase, connecting peptide and farnesyl pyrophosphate synthetase.

10. The application of the recombinant strain in any one of claims 1 to 7 in the production of β -elemene and/or germacrene A is characterized by comprising the following steps of (1) culturing the recombinant strain in any one of claims 1 to 7 by using a fermentation medium to obtain a fermentation product, (2) extracting the fermentation product by using an organic solution, collecting an organic phase to obtain the germacrene A, and (3) heating the organic phase to 300 ℃ at 250 ℃ to obtain β -elemene.